Estrogen Metabolism, Iodine, 2MEO Part Three

Ellen is a 56 year old physician doing well with her bioidentical hormone replacement program with full relief of menopausal symptoms of hot flashes and night sweats, and better sleep. She is taking an iodine supplement called Iodoral which is a tablet form of Lugol’s solution, and during a telephone follow up call she asked a question about the iodine supplement. “Is it really needed? The dosage seems excessive.” And, she read on the internet that Iodine can be harmful. In this chapter, we will discuss further the importance of iodine for breast cancer prevention, specifically, as iodine relates to estrogen metabolism. In short, Iodine is very beneficial for metabolism of estradiol into the “good” intermediates, and away from the “bad” ones.

Header Image: Le Canoeist Luncheon by Pierre-Auguste Renoir (1841–1919)
circa 1879, oil on canvas, Art Institute of Chicago, Potter Palmer Collection
Creative Commons Attribution-Share Alike 1.0 License or the GNU Free Documentation License, Version 1.2 Courtesy of Wikimedia Commons. Source. Permission

Estrogen Metabolism in a Nutshell

Estradiol Produced by Aromatase Conversion of Testosterone

The ovary is not the only organ which has the aromatase enzyme for conversion of testosterone to estradiol. Other sites with aromatase activity include the brain, retina, adrenal gland, testis, blood vessels (vasculature), fat cells (adipose tissue), skin and bone, all capable of converting testosterone to estrogen, thus producing estrogen. Where does all this estrogen go? Excess estrogen is metabolized by the liver into intermediates which are then excreted by the liver into the bile and into the lumen of the GI tract, and then excreted in the stools. The study of estrogen metabolism involves a detailed look at these estrogen metabolite intermediates, some of which are good, and some bad. This is also called Estrogen Detoxification, the elimination of toxic estrogen metabolites.

What are the Three Human Estrogens? E1, E2 and E3.

When we refer to estrogen, we must be specific about which type of estrogen we are referring to. There are three human estrogens, E1, E2 and E3. A fourth type of estrogen is derived from pregnant horses called equine estrogen or CEE (conjugated equine estrogen), trade name Premarin.

E1 (Estrone), weak estrogen, produced by body fat, placenta and ovaries. E1 and E2 are reversibly converted to each other by 17β-hydroxysteroid dehydrogenase in the liver.
E2 (Estradiol), produced by aromatase conversion of testosterone to estradiol. E2 is the strongest estrogen with very strong binding to estrogen receptors, 50% to ER alpha and 50% to ER beta. E2 is made in ovaries through aromatase conversion of testosterone.
E3 (Estriol) weak estrogen, is the main estrogen of pregnancy, secreted by the placenta. Estriol is produced by conversion of estradiol to estriol, and predominantly activates ER-beta, acting as a tumor suppressor. (1)

ER Alpha and ER Beta

Notice the above discussion of estrogen involves describing the ability to bind to and activate the two estrogen receptors, ER-alpha and ER-beta. This is important because ER-alpha is the proliferative receptor, associated with increased breast cancer risk, while ER-beta is the tumor suppressor receptor, with reduced risk for breast cancer. (2-4)

The Three Phases of Estrogen Detoxification

As mentioned above, Estradiol (E2) and Estrone (E1) are readily inter-converted in the liver by the17β-hydroxysteroid dehydrogenase enzyme, so estrogen metabolism starts with conversion of estradiol to estrone (E1) which enters the Phase One Detoxification pathway:

The Phase One Estrogen Metabolism Pathways

1) (CYP1A1 enzyme) acts on E1 => making 2-OH-E1 (2-hydroxy-estrone) => and then 2–MeO-E1 (2 methoxy-estrone) and 2 methoxy-estradiol (2MEO). This is Very GOOD ! This pathway requires a functioning COMT enzyme. 2MEO has anticancer activity as discussed below.

2) (CYP1B1 enzyme) converts E1=> 4-OH-E1 (4-hydroxy-estrone) => Quinones and DNA Adducts. These are carcinogenic and  BAD ! and is associated with higher risk for breast and endometrial cancer. This pathway is driven by CYP1B1 and becomes dominant when patient has SNP in COMT causing impaired COMT function. 4-Hydroxy Estrogens lead to 4-Hydroxy Quinones which produce carcinogenic DNA Adducts. If COMT is functional then 4-OH-E1 is methylated to 4-MeO-E1 (4 methoxy-estrone) This is Good !

3) Estrone (E1) =>16-OH-E1 (16-hydroxy-estrone) may ultimately be converted to estriol (E3), a weak estrogen with preferential ER-beta binding, this is a beneficial a tumor suppressor receptor. However, the 16-OH-estrone metabolites are generally regarded as genotoxic with high affinity for the estrogen receptor causing proliferative effects. This is BAD. (65-66)

Why is Methylation Important?

Adding a methoxy group to estrogen metabolites is called methylation, requiring a functioning COMT gene and a functioning MTHFR gene. SNPS (mutations) in either of these two genes causes poor methylation and increased levels of bad metabolites called quinones which lead to DNA adducts, increasing breast cancer risk. In 2016, Dr Samia Shouman writes:

Estradiol is metabolized into 2-OHE2 and 4-OHE2 by CYP1A1 and CYP1B1, respectively. These catechols undergo further oxidation into semiquinones and quinones that react with DNA to form depurinating adducts leading to mutations associated with breast cancer. (5)

See Estrogen Metabolism Chart below, courtesy of (Shouman, 2016)(5):

Above Chart: Green Ellipses show conversion of Estradiol by CYP 1A1 enzyme (P450 system) to beneficial metabolite 2-Hydroxy-Estradiol (2-OH-E2), which is then converted by COMT (Catechol-O-Methyl Transferase) into 2-methoxy-estradiol, a favorable metabolite which has anti-cancer effects. Red Ellipses show conversion of estradiol by CYP1B1 to 4-Hydroxy-Estradiol, a carcinogenic metabolite. If COMT is functioning, this will convert estradiol to 4-Methoxy-Estradiol. If the COMT gene is mutated, then COMT enzyme is impaired and 4-OH metabolites will be converted to Quinones and DNA Adducts which are carcinogenic. This is VERY BAD. 4-Hydroxy estrogens attach to estrogen receptors with very high affinity, and are considered carcinogenic metabolites.(5)

Below Chart: Phase One Estrogen Metabolism Simplified:

Above Chart Shows two major estrogen pathways denoted by Upper Green Arrows-CYP1A1  and Lower Red Arrows- CYP1B1.

Upper Panel: Estradiol is metabolized by the CYP1A1 enzyme (Green Arrows) to 2-Hydroxy-estradiol (2-OH-E2), which is then metabolized by COMT (catechol-O-Methyl-Transferase) enzyme to the anti-cancer agent, 2MEO-E2 (2 Methoxy-Estradiol) Shown in Green Ellipse.

Lower Panel: However, Estradiol can also be metabolized by the CYP1B1 enzyme (Red Arrows) to 4-Hydroxy-Estradiol (4-OH-E2) leading to Quinones and DNA Adducts which are carcinogenic (Red Ellipse and Arrows). Above chart courtesy of (Itoh,Toshimasa, 2010)(6).

Phase Two Estrogen Metabolism: Conjugation

Phase Two Metabolism is “conjugation” which turns these estrogen metabolites into water soluble compounds easily excreted by liver into the gut or kidney into urine. This involves methylation, sulfation or glucuronidation.

Phase Three estrogen metabolism involves excretion of estrogen metabolites into the gut, supported by prebiotics, probiotics, Calcium D Glucarate and fiber.

Above information courtesy of Sarah Nyrose ND and click here to watch the You Tube Video.

4-OH-Estrogens are Highly Estrogenic and Carcinogenic, and are Markers of Breast Cancer

In 2019, Dr. Suyu Miao studied the effects of 4-hydroxy-estrogens (4-OH-estrogens) finding they bind more strongly to estrogen receptors than estradiol (E2). In addition to this more estrogenic effect, Dr. Miao found direct evidence of carcinogenicity of 4 hydroxy-estrogens by inducing breast cancer in athymic nude mice. In Dr. Suyu Miao’s mouse study, normal breast cells were pretreated with 4-OH-E2 and then injected into athymic nude mice.  80 percent of the injected mice breast developed breast cancer within 2 weeks. Control mice injected with untreated breast cells did not develop breast cancer. Thus Dr. Suyu Miao showed 4-OH-E2 to reliably induce breast cancer in a mouse model, writing:

…although 2-hydroxy metabolite 2-OHE2 had limited estrogenic activity, 4-hydroxy metabolite 4-OHE2 had much stronger estrogenic activity, which was even more potent than that of E2 in MCF-7 cells…The tumorigenic effect of 4-OH-E2 was first evaluated in athymic nude mice models. As expected, while mice injected with MCF10A cells [normal breast cells] into the mammary fat pad of nude mice did not form tumors, [however] four out of five mice (80%) injected with MCF10A-H cells formed tumors after 2 weeks. (7)

CYP1B1 Enzyme Creates 4-OH-Estrogens

Dr. Suyu Miao also studied the effects of CYP1B1 enzyme in the transgenic female mouse model expressing the CYP1B1 gene which converts estradiol (E2) to 4-OH-E2.  The mice were supplemented with E2, estradiol slow release pellets, and compared to control mice with no E2 pellet treatment. The E2 pellet treated transgenic mice had greater breast proliferation and higher KI-67 when compared to control mice which had undetectable KI-67. In addition, the E2 treated trans-genic mice had multiple mammary tumors, while the controls had none. Thus Dr. Suyu Miao demonstrated the CYP1B1 enzyme converts estradiol to 4-OH estrogens which are carcinogenic, writing:

The results indicated that although 2-hydroxy metabolite 2-OHE2 had limited estrogenic activity, 4-hydroxy metabolite 4-OHE2 had much stronger estrogenic activity, which was even more potent than that of E2 in MCF-7 cells…The effects of 4-OH-E2 on the mammary glands and development of mammary tumors were evaluated in a more relevant C57BL/6J-CYP1B1 transgenic mice model expressing transgene CYP1B1, which is a cytochrome P450 superfamily enzyme converting E2 into 4-OH-E2. Female mice were supplemented with or without E2 pellets (0.25 mg/90-day slow release). The effect of transgene expression on the mammary glands was assayed by morphological analyses of the gland after 90 days of E2 exposure (Figure 4). Histological evaluation of hematoxylin & eosin (H&E)-stained mammary sections revealed a normal monolayer and similar morphology in the glands from the virgin transgenic mice and the control littermate, indicating that the expression of transgene did not alter the mammary gland development. However, a significant alteration of the gland morphology with the appearance of mammary carcinoma in the transgenic mouse was observed when the mice were supplemented with E2 (Figure 4B). The epithelium of transgenic glands exhibited a disorganized structure with respect to the ordinate arrangement of the wild-type epithelium. Furthermore, while a normal mammary gland had a single layer of epithelial cells, the glands from transgenic mouse displayed a highly proliferative stage characterized by areas of multilayers. The duct lumen was narrowed and blocked by the hyperplastic glandular epithelial cells in the mammary glands of C57BL/6J-CYP1B1 + E2 mice, indicating a highly proliferative capability of the cells in the gland. Indeed, a strong immunohistochemical staining of Ki67 was only observed in the C57BL/6J-CYP1B1 + E2 group (Figure 4B), whereas Ki67 signal was undetectable in the mammary glands from other groups (Figure 4A). No morphological differences were observed in the tissues of liver and lung among all groups of mice. Taken together, multiple mammary tumors were only present in mice of the C57BL/6J-CYP1B1 + E2 group, but not in mice of other groups. (7)

The stepwise regression analysis revealed 4-OH-E1 to be the most important factor of breast cancer risk… the most significant alteration was an increase in 4-hydroxy metabolites relative to other metabolites in EMs [estrogen metabolites] in patients with breast cancer. Second, the biological relevance of increased 4-OH-E metabolism to breast cancer development was investigated in mammary epithelial cells and in vivo experimental models. It was found that 4-OH-E2 not only induced malignant transformation of breast epithelial cells in vitro but also stimulated tumor growth in the xenograft model and induced mammary carcinomas in the transgenic mice model expressing CYP1B1, a key enzyme of 4-hydroxy metabolites. Third, the molecular mechanisms underlying 4-OH-E metabolites induced malignant transformation. At the molecular level, 4-OH-E2 compromised the function of SAC spindle-assembly checkpoint (SAC) and thus rendered genome instability…Among many alterations of EMs in the breast cancer group, the most significant one in this study was an increase in 4-hydroxy metabolites. Urine 4-OH-E1 in the patients with breast cancer was three times higher than that in healthy women, while other EMs changed less. The best indicator that reflected the risk of breast cancer was the ratio of 4-hydroxy metabolites to total estrogen. Conclusions: In this comprehensive study of sexual hormone metabolism and risk of breast cancer in premenopausal women, we consider the ratio of 2-OHE1:16α-OHE1 is not a clear marker of breast cancer risk in premenopausal women. However, the ratio of 4-hydroxy metabolites to total estrogen is the best indicator reflects the risk of breast cancer. Our study found 4-OH-E2 induced carcinogenesis by destroying the SAC [spindle-assembly checkpoint] and induced the abnormal mitosis. The malignant cells transformed by 4-OH-E2 was hard to kill by antitumor drug. Thus, the relative proportions of 4-hydroxy estrogenic metabolites and 4-hydroxy estrogens are predictors of breast cancer risk and are also important factors in the prognosis of breast cancer patients and the choice of treatment…The ratio of 4-hydroxy metabolites to total estrogen is the best indicator reflects the risk of breast cancer. Our study found 4-OH-E2 induced carcinogenesis by destroying the SAC [spindle-assembly checkpoint] and induced the abnormal mitosis. (7)

In 2007, Dr. Alexandra R. Belous showed direct proof CYP1B1-mediated, E2-induced DNA adduct formation as the basis for carcinogenesis. (8-9)

How to Divert Estrogen Metabolism Away from BAD (4-OH-estrogens to GOOD Metabolites (2-OH-estrogens =>2-MeO-E1)

In view of the above discussion, the next logical question is : How can metabolism be shifted away from the 4-OH-estrogens, and towards the 2-OH estrogen pathway? This can be accomplished by upregulating the CYP1A1 enzyme in Liver, thus preferentially converting E1 (estrone) to 2-OH-E1 (2-hydroxy-estrone).

Next, we discuss using nutritional supplements such as Iodine (Iodoral), I3C/DIM, and resveratrol to manipulate estrogen metabolism. One useful dietary modification is to increase cruciferous vegetable such as broccoli and brussel sprouts. Or one may supplment with Indole 3 Carbinol (I3C) and its more active dimer, DIM (Di-Indole-Methane). Of course, one must avoid Endocrine Disrupting Chemical (EDC’s) such as BPA (Bisphenol-A) and Phalates in plastics, insecticides and other EDC’s. (10-12)

Using Iodine (Iodoral) to Manipulate Estrogen Metabolism

Let us next review how iodine can be used to manipulate estrogen metabolism toward the beneficial pathways, thus preventing breast cancer.

In 1982) Jonathan V. Wright, MD was the first to develop and introduce the use of bio-identical hormones, estrogens, progesterone, DHEA and testosterone. Dr. Wright discovered the effect of iodine on estrogen metabolism. A graduate of Harvard University and the University of Michigan Medical School (1969). Dr. Wright established Tahoma Clinic in 1973 in Washington State, Meridian Valley Laboratory (1976).

In 2008, Dr. Frederick R Stoddard II of Bernard Eskin’s group studied how iodine supplementation alters gene expression in MCF7 breast cancer cells, finding Lugol’s iodine, (5% I2, 10% KI) useful for manipulation of estrogen metabolism.

 Iodine Decreased ER-alpha mRNA Levels (Bernard Eskin Group)

Left Image: Fig 2A from (Stoddard, 2008) showing mRNA changes after Lugol’s Iodine in MCF7 breast cancer cells in-vitro. Green Ellipses show marked decrease in estrogen response elements TFF and WISP2, as well as CCND1 (Cyclin D1) mRNA compared to controls RED Arrows.(17-21)

In 2012, Dr. Alexander Poor, a member of Bernard Eskin’s group studied iodine, estrogen and breast cancer, discussing the 2008 gene array study (above) explaining Iodine inhibits the expression of estrogen-responsive genes TFF1 and WISP2, and up-regulates estrogen metabolism (CYP1A1, CYP1B1, and AKR1C1).  Lastly, iodine decreases Cyclin D1 (a competitive inhibitor of BRCA1) mRNA levels which may functionally permit BRCA1 inhibition of estrogen responsive transcription. Finally, ERα [Estrogen Receptor Alpha] mRNA levels are decreased by Lugols’ Iodine in MCF-7 (breast cancer) cells in-vitro, writing:

We analyzed the effects of iodine on global gene expression in estrogen responsive MCF-7 breast cancer cell line. Microarray analysis and quantitative real time polymerase chain reaction (RT-PCR) indicated that iodine inhibits the expression of estrogen-responsive genes TFF1 and WISP2 [27]…Our data further provided three potential mechanisms to explain the observed decrease in estrogen response. First, iodine treatment results in decreased ERα mRNA levels; second, iodine up-regulates genes involved in estrogen metabolism (CYP1A1, CYP1B1, and AKR1C1), and finally, iodine decreases Cyclin D1 (a competitive inhibitor of BRCA1) mRNA levels which may functionally permit BRCA1 inhibition of estrogen responsive transcription. Thus the interaction between iodine and estrogen signaling may inhibit breast cancer growth by affecting an intermediate, perhaps the estrogen receptor system. (13)

Note: TFF1 is the classical target gene of the Estrogen Receptor and the most studied within the medical literature.

Note: Estrogen Receptor Alpha (ER-alpha) is proliferative, while Estrogen Receptor Beta (ER-beta) is Tumor Suppressor. Activation of ER-alpha activates transcription of the proliferative oncogene, Cyclin D1, implicated in carcinogenesis. The BRCA1 gene is a tumor suppressor gene, so any impairment of BRCA1, such as elevated cyclin D1 or a mutation in the BRCA1 gene is carcinogenic. According to Dr C. Wang in 2005, upregulation of Cyclin D1 prevents the BRCA1 gene from repressing the proliferative effect of ER-alpha (Estrogen Receptor alpha). The main activity of the BRCA1 gene is repair of oxidative damage, such as double strand breaks in DNA using the seleno-protein system. Thus, selenium supplementation has been found effective for reducing breast cancer risk associated with BRCA1 mutation. (14-16)

Iodine Increases the CYP1A1/CYP1B1 Ratio

The 2008 gene array study by Stoddard reveals iodine supplementation shifts the ratio in favor of CYP1A1 over CYP1B1, thus increasing 2-hydroxyestradiol (2-OH-E2) which is then converted to the favorable 2-methoxyestradiol, a favorable metabolite which has anti-cancer effects. As noted in the previous charts of estrogen metabolism above, CYP1B1 leads to formation of the bad metabolite, 4-hydroxy-estradiol (4-OH-E2) which converts to the quinones which form carcinogenic DNA adducts. Thus, even though CYP1B1 mRNA production is increased 40-Fold by Iodine (Lugol’s Solution), the increase in CYP1A1 is a much greater 250 Fold. See the chart at left: Fig 2B (Stoddard, 2008) showing  Fig 2C (Green Arrow) 250 fold increase in CYP1A1, while CYP1B1 mRNA (RED ARROW) is increased only 40 fold. (17-21)

In 2008, Dr. Stoddard revealed Lugol’s iodine increases the CYP1A1/CYP1B1 ratio which leads to formation of 2-methoxy-estradiol which has strong anti-cancer effects, acting  independent from ER Alpha and Beta. Secondly, Lugol’s iodine prevents BRCA1 gene inhibition, freeing the BRCA1 gene to inhibit ER-alpha signalling and thus downregulate proliferation. The BRCA1 gene is also responsible for DNA damage repair, writing:

The observed increase in the CYP1A1/CYP1B1 ratio may shift the direction of estrogen metabolism favoring 2-OH-E₂ which may either directly affect proliferation through increasing 2-methoxyestradiol, decreasing 3, 4-estradiol quinone or indirectly via the inactivation of E₂. The importance of the CYP1A1/CYP1B1 ratio in-vivo is evident in the increased presence of 4-OH-E₂ in breast cancer tissue compared to non breast cancer controls….Data presented suggests that iodine/iodide may inhibit the estrogen response through…1) up-regulating proteins involved in estrogen metabolism (specifically through increasing the CYP1A1/1B1 ratio), and…2) decreasing BRCA1 inhibition thus permitting its inhibition of estrogen responsive transcription [ER-alpha]. (17-21)

I3C/DIM Upregulates CYP1A1 Estrogen Metabolism

As mentioned above, upregulation of the CYP1A1 pathway is beneficial as this leads away from quinones and DNA adduct formation, and leads towards formation of the natural anti-cancer agent, 2-methoxy-estradiol. A number of animal studies have shown I3C treatment upregulates CYP1A1 gene expression. In 2010, Dr. N.V. Trusov found upregulated mRNA content in liver for CYP1A1, CYP1A2, and CYP3A1 enzymes in I3C treated mice. This effect was accompanied by an increased activity of phase II detoxification metabolism including the quinone reductase enzymes. (23-26)

I3C/DIM Anti-Cancer Effects

In 2008, Dr. Jing-Ru Weng studied I3C and its more active dimer, DIM, finding suppression of proliferation in breast, colon, prostate, and endometrial cancer cell lines.  DIM also inhibited spontaneous or chemical-induced cancer formation in breast , liver, lung, cervix, and gastrointestinal tract in various animal model studies. (27)

I3C Downregulates ER-alpha and Induces CYP1A1

In 2008, Dr. Jing-Ru Weng found I3C/DIM downregulates ER-alpha signalling, while at the same time increasing binding of ER-beta to ERE (estrogen response elements), resulting in strong anti-proliferative effects, writing:

Indole-3-carbinol is a negative regulator of ERα signaling in human tumor cells . In addition to altering estrogen metabolism through CYP1A1, indole-3-carbinol and its metabolites also affect ER signaling through two different mechanisms…indole-3-carbinol and DIM could suppress ERα expression in breast cancer cells … Moreover, indole-3-carbinol was reported to increase the binding of ERβ to the estrogen response element, resulting in a significantly higher ERβ/ERα ratio that is associated with an antiproliferative status in human breast cancer cells…(27)

I3C Reduces ER alpha by 60 Percent

In 2006, Dr. Thomas Wang used MCF-7 (breast cancer) cells in-vitro to show that I3C reduces ER-alpha mRNA expression by 60%. DIM is the more active dimer of I3C, showing a 20 fold greater potency than I3C, and is therefore the preferred supplement. (28)

I3C Induces 6-fold increase in binding of ER-Beta to the Estrogen Response Element

In 2006, Dr. Shyam Sundar studied two breast cancer cell lines, in-vitro, finding I3C strongly downregulated ER-alpha protein levels, and transcription. Even though ER-beta protein levels remains unchanged, I3C induced 6-fold increased binding of ER-beta to its ERE [Estrogen Response Element], thus acting as a tumor suppressor, writing:

Taken together, our results demonstrate that the expression and function of ERα and ERβ can be uncoupled by I3C with a key cellular consequence being a significantly higher ERβ:ERα ratio that is generally highly associated with antiproliferative status of human breast cancer cells. (29)

I3C Effective for Ulcerative Colitis in Animal study

Indole-3-Carbinol (I3C) has shown benefits for ulcerative colitis in animal models. In 2021, Dr. Shunting Peng writes:

I3C is a good candidate as a natural product to prevent and treat UC [Ulcerative Colitis]. (30-31)

DIM Increases 2-OH-Estrogen in Human Study

In 2024, Dr. Mark Newman studied the effect of DIM on estrogen metabolism in premenopausal women using dried urine collection showing DIM increases  2-OH-estrogens (2-OH-E1). Dim also increased the the 2/16 ratio (ratio of 2-OH-estrogens to 16-OH estrogens). (32)

More Charts on Estrogen Metabolism

Here is a more recent chart from 2023 showing the the enzymes involved in various estrogen conversions.(Al-Shami, Khayry, 2023). (33)

Notice estradiol (E2) is freely inter-converted to estrone (E1) by the 17 Beta HydroxySteroid Dehydrogenase enzyme in the Liver. This reversible conversion also applies to the 2-OH metabolites of E1 and E2. 2-Hydroxy-Estrone is converted by the COMT enzyme to the GOOD metabolite 2 methoxy-estrone, which is then converted to 2 methoxy-extradiol (not shown on this chart).

Above Image: Fig. 3 from (Al-Shami, Khayry, 2023) Estrogen Metabolism Chart: Notice Green Ellipse is 2 Methoxy estrone which made by converting 2-hydroxyestrone to 2-methoxyestrone and 2-methoxyestradiol (not shown) by the COMT enzyme (Catechol-O-Methyl-Transferase).  The other Green Ellipse is 4-Methoxy-Estrone which is also made by the COMT enzyme.  These are GOOD metabolites. However, If the COMT gene is mutated, and the COMT enzyme is impaired, then the result is the RED Ellipse, catechol estrogens made by CYP1B1 are converted to quinones which become DNA adducts (see below red ellipse) which then attach to DNA causing oxidative damage. This is carcinogenic. The catechol estrogens are BAD metabolites. Courtesy of: Al-Shami, Khayry, et al. “Estrogens and the risk of breast cancer: A narrative review of literature.” Heliyon 9.9 (2023). (33)

Another Chart of Estrogen Metabolism

See below chart of estrogen metabolism without the enzymes depicted. Note: 2-hydroxy estrone (2-OH-E1) is reversibly converted to 2 hydroxy-extradiol (2-OH-E2), and then to 2 methoxyestradiol (2MEO-E2), a natural anti-cancer agent:

Above Image: Notice Green Ellipse and Arrows. This is the pathway leading to 2-Methoxyestradiol, the GOOD Estrogen Metabolite, a natural anticancer agent. Chart on Estrogen Metabolism Courtesy of Dr. Barbara Fuhrman and NIH, 2012 (67)

Back to Medical School?

What is it like in medical school? Memorize the above three charts and be able to reproduce them on your final exam.

Predicting Breast Cancer Risk with Genetic Testing

SNPs (Single Nucleotide Polymorphism) COMT gene

Mutations in genes for the enzymes, COMT, CYP1A1, CYP1B1, ER-alpha, and ER-beta, also called SNPs. These are important causes of breast cancer because they impair estrogen metabolism. In 2015, Dr Hamed Samavat writes:

It has been postulated that genetic polymorphisms [also called SNPs] in genes encoding enzymes involved in estrogen metabolism pathways and the genes encoding the ERs [estrogen receptors] are associated with breast cancer risk. Polymorphic variations in genes encoding COMT, CYP1A1, CYP1B1, estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), CYP17A1, and CYP19A1 have received extensive attention within the last decade. (1)

The enzyme, COMT (Catechol O methyl Transferase gene) is involved in methylation of estrogen, and conversion of 2 hydroxy estrone to 2 methoxy estrone. Any SNP (single nucleotide polymorphism, also called mutation) in the COMT gene will reduce the activity of the COMT enzyme will increase catechol estrogen accumulation leading to catechol estrogens, quinone-estrogens and DNA adducts which are carcinogenic. Normally, estradiol binds to ER-alpha and ER-beta receptors in our DNA without causing any oxidative damage the DNA. However, catechol estrogens are highly oxidative creating DNA adducts and DNA mutations, thus inducing breast cancer. Preventiion inviolves supplements such as Iodine (Iodoral), I3C/DIM, resveratrol, and NAC. Methyl donors such as Vitamins B12, B6 and methylfolate assist the methylation pathways. As you might suggest from the above discussion, it is possible to predict breast cancer risk based on genetic testing for mutations in genes for the following enzymes: COMT, CYP1A1, and CYP1B1. In 2021, Dr. Feng Zhao did exactly this, finding good predictive ability.(34)

PolyMorphisms in Estrogen Detoxification Pathways Increase Cancer Risk

In 2021, Dr. Micaela Almeida studied polymorphisms in genes involving metabolic pathways in estrogen detoxification using samples from 157 women with breast cancer. Genes studied were GSTM1, GSTT1, CYP1B1 Val432Leu and MTHFR C677T. The genes for GSTM1 and GSTT1 code for phase II enzymes that detoxify catechol estrogen quinones through the conjugation of glutathione (GSH). The absence of these enzymes, due to the null polymorphism of GSTM1 and GSTT1, compromises the detoxification and allows accumulation of catechol estrogens, leading to carcinogenic DNA adduct formation. The polymorphism Val432Leu (CYP1B1) increases CYP1B1 activity, contributing to higher levels of 4-Hydroxy catechol estrogens that are detoxified by Phase II enzymes…The polymorphism of MTHFR C677T reduces MTHFR activity, leading to decreased activity of COMT (catechol-O-methyl-transferase) needed for prevention of catechol estrogen formation. Dr. Micaela Almeida found various combinations of polymorphisms increased breast cancer risk, writing:

GSTM1 and GSTT1 are phase II enzymes that detoxify catechol estrogen quinones through the conjugation of GSH. The absence of theseenzymes, due to the null polymorphism of GSTM1 and GSTT1, compromises the detoxification and allows the accumulation of catechol estrogens, leading to DNA adducts formation …We suppose that prolonged exposure to estrogen levels combined with an inefficient detoxification due to GSTM1 and GSTT1 null genotype are related to breast cancer development at later ages. This fact can be explained by the accumulation of catechol estrogens and DNA adducts formation during a lifetime, which culminate in breast cancer development. ..A two-way association of MTHFR C677T and GSTT1 null genotype was performed and we verified that the majority of women carriers of both altered T allele of MTHFR C677T and GSTT1 null genotype were 50 years old or more at the age of [breast cancer] diagnosis (p-value = 0.034). These results might be explained by the fact that the metabolic pathway is extremely compromised due to inexistent GSTT1 and low COMT activity; low levels of Phase II enzymes highly compromise 4-OH-E2 detoxification and eventually will contribute to tumor development due to inefficient estrogens detoxification during reproductive life. (35)

SNPs Increased Risk for Ovarian Cancer 6-Fold

In 2021, Dr. Ercole Cavalieri studied SNPs in estrogen metabolic pathways, finding SNPs in CYP1B1 and COMT increased ovarian cancer by either 300 percent, or 600 percent depending on whether there was one or two copies of the mutations, writing:

When women had one or two copies of the SNP for a more active CYP1B1 plus two copies of the SNP for a less active COMT, they were three times more likely to have ovarian cancer, and had approximately twice the ratio of estrogen-DNA adducts to estrogen metabolites and conjugates as women without the SNPs. When the women had two copies of both the CYP1B1 and COMT SNPs, they were six times more likely to have the disease and had even higher estrogen-DNA adduct ratios.(36)

Resveratrol and NAC Prevents Quinone and Adduct Formation

The next logical question is how can we prevent estrogen-quinone formation and thus reduce cancer risk. In 2021, Dr. Ercole,Cavalieri found resveratrol and NAC (N-acetyl-cysteine) useful for preventing estrogen quinones. Resveratrol is a a natural substance found in grape skins and peanuts having anti-inflammatory, antioxidant, antihyperlipidemic, and anticarcinogenic, immune-modulating, cardioprotective, hepatoprotective, and neuroprotective properties. Resveratrol has the ability to prevent estrogen quinone formation by reducing CYP1B1 activity, and thus reducing 4-hydroxy-quinones. Both NAC and Resveratrol convert semi-quinones back to catechol estrogens which can then be methylated to safer metabolites. NAC also forms conjugates with quinones, preventing formation of DNA adducts. Dr. Ercole,Cavalieri writes:

Both resveratrol and NAC have been shown to inhibit the formation of depurinating estrogen-DNA adducts in cultured mammalian cells. Resveratrol was found to inhibit the malignant transformation of the human MCF-10F breast epithelial cell line. NAC was found to inhibit the malignant transformation of both MCF-10F and immortalized mouse mammary cells. The two compounds work together additively to reduce the formation of depurinating estrogen-DNA adducts in MCF-10F cells treated with 4-OHE2. These results lay the foundation for investigating the ability of resveratrol and NAC to reduce estrogen-DNA adduct formation in humans as an approach to cancer prevention…Estrogens initiate cancer through metabolism to catechol estrogen-3,4-quinones. The quinones react with DNA to primarily form the depurinating DNA adducts 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade. The resulting apurinic sites in specific locations in DNA generate mutations that can initiate cancer; this has been shown in laboratory animals and human beings. Two compounds, resveratrol and NAC, are potential cancer-preventive compounds that can reduce the formation of estrogen-DNA adducts in people, thereby blocking the initiation of estrogen-induced cancer…Other evidence points to an ER-independent mechanism…The observation of high ratios of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates in women at high risk for breast cancer, as well as women with breast cancer, is consistent with an ER [Estrogen Receptor] -independent mechanism of initiation. In addition, the presence of SNPs in CYP1B1 and COMT that increase both the formation of depurinating estrogen-DNA adducts and the likelihood of ovarian cancer (six-fold) supports an ER-independent mechanism of cancer initiation by estrogens.(36)

Resveratrol Downregulates ER-Alpha

Another benefit of resveratrol is the down-regulation of ER-alpha, the proliferative estrogen receptor. In 2016, Dr. Julieta Saluzzo studied in-vitro breast cancer cells, finding resveratrol downregulated ER-alpha protein. (37)

Berberine Increases CYP1A1 over CYP1B1 

In 2014, Dr. Chun-Jie Wen studied berberine, a botanical used as a nutritional, in MCF-7 breast cancer cells in-vitro, finding preferential induction of CYPA1A over CYP1B1, suggesting anti-cancer effects, writing:

Previous reports suggested that 2-OH E2 have putative protective effects, while 4-OH E2 is genotoxic and has potent carcinogenic activity. Thus, the ratio of 2-OH E2/4-OH E2 is a critical determinant of the toxicity of E2 in mammary cells. In the present study, we investigated the effects of the berberine on the expression profile of the estrogen metabolizing enzymes CYP1A1 and CYP1B1 in breast cancer MCF-7 cells. Berberine treatment produced significant induction of both forms at the level of mRNA expression, but with increased doses produced 16~ to 52~fold greater inductions of CYP1A1 mRNA over CYP1B1 mRNA. Furthermore, berberine dramatically increased CYP1A1 protein levels but did not influence CYP1B1 protein levels in MCF-7 cells. In conclusion, we present the first report to show that berberine may provide protection against breast cancer by altering the ratio of CYP1A1/CYP1B1, could redirect E2 metabolism in a more protective pathway in the breast cancer MCF-7 cells. (51)

MethylFolate for Homozygous MTHFR carriers.

About two thirds (66%) of the population has at least one mutation in the MTHFR gene, either 677C or 1298A, the two predominant variants of the MTHFR mutation. About 8.5-13.5 percent of the population harbors the most clinically expressed homozygous variation with both matching chromosomes bearing a mutation, and about 2.25% of people have compound heterozygous variant, meaning mutations with two different variants which may or may not reach clinical expression. (38-39)

The MTHFR Mutation

MTHFR mutation has been associated with increased risk for breast and other cancer, coronary artery disease, neuro-psychiatric disorders, etc. Treatment involves supplementation with methyl-folate rather than the folate inactive form of the vitamin. A good quality multivitamin usually contains methyl-folate, but always check the label to make sure, and avoid the lower quality multivitamins which contain folic acid. (40-42)

2-Methoxy-Estradiol (2MEO) is a Natural AntiCancer Drug

What is the mechanism of 2MEO acting as a natural anti-cancer drug? 2ME0 inhibits tubulin polymerization, and serves as a microtubule inhibitor. As such, 2MEO bears a resemblance to many other anti-cancer microtubule inhibitor drugs. In this category, yopu will find the repurposed  anti-parasitic drugs fenbendazole (veterinary use), mebendazole (human use), and albendazole. Conventional chemotherapy drugs in the taxane family are microtublule inhibitors such as paclitaxel (Taxol), docetaxel (Taxotere) and cabazitaxel (Jevtana). Vinblastine and colchicine are also microtubule inhibitors. (43)

2MEO Synergy with Albendazole

In 2013, Dr. Anahid Ehteda studied 2MEO synergy with a second microtubule inhibitor, albendazole using the colorectal cancer cells (HCT-116) xenografted into nude mice, finding the two drugs were indeed synergistic anticancer agents. Both colchicine and 2MEO are ligands for the colchicine binding site on tubulin. Remember, the anticancer activity of 2MEO is independent of estrogen receptors alpha and beta. (44-47)

Above image: chemical structure of colchicine (left) and 2MEO (right). Both bind to the colchicine binding site on tubulin. Figure courtesy of (Zefirov, Nikolai A, 2019) (48)

Inhibiting tubulin formation prevents the spindle formation required for mitosis (cell division) thus causing cell cycle arrest in prometaphase (G₂/M phase). The guardian of the genome, P53, senses the cell cycle arrest and becomes activated. This upregulation of P53 takes as little as 10 nM of 2 MEO, resulting in apoptosis (programmed cell death) and inhibition of NFKappa-B transcriptional activity (Nuclear factor Kappa-B is the master inflammatory controller). (49)

Anti-Cancer Activity of 2MEO in Various Cancers

Potent anti-cancer activity of 2MEO in various cancer cell types has been known for over 25 years, and has been studied in many cancers. (52-64)

Here is the list:

breast cancer (Brueggemeier, Robert , 2001)
nasopharyngela carcinoma   (Zhou, Ning Ning 2004)
osteosarcoma (Tang, Xiaoyan, 2020)
colorectal cancer (Carothers, A. M, 2002) (Lee, Ji Young, 2014),
lung cancer (Mukhopadhyay, Tapas, 1997),
oral Cancer (Takata, Hidehiko, 2004),
uterine sarcoma (Amant, Frederic, 2003),
pancreatic cancer (Schumacher, Guido,1999),
T lymphoblastic leukemia (Zhang, Xueya, 2010),
prostate cancer (Kumar, Addanki, 2001),
anaplastic thyroid cancer (Roswall, Pernilla, 2006),
melanoma (Hua, Weitian, 2022).
(52-64)

2-MEO Remains Largely Unknown

2-methoxy-estradiol shows considerable promise as a safe highly effective cancer drug. One might ask why 25 years has gone by without any drug maker showing interest in making 2MEO widely available as a safe oncology drug? Unfortunately, since there is no patent protection for a natural substances, the drug will probably will never be formally studied in human trials, and will never receive FDA approval and will remain largely unknown. Despite its status outside of conventional oncology, 2methoxyestradiol is available for the brave at heart as an oral capsule from a few selected compounding pharmacies in the United States. I find this a sad commentary on the state of oncology drug discovery in the US.

Back to the WHI Second Arm Study

Remember the second arm of the Women’s Health Initiative which found 23 percent reduction in breast cancer in the estrogen (CEE) treated group? The mechanism described by V. Craig Jordan is the induction of apoptosis after LTED (long term estrogen deprivation). The second arm of the WHI used older post-menopausal women who had been estrogen deprived for longer than 5 years. In a 2023 study by Dr. Masayo Hirao-Suzuki 2-methoxyestradiol was found a selective inhibitor of LTED MCF-7 breast cancer cells. These are cells which had been long term estrogen deprived. In these cells, 2MEO induced cell cycle arrest at low concentrations (1 microMolar). None of the other microtubule agents had this type of selectivity for LTED breast cancer cells. Dr. Masayo Hirao-Suzuki writes:

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells…derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents … LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. ..2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells…Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation. (50)

The above study by Dr. Masayo Hirao-Suzuki suggests another mechanism of estrogen induction of apoptosis in LTED breast cancer cells as described by V. Craig Jordan. This mechanism involves the endogenous production of 2MEO which then induces apoptosis in LTED breast cancer cells. (50)

Conclusion: Study of estrogen metabolism and detoxification pathways provide a new level of understanding of breast cancer risk, highlighting the use of Iodine, DIM and other supplements to shift estrogen metabolism away from CYP1B1 and towards CYPA1A leading to 2-methoxy-estradiol, a natural anti-cancer drug. On the other hand, if CYP1B1 predominates, this leads to 4-hydroxy-estrogens and carcinogenic DNA adduct formation. Natural supplements such as Lugol’s iodine, I3C/DIM, resveratrol, NAC, and methylfolate assist in manipulating estrogen detoxification pathways towards the favorable 2 methoxy-estradiol and away from 4-hydroxy quinones, thus reducing breast cancer risk.

Estrogen Metabolism Testing

A number of commercially available laboratories offer urine testing for estrogen metabolites, including the 4-hydroxy estrogens. At the time of writing, these include Genova lab, ZRT lab, Doctor’s Data Lab, and Dutch Test. Note: I have no financial interest in any of these laboratories.

Articles with Related Interest:

Iodine Treats Breast Cancer Overwhelming Evidence

Iodine Prevents Breast cancer Part One

Iodine Prevents Breast Cancer  Part Two

All Articles on Bioidentical Hormones

Jeffrey Dach MD
7450 Griffin Road, Suite 180/190
Davie, Florida 33314
954-792-4663

References:

Iodine and Estrogen Metabolism

1) Samavat, Hamed, and Mindy S. Kurzer. “Estrogen metabolism and breast cancer.” Cancer letters 356.2 (2015): 231-243.

2) Mal, Rahul, et al. “Estrogen receptor beta (ERβ): a ligand activated tumor suppressor.” Frontiers in oncology 10 (2020): 587386.

3) Jia, Min, Karin Dahlman-Wright, and Jan-Åke Gustafsson. “Estrogen receptor alpha and beta in health and disease.” Best practice & research Clinical endocrinology & metabolism 29.4 (2015): 557-568.

4) Williams, Cecilia, et al. “A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells.” Oncogene 27.7 (2008): 1019-1032.

5) Shouman, Samia, Mohamed Wagih, and Marwa Kamel. “Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells.” Cancer Biology & Medicine 13.4 (2016): 505.

6) Itoh, Toshimasa, et al. “A 3D model of CYP1B1 explains the dominant 4-hydroxylation of estradiol.” Journal of chemical information and modeling 50.6 (2010): 1173-1178.

7) Miao, Suyu, et al. “4-Hydroxy estrogen metabolite, causing genomic instability by attenuating the function of spindle-assembly checkpoint, can serve as a biomarker for breast cancer.” American Journal of Translational Research 11.8 (2019): 4992.

8) Belous, Alexandra R., et al. “Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation.” Cancer research 67.2 (2007): 812-817.

9) Liehr, Joachim G., and Mary Jo Ricci. “4-Hydroxylation of estrogens as marker of human mammary tumors.” Proceedings of the National Academy of Sciences 93.8 (1996): 3294-3296.

10) Kawa, Iram Ashaq, et al. “Bisphenol A (BPA) acts as an endocrine disruptor in women with Polycystic Ovary Syndrome: Hormonal and metabolic evaluation.” Obesity Medicine 14 (2019): 100090.

11) Talpade, J., et al. “Bisphenol A: An endocrine disruptor.” J Entomol Zool Stud 6.3 (2018): 394-7.

12) Hafezi, Shirin A., and Wael M. Abdel-Rahman. “The endocrine disruptor bisphenol A (BPA) exerts a wide range of effects in carcinogenesis and response to therapy.” Current molecular pharmacology 12.3 (2019): 230-238.

13) Poor, Alexander E., et al. “Urine Iodine, Estrogen, and Breast Disease.” Journal of Cancer Therapy 3 (2012): 1164-1169. (Authors: Alexander E. Poor^1*, Bernard A. Eskin², Christine Georgiadis¹, Brian Hamzavi¹, Ari D. Brooks¹⁾

14) Wang C, Fan S, Li Z, Fu M, Rao M, Ma Y, Lisanti MP, Albanese C, Katzenellenbogen BS, Kushner PJ. et al. Cyclin D1 antagonizes BRCA1 repression of estrogen receptor alpha activity. Cancer Res. 2005;65:6557-6567

15) Kowalska, Elzbieta, et al. “Increased rates of chromosome breakage in BRCA1 carriers are normalized by oral selenium supplementation.” Cancer Epidemiology Biomarkers & Prevention 14.5 (2005): 1302-1306.

16) Dziaman, Tomasz, et al. “Selenium supplementation reduced oxidative DNA damage in adnexectomized BRCA1 mutations carriers.” Cancer epidemiology, biomarkers & prevention 18.11 (2009): 2923-2928.

17) Stoddard II, Frederick R., et al. “Iodine alters gene expression in the MCF7 breast cancer cell line: evidence for an anti-estrogen effect of iodine.” International journal of medical sciences 5.4 (2008): 189.

The observed increase in the CYP1A1/CYP1B1 ratio may shift the direction of estrogen metabolism favoring 2-OH-E2 which may either directly affect proliferation through increasing 2-methoxyestradiol, decreasing 3, 4-estradiol quinone or indirectly via the inactivation of E2.

To elucidate the role of iodine in breast cancer, the effect of Lugol’s iodine solution (5% I2, 10% KI) on gene expression was analyzed in the estrogen responsive MCF-7 breast cancer cell line. Microarray analysis identified 29 genes that were up-regulated and 14 genes that were down-regulated in response to iodine/iodide treatment.

Quantitative RT-PCR confirmed the array data demonstrating that iodine/iodide treatment increased the mRNA levels of several genes involved in estrogen metabolism (CYP1A1, CYP1B1, and AKR1C1) while decreasing the levels of the estrogen responsive genes TFF1 and WISP2.[Wnt-inducible signaling pathway protein 2 (WISP2)]

In addition to elucidating our understanding of the effects of iodine/iodide on breast cancer, this work suggests that iodine/iodide may be useful as an adjuvant therapy in the pharmacologic manipulation of the estrogen pathway in women with breast cancer.

Our data shows that treatment with iodine and iodide increases the mRNA levels of Cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), two estrogen phase I estrogen metabolizing enzymes that oxidizes 17β-estradiol to 2-hydoxyestradiol (2-OH-E2) and 4-hydoxyestradiol (4-OH-E2), respectively. These catechol estrogens can be further oxidized to quinones. 3,4-estradiol quinine, a metabolite of 4-OH-E2, has been shown to react with DNA forming de-purinating adducts resulting in genotoxicity 42, while data suggests that 2-OH-E2 can be metabolized to 2-methoxyestradiol, an estrogen metabolite with anti-proliferative effects 43

LaVallee, Theresa M., et al. “2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors α and β.” Cancer research 62.13 (2002): 3691-3697.

The observed increase in the CYP1A1/CYP1B1 ratio may shift the direction of estrogen metabolism favoring 2-OH-E2 which may either directly affect proliferation through increasing 2-methoxyestradiol, decreasing 3, 4-estradiol quinone or indirectly via the inactivation of E2. The importance of the CYP1A1/CYP1B1 ratio in-vivo is evident in the increased presence of 4-OH-E2 in breast cancer tissue compared to non breast cancer controls 44. However, the regulation and interplay between CYP1A1, CYP1B1, other Phase I and II enzymes and estrogen is complex, being influenced by multiple factors and multiple polymorphisms, thus more data is required to illuminate the importance of these changes in response to iodine.
————————————–

In addition to affecting estrogen metabolism, iodine/iodide may also inhibit estrogen induced transcription via increased BRCA1 activity. BRCA1 is a known inhibitor of ERα transcription while Cyclin D1 is thought to enhance the estrogen response via a competitive inhibition with BRCA1 41.

41) Wang, Chenguang, et al. “Cyclin D1 antagonizes BRCA1 repression of estrogen receptor α activity.” Cancer research 65.15 (2005): 6557-6567.

Our data demonstrates decreased Cyclin D1 mRNA which could result in decreased competitive inhibition of BRCA1 allowing BRCA1 to inhibit estrogen induced transcription. Increased transcription of GADD45A, CYP1B1, and CYP1A1 are consistent with increased BRCA1 activity 34, 45.

—————————–

We suggest that the protective effects of iodine/iodide on breast disease may be in part through the inhibition or modulation of estrogen pathways. Data presented suggests that iodine/iodide may inhibit the estrogen response through

1) up-regulating proteins involved in estrogen metabolism (specifically through increasing the CYP1A1/1B1 ratio), and

2) decreasing BRCA1 inhibition thus permitting its inhibition of estrogen responsive transcription.

The two estrogen responsive genes (TFF1 and WISP2) showed a significant
decrease in mRNA expression levels (Figure 2A) while the estrogen metabolism genes (CYP1A1, CYP1B1, and AKR1C1) demonstrated a significant increase in mRNA levels (Figures 2B and C).

In this study we provide the first gene array profiling of an estrogen responsive breast cancer cell line demonstrating that the combination of iodine and iodide alters gene expression. Among the list of altered genes were several genes documented to be estrogen responsive such as TFF1 and WISP2. Furthermore, the list contained several
genes involved in the estrogen response including Phase I estrogen metabolizing enzymes (CYP1A1 and CYP1B) and Cyclin D1, a competitive inhibitor of
BRCA1 [41].

Saxena, Neela, et al. “Differential expression of WISP-1 and WISP-2 genes in normal and transformed human breast cell lines.” Molecular and cellular biochemistry 228 (2001): 99-104.

WISP-2 mRNA transcription was identified in all 4 tumor derived cell lines, but the mRNA expression was undetected or minimally detected in normal breast epithelial cells. WISP-1 mRNA expression was identified in normal and transformed cell lines. However, the level of expression was higher in different breast tumor cell lines as compared to HMEC. The mRNA expression profiles of WISP genes in normal breast epithelial cells and breast tumor derived cell lines indicated a strong possibility of the involvement of WISP-signaling in the development of human breast tumors, and can be utilized as genetic markers of this disease.

WISP-2 expression is mediated through ER-α.In combination with estradiol, progesterone acted as an antagonist inhibiting the expression of WISP-2 mRNA.

Banerjee, Snigdha, et al. “WISP-2 gene in human breast cancer: estrogen and progesterone inducible expression and regulation of tumor cell proliferation.” Neoplasia 5.1 (2003): 63-73.

WISP-2 mRNA and protein was overexpressed in preneoplastic and cancerous cells of human breast. Statistical analyses show a significant association between WISP-2 expression and estrogen receptor (ER) positivity. In normal breast, the expression was virtually undetected. The studies showed that WISP-2 is an estrogen-induced early response gene in MCF-7 cells and the expression was continuously increased to reach a maximum level at 24 h. The estrogen effect was inhibited by a pure antiestrogen (ICI 182,780). Human mammary epithelial cells, in which WISP-2 expression was undetected or minimally detected, responded to 17β-estradiol by upregulating the WISP-2 gene after transfection with ER-α, providing further evidences that WISP-2 expression is mediated through ER-α. Overexpression of WISP-2 mRNA by estrogen may be accomplished by both transcriptional activation and stabilization. MCF-7 cells exposed to progesterone had a rapid but transient increase in WISP-2 expression, and PR antagonist RU38486 blocked this mRNA induction. In combination with estradiol, progesterone acted as an antagonist inhibiting the expression of WISP-2 mRNA. Moreover, disruption of WISP-2 signaling in MCF-7 cells by use of antisense oligomers caused a significant reduction in tumor cell proliferation. The results are consistent with the conclusion that WISP-2 expression is a requirement for breast tumor cells proliferation.

Stoddard Continued……………………………………..

CYP1A1/CYP1B1 ratio

Our data shows that treatment with iodine and iodide increases the mRNA levels of Cytochrome Cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), two estrogen phase I estrogen metabolizing enzymes that oxidizes 17β-estradiol to 2-hydoxyestradiol (2-OH-E2) and 4-hydoxyestradiol (4-OH-E2), respectively. These catechol estrogens can be further oxidized to quinones. 3,4-estradiol quinine, a metabolite of 4-OH-E2, has been shown to react with DNA forming de-purinating adducts resulting in genotoxicity [42], while data suggests that 2-OH-E2 can be metabolized to 2-methoxyestradiol, an estrogen metabolite with anti-proliferative effects [43]. The observed increase in the CYP1A1/CYP1B1 ratio may shift the direction of estrogen metabolism favoring 2-OH-E2 which may either directly affect proliferation through increasing 2-methoxyestradiol, decreasing 3, 4-estradiol quinone or indirectly via the inactivation of E2. The importance of the CYP1A1/CYP1B1 ratio in-vivo is evident in the increased presence of 4-OH-E2 in breast cancer tissue compared to non breast cancer controls [44]. However, the regulation and interplay between CYP1A1, CYP1B1, other Phase I and II enzymes and estrogen is complex, being influenced by multiple factors and multiple polymorphisms, thus more data is required to illuminate the importance of these changes in response to iodine.

In addition to affecting estrogen metabolism, iodine/iodide may also inhibit estrogen induced transcription via increased BRCA1 activity. BRCA1 is a known inhibitor of ERα transcription while Cyclin D1 is thought to enhance the estrogen response via a competitive inhibition with BRCA1 [41]. Our data demonstrates decreased Cyclin D1 mRNA which could result in decreased competitive inhibition of BRCA1 allowing BRCA1 to inhibit estrogen induced transcription. Increased transcription of GADD45A,
CYP1B1, and CYP1A1 are consistent with increased BRCA1 activity [34, 45].

It has been found that CCND1 over-expression plays an important role in the development
of Tamoxifen resistant breast cancer [25, 47-49]; our data shows that iodine/iodide treatment
can decrease mRNA levels of CCND1. This provides two potential mechanisms by which iodine/iodide could enhance the efficacy of Tamoxifen therapy:
1) having an additive effect on estrogen inhibition and
2) inhibiting the expression of CCND1 thus preventing or slowing the development of Tamoxifen resistance.

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Data presented suggests that iodine/iodide may inhibit the estrogen response through

1) up-regulating proteins involved in estrogen metabolism (specifically through increasing the CYP1A1/1B1 ratio), and

2) decreasing BRCA1 inhibition thus permitting its inhibition of estrogen responsive transcription.

!!!!!!!!!!!!!!!!!!!!!!!!!! END STODDARD !!!!!!!!!!!!!!!!!

18) LaVallee, Theresa M., et al. “2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors α and β.” Cancer research 62.13 (2002): 3691-3697.

19) Wang, Chenguang, et al. “Cyclin D1 antagonizes BRCA1 repression of estrogen receptor α activity.” Cancer research 65.15 (2005): 6557-6567.

20) Saxena, Neela, et al. “Differential expression of WISP-1 and WISP-2 genes in normal and transformed human breast cell lines.” Molecular and cellular biochemistry 228 (2001): 99-104.

21) Banerjee, Snigdha, et al. “WISP-2 gene in human breast cancer: estrogen and progesterone inducible expression and regulation of tumor cell proliferation.” Neoplasia 5.1 (2003): 63-73.

22) Rajan, Arathi, et al. “Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers.” American Journal of Cancer Research 12.1 (2022): 17.

23) Trusov, N. V., et al. “Effects of combined treatment with resveratrol and indole-3-carbinol.” Bulletin of experimental biology and medicine 149 (2010): 213-218.

24) Mohammadi, Saeed, et al. “Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.” Journal of receptors and signal transduction 37.5 (2017): 506-514.

25) Tutelyan, V. A., et al. “Indole-3-carbinol induction of CYP1A1, CYP1A2, and CYP3A1 activity and gene expression in rat liver under conditions of different fat content in the diet.” Bulletin of experimental biology and medicine 154 (2012): 250-254.

26) Ociepa-Zawal, Marta, et al. “The effect of indole-3-carbinol on the expression of CYP1A1, CYP1B1 and AhR genes and proliferation of MCF-7 cells.” Acta Biochimica Polonica 54.1 (2007): 113-117.

27) Weng, Jing-Ru, et al. “Indole-3-carbinol as a chemopreventive and anti-cancer agent.” Cancer letters 262.2 (2008): 153-163.

28) Wang, Thomas TY, et al. “Estrogen receptor α as a target for indole-3-carbinol.” The Journal of nutritional biochemistry 17.10 (2006): 659-664.

29) Sundar, Shyam N., et al. “Indole-3-carbinol selectively uncouples expression and activity of estrogen receptor subtypes in human breast cancer cells.” Molecular Endocrinology 20.12 (2006): 3070-3082.

30) Peng, Chunting, et al. “Indole-3-carbinol ameliorates necroptosis and inflammation of intestinal epithelial cells in mice with ulcerative colitis by activating aryl hydrocarbon receptor.” Experimental Cell Research 404.2 (2021): 112638.

31) Busbee, Philip B., et al. “Indole-3-carbinol prevents colitis and associated microbial dysbiosis in an IL-22–dependent manner.” JCI insight 5.1 (2020).

32) Newman, Mark, and Jaclyn Smeaton. “Exploring the Impact of 3, 3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women.” (2024).

33) Al-Shami, Khayry, et al. “Estrogens and the risk of breast cancer: A narrative review of literature.” Heliyon 9.9 (2023).

34) Zhao, Feng, et al. “Discovery of breast cancer risk genes and establishment of a prediction model based on estrogen metabolism regulation.” BMC cancer 21 (2021): 1-11.

35) Almeida, Micaela, et al. “Influence of estrogenic metabolic pathway genes polymorphisms on postmenopausal breast cancer risk.” Pharmaceuticals 14.2 (2021): 94.

36) Cavalieri, Ercole, and Eleanor Rogan. “The 3, 4-Quinones of Estrone and Estradiol Are the Initiators of Cancer whereas Resveratrol and N-acetylcysteine Are the Preventers.” International Journal of Molecular Sciences 22.15 (2021).

37) Saluzzo, Julieta, et al. “The regulation of tumor suppressor protein, p53, and estrogen receptor (ERα) by resveratrol in breast cancer cells.” Genes & Cancer 7.11-12 (2016): 414.

38) Long, Sarah, and Jack Goldblatt. “MTHFR genetic testing: Controversy and clinical implications.” Australian family physician 45.4 (2016): 237-240.

39) Graydon, James S., et al. “Ethnogeographic prevalence and implications of the 677C> T and 1298A> C MTHFR polymorphisms in US primary care populations.” Biomarkers in Medicine 13.8 (2019): 649-661.

40) Cristalli, Carlotta Pia, et al. “Methylenetetrahydrofolate reductase, MTHFR, polymorphisms and predisposition to different multifactorial disorders.” Genes & Genomics 39 (2017): 689-699.

41) Petrone, Igor, et al. “MTHFR C677T and A1298C polymorphisms in breast cancer, gliomas and gastric cancer: a review.” Genes 12.4 (2021): 587.

42) Li, Z., et al. “The methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism is associated with breast cancer subtype susceptibility in southwestern China.” PLoS ONE 16.7 (2021): e0254267.

43) Romero, Yair, et al. “Antitumor Therapy under Hypoxic Microenvironment by the Combination of 2‐Methoxyestradiol and Sodium Dichloroacetate on Human Non‐Small‐Cell Lung Cancer.” Oxidative Medicine and Cellular Longevity 2020.1 (2020): 3176375.

44) LaVallee, Theresa M., et al. “2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors α and β.” Cancer research 62.13 (2002): 3691-3697.

45) Ehteda, Anahid, et al. “Combination of albendazole and 2-methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice.” BMC cancer 13 (2013): 1-13.

46) Zefirov, Nikolai A., et al. “Adamantyl-substituted ligands of colchicine binding site in tubulin: different effects on microtubule network in cancer cells.” Structural Chemistry 30 (2019): 465-471.

47) D’Amato, ROBERT J., et al. “2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.” Proceedings of the National Academy of Sciences 91.9 (1994): 3964-3968.

48) Zefirov, Nikolai A., et al. “Adamantyl-substituted ligands of colchicine binding site in tubulin: different effects on microtubule network in cancer cells.” Structural Chemistry 30 (2019): 465-471.

49) Siebert, Amy E., et al. “Effects of estrogen metabolite 2-methoxyestradiol on tumor suppressor protein p53 and proliferation of breast cancer cells.” Systems Biology in Reproductive Medicine 57.6 (2011): 279-287.

50) Hirao-Suzuki, Masayo, et al. “2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.” Current Issues in Molecular Biology 45.9 (2023): 7336-7351.

51) Wen, Chun-Jie, et al. “Preferential induction of CYP1A1 over CYP1B1 in human breast cancer MCF-7 cells after exposure to berberine.” Asian Pacific Journal of Cancer Prevention 15.1 (2014): 495-499.

52) Brueggemeier, Robert W., et al. “2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics.” The Journal of steroid biochemistry and molecular biology 78.2 (2001): 145-156.

53) Zhou, Ning Ning, et al. “2-Methoxyestradiol induces cell cycle arrest and apoptosis of nasopharyngeal carcinoma cells.” Acta Pharmacologica Sinica 25 (2004): 1515-1520.

54) Tang, Xiaoyan, et al. “Anticancer effects and the mechanism underlying 2‑methoxyestradiol in human osteosarcoma in vitro and in vivo.” Oncology Letters 20.4 (2020): 1-1.

55) Carothers, A. M., et al. “2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells.” Cancer letters 187.1-2 (2002): 77-86.

56) Lee, Ji Young, et al. “Tumor suppressor protein p53 promotes 2-methoxyestradiol-induced activation of Bak and Bax, leading to mitochondria-dependent apoptosis in human colon cancer HCT116 cells.” Journal of Microbiology and Biotechnology 24.12 (2014): 1654-1663.

57) Mukhopadhyay, Tapas, and Jack A. Roth. “Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol.” Oncogene 14.3 (1997): 379-384.

58) Takata, Hidehiko, et al. “2-methoxyestradiol enhances p53 protein transduction therapy-associated inhibition of the proliferation of oral cancer cells through the suppression of NFkappaB activity.” Acta Medica Okayama 58.4 (2004): 181-187.

59) Amant, Frederic, et al. “2-Methoxyestradiol strongly inhibits human uterine sarcomatous cell growth.” Gynecologic oncology 91.2 (2003): 299-308.

60) Schumacher, Guido, et al. “Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines.” Clinical cancer research 5.3 (1999): 493-499.

61) Zhang, Xueya, et al. “2-Methoxyestradiol blocks cell-cycle progression at the G2/M phase and induces apoptosis in human acute T lymphoblastic leukemia CEM cells.” Acta Biochim Biophys Sin 42.9 (2010): 615-622.

62) Kumar, Addanki P., Gretchen E. Garcia, and Thomas J. Slaga. “2‐methoxyestradiol blocks cell‐cycle progression at G2/M phase and inhibits growth of human prostate cancer cells.” Molecular Carcinogenesis: Published in cooperation with the University of Texas MD Anderson Cancer Center 31.3 (2001): 111-124.

63) Roswall, Pernilla, et al. “2-methoxyestradiol induces apoptosis in cultured human anaplastic thyroid carcinoma cells.” Thyroid 16.2 (2006): 143-150.

64) Hua, Weitian, et al. “2-methoxyestradiol inhibits melanoma cell growth by activating adaptive immunity.” Immunopharmacology and Immunotoxicology 44.4 (2022): 541-547.

65) Starek-Świechowicz, Beata, Bogusława Budziszewska, and Andrzej Starek. “Endogenous estrogens—breast cancer and chemoprevention.” Pharmacological Reports 73.6 (2021): 1497-1512.

The CYP3A4 hydroxylates E1 to 16α-hydroxyestrone, a metabolite involved in breast cancer induction [45]. 45. Shou M, Korzekwa KR, Brooks EN, Krausz KW, Gonzales FJ, Gelboin HV. Role of human hepatic cytochrome P450 1A2 and 3A4 in the metabolic activation of estrone. Carcinogenesis. 1997;18(1):207–214.

The mechanisms of estrogen carcinogenesis include the participation of estrogen receptors, the genotoxic effect of the estrogen metabolites, and epigenetic processes that are also presented.

an enhanced 2-hydroxylation of estrogens, E1 and E2, was associated with reduced risk of postmenopausal breast cancer [22].
22. Ziegler RG, Fuhrman BJ, Moore SC, Mattews CE. Epidemiologic studies of estrogen metabolism and breast cancer. Steroids. 2015;99(Pt A):67–75

There is a suggestion that more extensive 2-hydroxylation of estrogens is associated with lower risk, whereas less extensive methylation of 4-hydroxylated catechols is associated with higher risk of postmenopausal breast cancer [21].21. Fuhrman BJ, Schairer C, Gail MH, Boyd-Morin J, Xu X, Sue LY, et al. Estrogen metabolism and risk of breast cancer in postmenopausal women. J Natl Cancer Inst. 2012;104(4):326–339

The lack of carcinogenic activity of 2-hydroxyestrogens has been attributed to a high rate of clearance, more rapid rate of O-methylation and lower hormonal potency in estrogen target tissues, plus produces 2-methoxyestradiol, which suppress tumor cell proliferation and angiogenesis [19]. Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis. 1998;19:1–27.

2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells and possesses antiangiogenic action. This metabolite has a lower binding affinity for both ERα and ERβ compared with that of E2 [40].

In Taiwanese women it was observed that of the three gens, i.e., CYP17, CYP1A1, and COMT, low activity of the COMT genotype was associated with the highest relative risk of breast cancer (RR = 4.0; 95% CI 1.12–19.8) [48].48. Huang CS, Chern HD, Chang KJ, Cheng CW, Hsu SM, Shen CY. Breast cancer risk associated with genotype polymorphism of the estrogen-metabolising genes CYP17, CYP1A1, and COMT: a multigenetic study on cancer susceptibility. Cancer Res. 1999;59:4870–4875.

66)Telang, Nitin T., et al. “Induction by estrogen metabolite 16α;-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells.” JNCI: Journal of the National Cancer Institute 84.8 (1992): 634-638.

67) Fuhrman BJ, Schairer C, Gail MH, Boyd-Morin J, Xu X, Sue LY, et al. Estrogen metabolism and risk of breast cancer in postmenopausal women. J Natl Cancer Inst. 2012;104(4):326–339

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68) Zhu, Bao Ting. “Is it necessary to control the level of estrogen receptor α and β activation in postmenopausal hormone replacement therapy in order to achieve the optimal outcome?.” Molecular Medicine Reports 1.1 (2008): 15-20.

Abstract : Endogenous estrogens exert an array of biological actions on women, many of which are mediated by the estrogen receptors (ERs) α and β. Results from our recent studies suggest that the human ERα and ERβ systems are differentially activated under different physiological conditions. In non-pregnant young women, the ERα system is preferentially activated over the ERβ system, mainly by estrone (E1) and its major oxidative metabolite, 2-hydroxy-E1. These two estrogens are among the quantitatively major estrogens present in young women, and have approximately 4-fold preferential activity for ERα over ERβ. During pregnancy, however, there is a preponderance of activation of ERβ over ERα conferred by various pregnancy estrogens such as estriol and other D-ring derivatives of 17β-estradiol (E2). These estrogens have an up to 18-fold preference for binding to ERβ than for ERα, and some of them are produced in unusually large quantities. Given this new information, it is hypothesized that the estrogens ideal for female hormone replacement therapy (HRT) would be those which produce a hormonal condition mirroring that found in non-pregnant young women rather than in pregnant women. Endogenous estrogen derivatives, such as the sulfated conjugates of E1, may be among the ideal candidates for achieving this clinical purpose. In comparison, Premarin, the most commonly-used HRT containing a mixture of conjugated estrogens isolated from pregnant mare’s urine, is less suitable because several of its estrogenic components can produce a strong preferential over-stimulation of the human ERβ signaling system.

69) Zhu, Bao Ting, et al. “Quantitative structure-activity relationship of various endogenous estrogen metabolites for human estrogen receptor α and β subtypes: Insights into the structural determinants favoring a differential subtype binding.” Endocrinology 147.9 (2006): 4132-4150.

To search for endogenous estrogens that may have preferential binding affinity for human estrogen receptor (ER) alpha or beta subtype and also to gain insights into the structural determinants favoring differential subtype binding, we studied the binding affinities of 74 natural or synthetic estrogens, including more than 50 steroidal analogs of estradiol-17beta (E2) and estrone (E1) for human ER alpha and ER beta. Many of the endogenous estrogen metabolites retained varying degrees of similar binding affinity for ER alpha and ER beta, but some of them retained differential binding affinity for the two subtypes. For instance, several of the D-ring metabolites, such as 16 alpha-hydroxyestradiol (estriol), 16 beta-hydroxyestradiol-17 alpha, and 16-ketoestrone, had distinct preferential binding affinity for human ER beta over ER alpha (difference up to 18-fold). Notably, although E2 has nearly the highest and equal binding affinity for ER alpha and ER beta, E1 and 2-hydroxyestrone (two quantitatively predominant endogenous estrogens in nonpregnant woman) have preferential binding affinity for ER alpha over ER beta, whereas 16 alpha-hydroxyestradiol (estriol) and other D-ring metabolites (quantitatively predominant endogenous estrogens formed during pregnancy) have preferential binding affinity for ER beta over ER alpha.

70) Bhavnani, Bhagu R., Shui-Pang Tam, and XiaoFeng Lu. “Structure activity relationships and differential interactions and functional activity of various equine estrogens mediated via estrogen receptors (ERs) ERα and ERβ.” Endocrinology 149.10 (2008): 4857-4870.

In conclusion, our results show that the effects of ring B unsaturated estrogens are mainly mediated via ERβ and that the presence of both ER subtypes further enhances their activity. It is now possible to develop hormone replacement therapy using selective ring B unsaturated estrogens for target tissues where ERβ is the predominant ER.

 

Gustafsson and colleagues (9) have demonstrated an androgen 5α androstane, 3β,17β-diol (3β-diol), a metabolite of 5α-dihydrotestosterone formed in the prostate, has estrogenic activity that is mediated via ERβ. Whether 3β-diol is the unique endogenous ligand for ERβ remains to be established. However, 3β-diol binds to both ERα and ERβ, albeit with slightly higher affinity with the latter (23, 29–33). Because 3β-diol also binds to ERα, the specificity of its activity is most likely due to its site of formation (prostate), and this has been recently reviewed (9, 33). A number of these and other (34) studies with 3β-diol suggest that ERβ ligands may be useful in the inhibition of prostatic epithelial cell proliferation. Our data indicate that some natural estrogens such as the ring B unsaturated equine estrogens of the type present in the drug CEE have the characteristics that can be useful as selective ERβ ligands.

71) Liu, Xiaohui, et al. “Bisphenol-C is the strongest bifunctional ERα-agonist and ERβ-antagonist due to magnified halogen bonding.” PloS one 16.2 (2021): e0246583.

72) Mal, Rahul, et al. “Estrogen receptor beta (ERβ): a ligand activated tumor suppressor.” Frontiers in oncology 10 (2020): 587386.

Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) belong to a superfamily of nuclear receptors called steroid hormone receptors, which, upon binding ligand, dimerize and translocate to the nucleus where they activate or repress the transcription of a large number of genes, thus modulating critical physiologic processes. ERβ has multiple isoforms that show differing association with prognosis. Expression levels of the full length ERβ1 isoform are often lower in aggressive cancers as compared to normal tissue. High ERβ1 expression is associated with improved overall survival in women with breast cancer. The promise of ERβ activation, as a potential targeted therapy, is based on concurrent activation of multiple tumor suppressor pathways with few side effects compared to chemotherapy. Thus, ERβ is a nuclear receptor with broad-spectrum tumor suppressor activity, which could serve as a potential treatment target in a variety of human cancers including breast cancer. Further development of highly selective agonists that lack ERα agonist activity, will be necessary to fully harness the potential of ERβ.

As with ERα, estrogenic compounds including estradiol, estrone, and estriol activate ERβ. Relative to ERα, ERβ binds estriol and ring B unsaturated estrogens with higher affinity, while the reverse is true of 17β-estradiol and estrone (7–10). On the other hand, the dihydrotestosterone metabolites 5-androstenediol and 3β androstanediol are relatively selective (3-fold) for ERβ over ERα (11)

 

The cell division protein cyclin D1 (CCND1), one target of AP-1 and SP1 mediated transcription, is upregulated by ERα and induces estrogen-mediated proliferation (48). The CCND1 promoter contains a cAMP response element and an AP-1 binding site, both of which play partially redundant roles in ERα mediated transcriptional up-regulation of cyclin D1. Surprisingly, activation of this promoter by ERβ was shown only to occur with antiestrogens. Estradiol, which up-regulates cyclin D1 transcription in cells that over-express ERα, inhibits its transcription in cells that over-express ERβ. Additionally, it was found that the presence of ERβ inhibits transcriptional up-regulation of cyclin D1 by ERα in the presence of estradiol, suggesting that ERβ may exert dominant negative effects on ERα mediated cyclin D1 transcription. Opposing actions and dominance of ERβ over ERα with respect to activation of cyclin D1 gene expression may explain why ERβ is a negative regulator of the proliferative effects of estrogen.

 

Estradiol increases the proliferation of and causes tumor formation of MCF-7 breast cancer cell line xenografts in an ERα dependent manner (54). In contrast, the over-expression of ERβ in MCF7 cells reduces proliferation in vitro and prevents tumor formation in mice in the presence of supplemental estradiol. Furthermore, ERβ was shown to repress c-myc and cyclin D1 expression, and to increase the expression of p21 and p27Kip1, leading to G2 cell cycle arrest in this model. In addition, ERβ regulates proliferation and migration through modulation of mitofusin 2 expression in MCF7 cells (54). Moreover, estrogen up-regulates the expression of LRP16 mRNA through the activation of ERα, but not ERβ, which promotes MCF-7 cell proliferation (55). Thus, ERβ and ERα have shown opposing effects on proliferation and the expression of various oncogenes and tumor suppressors in breast cancer cell lines in the presence of estradiol.

 

ERβ is unique in that it functions as a tumor suppressor in diverse biologic contexts. ERβ has been implicated in various cancer types, including breast, prostate, lung, glioblastoma, thyroid, and ovarian cancer (15–19).

 

Concerning breast cancer, ERβ expression by IHC is detectable in 20–30% of invasive breast cancers (127). In regard to association with patient outcomes in breast cancer, ERβ expression is an independent prognostic marker in ERα+-progesterone receptor positive breast cancer (128). Mann et al. (129) found that ERβ status is a predictor of survival in women with breast cancer when treated with adjuvant hormonal therapy. They also found that expression of ERβ in more than 10% of the cancer cells was associated with improved survival. This association has been confirmed in a meta-analysis showing improved disease-free survival in patients with tumors positive for ERβ1 and ERβ2 isoforms (130).

 

The promise of ERβ activation lies in the possibility of a targeted therapy that concurrently activates multiple tumor suppressor pathways while causing relatively few side effects. Thus, ERβ is a nuclear receptor with broad-spectrum tumor suppressor activity that could serve as a potential treatment target in a variety of human cancers.

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Duplicates Below this

Later Study by Eskin Group

2012: iodine treatment results in decreased ERα mRNA levels; (Eskin Group)

Poor, Alexander E., et al. “Urine Iodine, Estrogen, and Breast Disease.” Journal of Cancer Therapy 3 (2012): 1164-1169. Authors: Alexander E. Poor^1*, Bernard A. Eskin², Christine Georgiadis¹, Brian Hamzavi¹, Ari D. Brooks¹

Few studies had described iodine’s mechanism of action or its target pathways. Recently researchers have suggested that iodine inhibits breast cancer growth via cyclin-independent apoptosis; however there is evidence that a different pathway is involved. We analyzed the effects of iodine on global gene expression in estrogen responsive MCF-7 breast cancer cell line. Microarray analysis and quantitative real time polymerase chain reaction (RT-PCR) indicated that iodine inhibits the expression of estrogen-responsive genes TFF1 and WISP2 [27].

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Our data further provided three potential mechanisms to explain the observed decrease in estrogen response. First, iodine treatment results in decreased ERα mRNA levels; second, iodine up-regulates genes involved in estrogen metabolism (CYP1A1, CYP1B1, and AKR1C1), and finally, iodine decreases Cyclin D1 (a competitive inhibitor of BRCA1) mRNA levels which may functionally permit BRCA1 inhibition of estrogen responsive transcription. Thus the interaction between iodine and estrogen signaling may inhibit breast cancer growth by affecting an intermediate, perhaps the estrogen receptor system.

Iodine Downregulates Cyclin D1

Wang, Chenguang, et al. “Cyclin D1 antagonizes BRCA1 repression of estrogen receptor α activity.” Cancer research 65.15 (2005): 6557-6567.
The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor α (ERα)–dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERα binding. Cyclin D1 and BRCA1 were both capable of binding ERα in a common region of the ERα hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERα and for rescue of BRCA1-mediated ERα transcriptional repression. In chromatin immunoprecipitation assays, 17β-estradiol (E2) enhanced ERα and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERα recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERα recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERα recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERα repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

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Berberine CYP1A1 over CYP1B1 

Wen, Chun-Jie, et al. “Preferential induction of CYP1A1 over CYP1B1 in human breast cancer MCF-7 cells after exposure to berberine.” Asian Pacific Journal of Cancer Prevention 15.1 (2014): 495-499.

Previous reports suggested that 2-OH E2 have putative protective effects, while 4-OH E2 is genotoxic and has potent carcinogenic activity. Thus, the ratio of 2-OH E2/4-OH E2 is a critical determinant of the toxicity of E2 in mammary cells. In the present study, we investigated the effects of the berberine on the expression profile of the estrogen metabolizing enzymes CYP1A1 and CYP1B1 in breast cancer MCF-7 cells. Berberine treatment produced significant induction of both forms at the level of mRNA expression, but with increased doses produced 16~ to 52~fold greater inductions of CYP1A1 mRNA over CYP1B1 mRNA. Furthermore, berberine dramatically increased CYP1A1 protein levels but did not influence CYP1B1 protein levels in MCF-7 cells. In conclusion, we present the first report to show that berberine may provide protection against breast cancer by altering the ratio of CYP1A1/CYP1B1, could redirect E2 metabolism in a more protective pathway in the breast cancer MCF-7 cells.

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Mal, Rahul, et al. “Estrogen receptor beta (ERβ): a ligand activated tumor suppressor.” Frontiers in oncology 10 (2020): 587386.

Thus, ERβ is a nuclear receptor with broad-spectrum tumor suppressor activity, which could serve as a potential treatment target in a variety of human cancers including breast cancer.

As with ERα, estrogenic compounds including estradiol, estrone, and estriol activate ERβ. Relative to ERα, ERβ binds estriol and ring B unsaturated estrogens with higher affinity, while the reverse is true of 17β-estradiol and estrone (7–10). On the other hand, the dihydrotestosterone metabolites 5-androstenediol and 3β androstanediol are relatively selective (3-fold) for ERβ over ERα (11). The structures of these natural ligands and their binding affinities to ERα and ERβ have been shown in Table 1.

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Daidzein

Kumar, Vinod, and Shyam S. Chauhan. “Daidzein induces intrinsic pathway of apoptosis along with ER α/β ratio alteration and ROS production.” Asian Pacific Journal of Cancer Prevention: APJCP 22.2 (2021): 603.

Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC₅₀ value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER β has been observed which are conducive indicator of apoptosis.

Jin, S., et al. “Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway.” Annals of Oncology 21.2 (2010): 263-268.

Choi, Eun Jeong, and Gun-Hee Kim. “Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDA-MB-453 cells.” Phytomedicine 15.9 (2008): 683-690.

Prostate

Ranjithkumar, R., et al. “Novel daidzein molecules exhibited anti-prostate cancer activity through nuclear receptor ERβ modulation, in vitro and in vivo studies.” Journal of Chemotherapy 33.8 (2021): 582-594.

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May F. Novel drugs that target the estrogen-related receptor alpha: their therapeutic potential in breast cancer. Cancer Manag Res. 2014;6:225-252

Estrogen-related receptor alpha is expressed in the later stages of embryonic development and is abundant in heart, skeletal muscle, and the nervous system. The physiological role of estrogen-related receptor alpha, and of estrogen-related receptor gamma, is to act as an energy sensor to control cellular adaptation to energy demand and stress. To this end, estrogen-related receptor alpha is expressed at high levels in tissues with high energy demands, such as muscle and brown adipose tissue. Cells that do not express active estrogen-related receptor alpha cannot produce sufficient energy in times of peak demand.

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Aceves, Carmen, Brenda Anguiano, and Guadalupe Delgado. “The extrathyronine actions of iodine as antioxidant, apoptotic, and differentiation factor in various tissues.” Thyroid 23.8 (2013): 938-946.

We propose that the International Council for the Control of Iodine Deficient Disorders recommend that iodine intake be increased to at least 3 mg/day of I2 in specific pathologies to obtain the potential extrathyroidal benefits described in the present review.

Moreover, in an early phase clinical study conducted in 22 patients with mammary cancer, we found that 2–5 weeks of 5 mg/day I2 treatment caused significant increases of apoptotic rate and PPARγ expression, whereas there was a significant decrease in proliferation and diminished translocation of the estrogen receptor alpha to the nucleus, suggesting that the antineoplastic effect of iodine involves PPARγ activation (18).

In accordance with these findings, a recent report of Eskin’s group showed that in MCF-7 cells, iodine treatment may inhibit the estrogen response through upregulating proteins such as CYP1A1 and BRCA1 (76), both of which are also modulated by PPARγ (77,78).

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I2 decreases Nuclear translocation of ER -Alpha

Vega-Riveroll, Laura, et al. “Abstract P6-14-15: Impaired Nuclear Translocation of Estrogen Receptor Alpha Could Be Associated with the Antineoplastic Effect of Iodine in Premenopausal Breast Cancer.” Cancer Research 70.24_Supplement (2010): P6-14.

In conclusion, our data show that, in addition to the apoptotic and anti-invasive effects on mammary cancer exhibited by I2 that are independent of hormonal status, in pre-M women, I2 also acts to diminish the effects of estrogen (decreases nuclear translocation of ERα).

Patients were divided according to hormonal status into pre-M and post-M groups and were supplemented with I2 (5 mg daily) or placebo (vegetable dye) for 2-5 weeks before surgery. Thyroid status (T3 and TSH; RIA) and iodine intake (urine, HPLC) were analyzed. In biopsies (beginning) and/or tumors (final), the level and location (cytosol or nuclear) of estrogen receptor alfa (ERα) and beta (ERb) were analyzed by immunohistochemistry. In tumors, we measured PPARg expression, invasive potential [hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) by real time PCR] and fibrosis content (Masson’s trichromic method). The results show that I2 supplementation in pre-M women is accompanied by a significant increase in the concentration of total ERα and PPARg and no change in total ERb; however, the fraction of ERα in the nucleus was significant lower, and the fibrosis content was higher (70%) than in the placebo group (40%).

In contrast, I2 supplements significantly decreased HIF and VEGF expression in both pre-and post-M women. In conclusion, our data show that, in addition to the apoptotic and anti-invasive effects on mammary cancer exhibited by I2 that are independent of hormonal status, in pre-M women, I2 also acts to diminish the effects of estrogen (decreases nuclear translocation of ERα). Experiments that analyze the possible role of PPARg in these actions are in progress.

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Werner, Haim. “BRCA1: an endocrine and metabolic regulator.” Frontiers in endocrinology 13 (2022): 844575.

In classical terms, BRCA1 fits the criteria of a candidate tumor suppressor
gene…Among other physiological activities, BRCA1 was shown to induce the metabolic reprogramming of breast cancer cells with ensuing reversal of the Warburg effect. BRCA1 governs important steps of the lipogenetic pathway and has a key role in energy metabolism. BRCA1 interacts with several hormones, including IGF1, estrogens and androgens, cortisol, etc. In addition, BRCA1 seems to be involved in the process of reproduction.

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I would say researchers should be looking at iodine’s effect on it. Iodine decreases the inhibition of BRCA one.

Kiljańczyk, Adam, et al. “Blood Iodine as a Potential Marker of the Risk of Cancer in BRCA1 Carriers.” Nutrients 16.11 (2024): 1788.

We conducted a prospective study among 989 BRCA1 carriers to assess the association between blood iodine levels and breast and ovarian cancer risk. Using inductively coupled plasma mass spectrometry, we measured blood iodine levels and observed a negative association with breast cancer risk, with a significantly lower risk observed in quartile 4 (iodine > 38.0 µg/L) compared with quartile 1 (iodine < 30 µg/L) (HR = 0.49; 95%CI: 0.27–0.87; p = 0.01). Conversely, a suggestive increase in ovarian cancer risk was observed at higher iodine levels (HR = 1.91; 95%CI: 0.64–5.67; p = 0.25). No significant association was found between iodine levels and overall cancer risk.

Women are being checked for BRCCA polymorphisms and then getting breast and ovarian surgery. Iodine could help prevent that inhibition of BRCCA1.

estrogen receptor positive BR cancer 2-fold more sensitive to apoptotic effects of iodine that is ER negative Br Cancer

Iodine increases phase one estrogen metabolizing enzymes.
2-OH estradiol can be converted into 2MEO. Lindsey is the worlds expert on 2MEO.
Multiple anticancer effects.

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Estrogen Metabolism

• Parent estrogens (E1, E2, E3)
• Kid estrogens (catechol estrogens, 2, 4, 16 series)
• Grandkids estrogens (methylated estrogens, along with ER Beta are our natural cancer
fighters)
• Iodine helps take 2-OH series to 2 MEO series for cancer protection. “Growth out of control” protection.
• 4-OH is a minor disruptive series that can damage DNA when higher than 2 series, need
to give more iodine. And brassica family.

2-MEO
2-Methoxy-Estradiol – Big Gun anticancer metabolite

Growth control- More 2 series than 4 series to make 2-methoxy estradiol.
2-MEO is MAde in presence of COMT enzyme And IODINE.

IODINE COMT 2-methoxy estradiol

Theory about why DES offspring grow so many tumors: in utero exposure to DES suppresses Tumor Suppressor Gene P53.

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DCA and 2MEO

Romero, Yair, et al. “Antitumor Therapy under Hypoxic Microenvironment by the Combination of 2‐Methoxyestradiol and Sodium Dichloroacetate on Human Non‐Small‐Cell Lung Cancer.” Oxidative Medicine and Cellular Longevity 2020.1 (2020): 3176375.

DCA Turns Off the Glycolytic Flux and 2-ME Reduces in Part the Glutaminolysis under Hypoxia…The effect of 2-ME on growth and migration is predominant, since 2-ME has already been reported to be able to induce the arrest of the cell cycle [15] and as an inhibitor of tubulin polymerization [16].

2MEO – Cell cycle Arrest (Duplicate)
15.Zhou, Ning Ning, et al. “2-Methoxyestradiol induces cell cycle arrest and apoptosis of nasopharyngeal carcinoma cells.” Acta Pharmacologica Sinica 25 (2004): 1515-1520.

2 MEO inhibits tubulin polymerization
16. D’Amato, ROBERT J., et al. “2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.” Proceedings of the National Academy of Sciences 91.9 (1994): 3964-3968.

Ehteda, Anahid, et al. “Ehteda, Anahid, et al. “Combination of albendazole and 2-methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice.” BMC cancer 13 (2013): 1-13.

the interaction between ABZ [albendazole] and 2-methoxyestradiol (2ME), a related and a structurally similar compound to CLC [colchicine] was tested and found to be synergistic with ABZ. The mechanism underlying the synergistic interaction between ABZ and 2ME was then investigated and the antitumor effect of the combination therapy in mice bearing colorectal cancer xenograft was evaluated.

2MEO Anticancer Effects are Independent of ER-alpha and ER Beta

(duplicate)  Reference: LaVallee, Theresa M., et al. “2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors α and β.” Cancer research 62.13 (2002): 3691-3697.

2ME₂ is distinct among estradiol metabolites because of its inability to engage ERs as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of ERα and ERβ.

 2‐ME increased tumor mass when compared to the untreated animals!!!!!

Peta, Kimberly T., et al. “Effect of 2‐methoxyestradiol on mammary tumor initiation and progression.” Cancer Reports 7.4 (2024): e2068.

Each mouse received 100 mg/kg 2‐ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59.

2‐ME increased tumor mass when compared to the untreated animals (p = .0139). The pro‐tumorigenic activity of 2‐ME was accompanied by lower CD3+ T‐cell numbers in the tumor microenvironment (TME) and high levels of the pro‐inflammatory cytokine interleukin (IL)‐1β. Conversely, 2‐ME‐treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL‐10 plasma levels, and low IL‐6 and IL‐27 plasma levels.
Conclusion  Taken together, these findings suggest that 2‐ME promotes early‐stage BC development.

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2-MEO upregulates, Activates P53.

Breast Cancer cells

Siebert, Amy E., et al. “Effects of estrogen metabolite 2-methoxyestradiol on tumor suppressor protein p53 and proliferation of breast cancer cells.” Systems Biology in Reproductive Medicine 57.6 (2011): 279-287.
Addition of 10 nM – 1 μM 2-ME2 induced significant up-regulation in p53,

Osteosarcoma in vitro and in vivo 

Tang, Xiaoyan, et al. “Anticancer effects and the mechanism underlying 2‑methoxyestradiol in human osteosarcoma in vitro and in vivo.” Oncology Letters 20.4 (2020): 1-1.

The present study investigated the effects of 2-ME on the proliferation and apoptosis of human MG63 OS cells. The potential biological mechanisms by which 2-ME exerts its biological effects were also investigated in the present study. The results of the present study demonstrated that 2-ME inhibited the proliferation of OS cells in a time- and dose-dependent manner, induced G₂/M phase cell cycle arrest and early apoptosis. The expression levels of vascular endothelial growth factor (VEGF), Bcl-2 and caspase-3 were measured via western blotting and reverse transcription-quantitative PCR. As the concentration of 2-ME increased, the RNA and protein expression levels of VEGF and Bcl-2 decreased gradually, whereas the expression of caspase-3 increased gradually. In addition, tumor growth in nude mice was suppressed by 2-ME with no toxic side effects observed in the liver or kidney. Immunohistochemistry demonstrated that the expression levels of Bcl-2 and VEGF were significantly lower, and those of caspase-3 were significantly higher in test mice compared with the control group. TUNEL staining of xenograft tumors revealed that with increased 2-ME concentration, the number of apoptotic cells also gradually increased. Thus, 2-ME effectively inhibited the proliferation and induced apoptosis of MG63 OS cells in vitro and in vivo with no obvious side effects. The mechanism of the anticancer effect of 2-ME may be associated with the actions of Bcl-2, VEGF and caspase-3.

Colorectal Cancer in vivo and in vitro

Carothers, A. M., et al. “2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells.” Cancer letters 187.1-2 (2002): 77-86.

Menopausal estrogen replacement therapy is thought to be responsible for the recent decline in colorectal cancer (CRC) incidence among women. In the C57BL/6J-Min/1 mouse, an animal model of CRC, 17b-estradiol (E2) prevents tumor formation in ovariectomized females. We examined human CRC intestinal cell lines to determine whether particular E2 metabolites produced anti-tumor effects. Treatment of CRC cells with 2-methoxyestradiol (2-MeOE2) increased expression of p53 and p21WAF1/CIP1 proteins and induced apoptosis, but did not produce changes in expression of estrogen receptor (ER)a or ERb. The finding that 2-MeOE2 induces p53-mediated colon cell apoptosis in vitro supports a role for 2-MeOE2 as an endogenous mediator of intestinal tumor suppression.

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Colon cancer cells- 2MEO Upregulates P53, and impairs mitotic spindle network resulting in prometaphase arrest [the early phase of cell division when chromosomes separate and form duplicate centers to make two new cells. This requires intact spindle microtubule system]  ….among 2-MeO-E2-induced apoptotic events, including prometaphase arrest, up-regulation of Bax level, down-regulation of Bcl-2 level , activation of both Bak and Bax, and mitochondria-dependent caspase activation, the modulation of Bax and Bcl-2 levels is the target of the pro-apoptotic action of p53. P53 is activated by prolonged prophase [prophase arrest].

Lee, Ji Young, et al. “Tumor suppressor protein p53 promotes 2-methoxyestradiol-induced activation of Bak and Bax, leading to mitochondria-dependent apoptosis in human colon cancer HCT116 cells.” Journal of Microbiology and Biotechnology 24.12 (2014): 1654-1663.

To examine the effect of tumor suppressor protein p53 on the antitumor activity of 2-
methoxyestradiol (2-MeO-E2), 2-MeO-E2-induced cell cycle changes and apoptotic events were compared between the human colon carcinoma cell lines HCT116 (p53+/+) and HCT116 (p53-/-).When both cell types were exposed to 2-MeO-E2, a reduction in the cell viability and an enhancement in the proportions of G2/M cells and apoptotic sub-G1 cells commonly occurred dose-dependently. These 2-MeO-E2-induced cellular changes, except for G2/M arrest, appeared to be more apparent in the presence of p53. Immunofluorescence microscopic analysis using anti-α-tubulin and anti-lamin B2 antibodies revealed that after 2-MeO-E2 treatment, impaired mitotic spindle network and prometaphase arrest occurred similarly in both cell types. Following 2-MeO-E2 treatment, only HCT116 (p53+/+) cells exhibited an enhancement in the levels of p53, p-p53 (Ser-15), p21WAF1/CIP1, and Bax; however, the Bak level remained relatively constant in both cell types, and the Bcl-2 level decreased only in HCT116 (p53+/+) cells. Additionally, mitochondrial apoptotic events, including the activation of Bak and Bax, loss of Δψm, activation of caspase-9 and -3, and cleavage of lamin A/C, were more dominantly induced in the presence of p53. The Bak-specific and Bax-specific siRNA approaches confirmed the necessity of both Bak and Bax activations for the 2-MeO-E2-induced apoptosis in HCT116 cells. These results show that among 2-MeO-E2-induced apoptotic events, including prometaphase arrest, up-regulation of Bax level, down-regulation of Bcl-2 level, activation of both Bak and Bax, and mitochondria-dependent caspase activation, the modulation of Bax and Bcl-2 levels is the target of the pro-apoptotic action of p53.

Lung Cancer Cells

Mukhopadhyay, Tapas, and Jack A. Roth. “Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol.” Oncogene 14.3 (1997): 379-384.

2-Methoxyestradiol (2-MeOE2) treatment caused significant growth inhibition of H460 and A549 human lung cancer cell lines which contain wild-type p53. However, 2-MeOE2 had a little effect on the p53 negative H358 and p53 mutated H322 cell lines. Western blot analysis indicated that 2-MeOE2 treatment resulted in an eightfold increase in the endogenous wild-type p53 protein, while the level of the mutant p53 protein remained unchanged. TdT staining indicated that following 2-MeOE2-mediated increases in wildtype p53 protein, cells bypass the G1-S checkpoint of the cell cycle with 30 to 40% undergoing apoptosis. Introduction of anti-sense wt-p53 into wt-p53 cells abrogated the 2-MeOE2 effect. A significant portion of lung cancer retains the wild-type p53 gene therefore, 2-MeOE2 may have therapeutic application.

Oral Cancer inhibition of NFK-B

Takata, Hidehiko, et al. “2-methoxyestradiol enhances p53 protein transduction therapy-associated inhibition of the proliferation of oral cancer cells through the suppression of NFkappaB activity.” Acta Medica Okayama 58.4 (2004): 181-187.

2MEO Strong Inhibition of Uterine Sarcoma – Interference with Microtubule System

Amant, Frederic, et al. “2-Methoxyestradiol strongly inhibits human uterine sarcomatous cell growth.” Gynecologic oncology 91.2 (2003): 299-308.

Inhibition occurred after exposure to 2-methoxyestradiol and was accompanied by a threefold increase in the G2/M population, with a concomitant decrease in the G1 population, as shown by cell cycle analysis. SK-UT-1 cells exposed to 2-methoxyestradiol showed morphological changes indicative of apoptosis. Examination of signaling pathways that mediate 2-methoxyestradiol-induced apoptosis showed p53-independent growth inhibition. The inhibition of SK-UT-1 cell growth by arresting the cells during G2/M progression could be attributed to interference with the microtubule system, as determined by fluorescence immunohistochemistry.

2MEO – Potent AntiTumor Activity Pancreatic Cancer

Schumacher, Guido, et al. “Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines.” Clinical cancer research 5.3 (1999): 493-499.

nasopharyngeal cancer- 2-ME suppressed the transcriptional activity of NFκB,

Zhou, Ning Ning, et al. “2-Methoxyestradiol induces cell cycle arrest and apoptosis of nasopharyngeal carcinoma cells.” Acta Pharmacologica Sinica 25 (2004): 1515-1520.

In the present study, we examined whether 2-ME induced the stabilization of 11R-p53 and had an inhibitory effect on the proliferation of oral cancer cells. The application of 2-ME significantly enhanced the inhibitory effect of 11R-p53 on the proliferation of oral cancer cells. However, 2-ME had no effect on the intracellular half-life of 11R-p53 in oral cancer cells. Of interest is the finding that 2-ME suppressed the transcriptional activity of NFκB, which has an important role in tumorigenesis, but did not affect p53 transcriptional activity. These results suggest that 2-ME synergistically enhances the 11R-p53-induced inhibition of the proliferation of oral cancer cells through the suppression of NFkB transcription.

Lee, Yee-Man, et al. “Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells.” Cancer letters 268.2 (2008): 295-307.

Lung Cancer – wild-type p53 activation

Mukhopadhyay, Tapas, and Jack A. Roth. “Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol.” Oncogene 14.3 (1997): 379-384.

Solid Tumors

Schumacher, Guido, and Peter Neuhaus. “The physiological estrogen metabolite 2-methoxyestradiol reduces tumor growth and induces apoptosis in human solid tumors.” Journal of cancer research and clinical oncology 127 (2001): 405-410.

T lymphoblastic leukemia cells

Zhang, Xueya, et al. “2-Methoxyestradiol blocks cell-cycle progression at the G2/M phase and induces apoptosis in human acute T lymphoblastic leukemia CEM cells.” Acta Biochim Biophys Sin 42.9 (2010): 615-622.

Altered Tubulin Dynamics – 2-Methoxymethylestradiol Analog More Effective:

Brueggemeier, Robert W., et al. “2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics.” The Journal of steroid biochemistry and molecular biology 78.2 (2001): 145-156.

Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.

Prostate Cancer

Kumar, Addanki P., Gretchen E. Garcia, and Thomas J. Slaga. “2‐methoxyestradiol blocks cell‐cycle progression at G2/M phase and inhibits growth of human prostate cancer cells.” Molecular Carcinogenesis: Published in cooperation with the University of Texas MD Anderson Cancer Center 31.3 (2001): 111-124.

Anaplastic Thyroid Cancer

Roswall, Pernilla, et al. “2-methoxyestradiol induces apoptosis in cultured human anaplastic thyroid carcinoma cells.” Thyroid 16.2 (2006): 143-150.

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LTED Breast Cancer – 2-methoxyestradiol (2-MeO-E2),  microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1).

Hirao-Suzuki, Masayo, et al. “2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.” Current Issues in Molecular Biology 45.9 (2023): 7336-7351.
To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells. Among the β-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

Melanoma

Hua, Weitian, et al. “2-methoxyestradiol inhibits melanoma cell growth by activating adaptive immunity.” Immunopharmacology and Immunotoxicology 44.4 (2022): 541-547.

2‐ME increased tumor mass

(duplicatre)
Peta, Kimberly T., et al. “Effect of 2‐methoxyestradiol on mammary tumor initiation and progression.” Cancer Reports 7.4 (2024): e2068.
Taken together, these findings suggest that 2‐ME promotes early‐stage BC development.

Peta, Kimberly T., et al. “Effect of 2‐methoxyestradiol on mammary tumor initiation and progression.” Cancer Reports 7.4 (2024): e2068.

2‐ME increased tumor mass when compared to the untreated animals (p = .0139). The pro‐tumorigenic activity of 2‐ME was accompanied by lower CD3+ T‐cell numbers in the tumor microenvironment (TME) and high levels of the pro‐inflammatory cytokine interleukin (IL)‐1β. Conversely, 2‐ME‐treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL‐10 plasma levels, and low IL‐6 and IL‐27 plasma levels. Conclusion Taken together, these findings suggest that 2‐ME promotes early‐stage BC development.

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Lakhani, Nehal J., et al. “2‐Methoxyestradiol, a promising anticancer agent.” Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 23.2 (2003): 165-172.

Mueck, A. O., and H. Seeger. “2-Methoxyestradiol—biology and mechanism of action.” Steroids 75.10 (2010): 625-631.

Mooberry, Susan L. “Mechanism of action of 2-methoxyestradiol: new developments.” Drug Resistance Updates 6.6 (2003): 355-361.

Parada-Bustamante, Alexis, et al. “Role of 2-methoxyestradiol, an endogenous estrogen metabolite, in health and disease.” Mini reviews in medicinal chemistry 15.5 (2015): 427-438.

LaVallee, Theresa M., et al. “2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptors α and β.” Cancer research 62.13 (2002): 3691-3697.

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!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 4-OH-E1 4-OH-E2  IMPORTANT !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

4-Hydroxy Estrogens – CYP1B1, which is a cytochrome P450 superfamily enzyme converts E2 into 4-OH-E2.

4-OH-E1 to be the most important factor of breast cancer risk.

Urine 4-OH-E1 in the patients with breast cancer was three times higher than that in healthy women,

4-OH-Estrogens are highly estrogen and carcinogenic

and depurinating estrogen-DNA adducts

Miao, Suyu, et al. “4-Hydroxy estrogen metabolite, causing genomic instability by attenuating the function of spindle-assembly checkpoint, can serve as a biomarker for breast cancer.” American Journal of Translational Research 11.8 (2019): 4992.

an elevated ratio of the 4-hydroxylation pathway to parent estrogens, 4-OH-E1 + 4-OH-E2:(E1 + E2), was found to be significantly associated with an increment in risk (P-trend <0.001)

Compared with previous studies, this study indicated that breast cancer risk increased with increasing levels of 2-OHE1:16α-OHE1 ratio (P-trend <0.001)

although 2-hydroxy metabolite 2-OHE2 had limited estrogenic activity, 4-hydroxy metabolite 4-OHE2 had much stronger estrogenic activity, which was even more potent than that of E2 in MCF-7 cells.

4-OH-E2 induced tumorigenesis

The tumor growth of 4-OH-E2-treated MCF10A-H cells versus parental nontreated MCF10A cells (normal cells) was compared…The tumorigenic effect of 4-OH-E2 was first evaluated in athymic nude mice models (Figure 3). The tumor growth of 4-OH-E2-treated MCF10A-H cells versus parental nontreated MCF10A cells was compared. As expected, while mice injected with MCF10A cells into the mammary fat pad of nude mice did not form tumors, four out of five mice (80%) injected with MCF10A-H cells formed tumors after 2 weeks.

The effects of 4-OH-E2 on the mammary glands and development of mammary tumors were evaluated in a more relevant C57BL/6J-CYP1B1 transgenic mice model expressing transgene CYP1B1, which is a cytochrome P450 superfamily enzyme converting E2 into 4-OH-E2. Female mice were supplemented with or without E2 pellets (0.25 mg/90-day slow release). The effect of transgene expression on the mammary glands was assayed by morphological analyses of the gland after 90 days of E2 exposure (Figure 4). Histological evaluation of hematoxylin & eosin (H&E)-stained mammary sections revealed a normal monolayer and similar morphology in the glands from the virgin transgenic mice and the control littermate, indicating that the expression of transgene did not alter the mammary gland development. However, a significant alteration of the gland morphology with the appearance of mammary carcinoma in the transgenic mouse was observed when the mice were supplemented with E2 (Figure 4B). The epithelium of transgenic glands exhibited a disorganized structure with respect to the ordinate arrangement of the wild-type epithelium. Furthermore, while a normal mammary gland had a single layer of epithelial cells, the glands from transgenic mouse displayed a highly proliferative stage characterized by areas of multilayers. The duct lumen was narrowed and blocked by the hyperplastic glandular epithelial cells in the mammary glands of C57BL/6J-CYP1B1 + E2 mice, indicating a highly proliferative capability of the cells in the gland. Indeed, a strong immunohistochemical staining of Ki67 was only observed in the C57BL/6J-CYP1B1 + E2 group (Figure 4B), whereas Ki67 signal was undetectable in the mammary glands from other groups (Figure 4A). No morphological differences were observed in the tissues of liver and lung among all groups of mice. Taken together, multiple mammary tumors were only present in mice of the C57BL/6J-CYP1B1 + E2 group, but not in mice of other groups.

As a result of the changes in EM (estrogen metabolites) levels, the ratio of 4-hydroxylation metabolites of 4-OH-E (4-OH-E1 + 4-OH-E2):E (E1 + E2) showed a significant rise in the patient group versus the control group. In the C-2 pathway, 2-OH-E (2-OH-E1 + 2-OH-E2):E (E1 + E2) ratio was statistically significant. Interestingly, compared with the previous reports [14,25-27], the 2-OH-E1: 16α-OH-E1 ratio was higher in the patient group versus the control group, while the ratio of 2-OH-E (2-OH-E1 + 2-OH-E2):4-OH-E (4-OH-E1 + 4-OH-E2) was lower in the patient group versus control group

The stepwise regression analysis revealed 4-OH-E1 to be the most important factor of breast cancer risk. At subsequent steps of the forward-stepping procedure, E1 (P = 0.0023), 2-OH-E2, and 2-methoxy-E1 were also found to be independent and statistically significant factors

The results indicated that although 2-hydroxy metabolite 2-OHE2 had limited estrogenic activity, 4-hydroxy metabolite 4-OHE2 had much stronger estrogenic activity, which was even more potent than that of E2 in MCF-7 cells.\\\\\\

Discussion   the most significant alteration was an increase in 4-hydroxy metabolites relative to other metabolites in EMs [estrogen metabolites] in patients with breast cancer. Second, the biological relevance of increased 4-OH-E metabolism to breast cancer development was investigated in mammary epithelial cells and in vivo experimental models. It was found that 4-OH-E2 not only induced malignant transformation of breast epithelial cells in vitro but also stimulated tumor growth in the xenograft model and induced mammary carcinomas in the transgenic mice model expressing CYP1B1, a key enzyme of 4-hydroxy metabolites. Third, the molecular mechanisms underlying 4-OH-E metabolites induced malignant transformation. At the molecular level, 4-OH-E2 compromised the function of SAC spindle-assembly checkpoint (SAC) and thus rendered genome instability.

The lower ratio of 2-OHE1:16α-OHE1 had been hypothesized to be associated with breast cancer risk. Compared with previous findings [14,25-27], a significant higher urinary 2/16 ratio was observed in patients with breast cancer in this study. 2-OH-E2 was found to increase in the breast cancer group. The levels of 16α-OH-E1 did not change significantly in the breast cancer group versus normal controls.

Among many alterations of EMs in the breast cancer group, the most significant one in this study was an increase in 4-hydroxy metabolites. Urine 4-OH-E1 in the patients with breast cancer was three times higher than that in healthy women, while other EMs changed less. The best indicator that reflected the risk of breast cancer was the ratio of 4-hydroxy metabolites to total estrogen. The findings of this study were consistent with the assumption that the metabolic conversion of E2 to 4-OH-E2 and its additional activation to reactive semiquinone/quinone intermediates may be genotoxic, leading to various types of DNA damage, and have a direct role as tumor initiators [15-20].

Conclusions: In this comprehensive study of sexual hormone metabolism and risk of breast cancer in premenopausal women, we consider the ratio of 2-OHE1:16α-OHE1 is not a clear marker of breast cancer risk in premenopausal women. However, the ratio of 4-hydroxy metabolites to total estrogen is the best indicator reflects the risk of breast cancer. Our study found 4-OH-E2 induced carcinogenesis by destroying the SAC [spindle-assembly checkpoint] and induced the abnormal mitosis. The malignant cells transformed by 4-OH-E2 was hard to kill by antitumor drug. Thus, the relative proportions of 4-hydroxy estrogenic metabolites and 4-hydroxy estrogens are predictors of breast cancer risk and are also important factors in the prognosis of breast cancer patients and the choice of treatment.

CYP 1B1 Leads to DNA Adduct formation  2007

Belous, Alexandra R., et al. “Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation.” Cancer research 67.2 (2007): 812-817.

The results present direct proof of CYP1B1-mediated, E2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis

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Resveratrol and N-acetylcysteine 2021

Cavalieri, Ercole, and Eleanor Rogan. “The 3, 4-Quinones of Estrone and Estradiol Are the Initiators of Cancer whereas Resveratrol and N-acetylcysteine Are the Preventers.” International Journal of Molecular Sciences 22.15 (2021).

It is not surprising, therefore, that the 4-OHE1(E2) are far more potent as carcinogens than the 2-OHE1(E2) [38,39,40]. The 4-OHE1(E2) induce kidney tumors in male Syrian golden hamsters, but the 2-OHE1(E2) do not [38,39]. Neonatal exposure of CD-1 mice to 4-OHE2 led to uterine adenocarcinomas, but 2-OHE2 had only borderline ability to induce the tumors [40]. …the greater [carcinogenic] potency arises because E1(E2)-3,4-Q form depurinating DNA adducts more efficiently than E1(E2)-2,3-Q (Figure 2) [37].

Depurinating estrogen-DNA adducts can be detected in humans [13,14]. These adducts migrate out of cells and tissues, travel through the bloodstream, and are excreted in urine….However, the level of the adducts itself is not predictive of cancer risk since the level of estrogens in humans continuously varies. Instead, the molar ratio of depurinating estrogen-DNA adducts to estrogen metabolites and estrogen conjugates defines the balance of estrogen metabolism in a person and thus predicts that person’s risk for developing cancer [13,14,45].

The ratio in women at normal risk of developing breast cancer was significantly lower than the ratio in women at high risk or diagnosed with breast cancer (p < 0.001) in three different studies [44,45,46]. Similar estrogen-DNA adduct ratios are found in postmenopausal women and premenopausal women [46], establishing that the balance of estrogen metabolism does not depend on the level of circulating estrogens in a person. Instead, the balance of estrogen metabolism depends on a person’s enzymological makeup and can be affected by environmental factors that alter the balance of enzyme activities.

The critical observation from these studies is that women at high risk of developing breast cancer have a high ratio of adducts to metabolites and conjugates. This finding indicates that the formation of depurinating estrogen-DNA adducts begins before breast cancer develops and suggests that the formation of these adducts is a critical event in the initiation of cancer.

a significant difference was observed in the estrogen-DNA adduct ratios in the two groups of women. The women not diagnosed with breast cancer had an average ratio of 38.5, whereas those diagnosed with breast cancer had an average ratio of 92.4 (p < 0.001) [47]. These results support the role of estrogen-DNA adducts in the initiation of breast cancer.

The ratio of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates are also found to be significantly higher in women with ovarian (p < 0.0001) or thyroid (p < 0.0001) cancer [48,49].

single nucleotide polymorphisms (SNPs) in several genes for the enzymes involved in estrogen metabolism, CYP1B1 and COMT [49].

The study of women with and without ovarian cancer included urine samples for analysis of estrogen metabolites, conjugates and depurinating DNA adducts, as well as saliva samples for purification of DNA and analysis of single nucleotide polymorphisms (SNPs) in several genes for the enzymes involved in estrogen metabolism, CYP1B1 and COMT [49]. The selected SNPs resulted in a more active CYP1B1, which increased the formation of E1(E2)-3,4-Q, and a less active COMT, which decreased methylation of 4-OHE1(E2), thereby increasing the amount of E1(E2)-3,4-Q formed. Both of these SNPs thus resulted in more E1(E2)-3,4-Q available to react with DNA. When women had one or two copies of the SNP for a more active CYP1B1 plus two copies of the SNP for a less active COMT, they were three times more likely to have ovarian cancer, and had approximately twice the ratio of estrogen-DNA adducts to estrogen metabolites and conjugates as women without the SNPs. When the women had two copies of both the CYP1B1 and COMT SNPs, they were six times more likely to have the disease and had even higher estrogen-DNA adduct ratios [49].

In summary, these results in both men and women strongly suggest that the formation of depurinating estrogen-DNA adducts is a critical event in the initiation of cancer.

Estrogens need to be metabolized to the reactive catechol estrogen-3,4-quinones to be carcinogenic. Therefore, it should be possible to prevent cancer by inhibiting this metabolic process and/or blocking reaction of the quinones with DNA. Two compounds are particularly good at blocking the metabolism of estrogens to quinones and/or scavenging the reactive quinones, they are: resveratrol and N-acetylcysteine (NAC) [13,14,30,31,32,53].

resveratrol and N-acetylcysteine (NAC)

Resveratrol is a natural product found in grape skins, red wine, and peanuts, and it has various biological actions. Resveratrol is found to have several therapeutic effects; it is known to be anti-inflammatory, antioxidant, antihyperlipidemic, and anticarcinogenic, possessing immune-modulating, cardioprotective, hepatoprotective, and neuroprotective properties [54]. Among these effects are specific abilities to reduce catechol estrogen semiquinones back to catechol estrogens [30,53] and induce the estrogen-protective enzyme quinone reductase [30,31,32,53]. Resveratrol also modulates CYP1B1, thereby reducing its activity and thus the formation of 4-OHE1(E2). NAC has somewhat different properties. It, too, can reduce estrogen semiquinones back to catechol estrogens, but its primary effect is to react with quinones to form conjugates [30,31,32,53], thus preventing the formation of estrogen-DNA adducts.

Both resveratrol and NAC have been shown to inhibit the formation of depurinating estrogen-DNA adducts in cultured mammalian cells [30,31,32]. Resveratrol was found to inhibit the malignant transformation of the human MCF-10F breast epithelial cell line [31]. NAC was found to inhibit the malignant transformation of both MCF-10F and immortalized mouse mammary cells [33,55]. The two compounds work together additively to reduce the formation of depurinating estrogen-DNA adducts in MCF-10F cells treated with 4-OHE2 (Figure 4A) [30]. These results lay the foundation for investigating the ability of resveratrol and NAC to reduce estrogen-DNA adduct formation in humans as an approach to cancer prevention.

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!  BEST !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

4. Conclusions: Estrogens initiate cancer through metabolism to catechol estrogen-3,4-quinones. The quinones react with DNA to primarily form the depurinating DNA adducts 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade. The resulting apurinic sites in specific locations in DNA generate mutations that can initiate cancer; this has been shown in laboratory animals and human beings. Two compounds, resveratrol and NAC, are potential cancer-preventive compounds that can reduce the formation of estrogen-DNA adducts in people, thereby blocking the initiation of estrogen-induced cancer.

Other evidence points to an ER-independent mechanism. The development of estrogen-dependent mammary tumors in ERKO/Wnt-1 mice, even in the presence of an anti-estrogen, constitutes some of the strongest evidence for an ER-independent mechanism of cancer initiation by estrogens. The observation of high ratios of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates in women at high risk for breast cancer, as well as women with breast cancer [45,46,47], is consistent with an ER-independent mechanism of initiation. In addition, the presence of SNPs in CYP1B1 and COMT that increase both the formation of depurinating estrogen-DNA adducts and the likelihood of ovarian cancer (six-fold) [50] supports an ER-independent mechanism of cancer initiation by estrogens.

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2017
Cavalieri, Ercole L., and Eleanor G. Rogan. “Etiology and prevention of prevalent types of cancer.” Journal of rare diseases research & treatment 2.3 (2017): 22.

2016
Cavalieri, Ercole L., and Eleanor G. Rogan. “Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention.” Clinical and translational medicine 5 (2016): 1-15.

4 Hydroxy estrogens are Markers of breast cancer

Liehr, Joachim G., and Mary Jo Ricci. “4-Hydroxylation of estrogens as marker of human mammary tumors.” Proceedings of the National Academy of Sciences 93.8 (1996): 3294-3296.

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Russo

Estrogen Causes Breast Cancer

Yue, Wei, et al. “Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis.” Steroids 78.2 (2013): 161-170.

Russo, Jose, et al. “17β-Estradiol is carcinogenic in human breast epithelial cells.” The Journal of steroid biochemistry and molecular biology 80.2 (2002): 149-162.

only breast cells with the  4p15.3-16 deletion mutation were tumorogenic in SCID mice 

Russo, Jose, et al. “17‐Beta‐estradiol induces transformation and tumorigenesis in human breast epithelial cells.” The FASEB journal 20.10 (2006): 1622-1634.

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17beta-estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER-alpha) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage-independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11-13. Only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor-negative adenocarcinomas that expressed keratins, EMA, and E-cadherin. Tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of MCF-10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

These cells, if containing a 4p15.3-16 deletion, are tumorigenic in SCID mice (Russo et al.,. 2006).

Russo, J. I. H. R., and Irma H. Russo. “The role of estrogen in the initiation of breast cancer.” The Journal of steroid biochemistry and molecular biology 102.1-5 (2006): 89-96.

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17β-estradiol (E2) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E2, 4-OH-E2, and 16-α-OH E2) respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither TAMOXYFEN (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E2 induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-α and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E2 induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

Yue, Wei, et al. “Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis.” Steroids 2.78 (2013): 161-170.
Long term exposure to estrogens is associated with an increased risk of breast cancer. The precise mechanisms responsible for estrogen mediated carcinogenesis are not well understood. The most widely accepted theory holds that estradiol (E(2)), acting through estrogen receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E(2) then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development. This manuscript critically reviews existing data implicating both ER-dependent and -independent mechanisms. The weight of evidence supports the possibility that both mechanisms are involved in the carcinogenic process. In addition, estrogen metabolites likely modulate stem cell functionality and cancer progression. The roles of ER dependent and independent actions in the carcinogenic process are pertinent to the consideration of breast cancer preventative agents as anti-estrogens block only receptor mediated pathways whereas the aromatase inhibitors block both.

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Ke, Bin, Chunyu Li, and Huifang Shang. “Sex hormones in the risk of breast cancer: a two-sample Mendelian randomization study.” American Journal of Cancer Research 13.3 (2023): 1128.

no association was identified between estradiol, progesterone and the risk of breast cancer.

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2020 Serum Iodine and Breast Cancer Risk, Malmö Diet and Cancer Study

24) Manjer, Jonas, Malte Sandsveden, and Signe Borgquist. “Serum Iodine and Breast Cancer Risk: A Prospective Nested Case–Control Study Stratified for Selenium Levels.” Cancer Epidemiology, Biomarkers & Prevention 29.7 (2020): 1335-1340.

Iodine has been suggested to protect against breast cancer, but there are no epidemiologic studies on individual risk. An interesting finding is that in areas where the exposure to both selenium and iodine are high (e.g., Japan), the risk of breast cancer is lower than in areas where selenium is high and iodine low (e.g., United States), or in areas where both are low (e.g., Northern Europe). The aim of this study was to investigate the association between prediagnostic serum iodine levels and subsequent breast cancer risk, and to investigate if this potential association was modified by selenium levels.

Methods: The Malmö Diet and Cancer Study provided prediagnostic serum samples and the current analysis included 1,159 breast cancer cases and 1,136 controls. Levels of baseline serum iodine and selenium were analyzed. A logistic regression analysis yielded ORs with 95% confidence intervals adjusted for potential confounders.

Results: There was no evidence of an overall association between iodine levels and risk of breast cancer. Among women with high selenium levels (above the median), high iodine levels were associated with a lower risk of breast cancer; the OR for above versus below the median was 0.75 (0.57-0.99). The corresponding OR for women with low selenium was 1.15 (0.87-1.50), and the P interaction was 0.06.

Conclusions: The combination of high serum iodine levels and high selenium levels was associated with a lower risk of breast cancer.

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selenium breast cancer

Szwiec, Marek, et al. “Serum selenium level predicts 10-year survival after breast cancer.” Nutrients 13.3 (2021): 953.

We now have updated the cohort to include 10-year survival rates. A blood sample was obtained from 538 women diagnosed with first primary invasive breast cancer between 2008 and 2015 in the region of Szczecin, Poland. Blood was collected before initiation of treatment. Serum selenium levels were quantified by mass spectroscopy. Each patient was assigned to one of four quartiles based on the distribution of serum selenium levels in the whole cohort. Patients were followed from diagnosis until death or last known alive (mean follow-up 7.9 years). The 10-year actuarial cumulative survival was 65.1% for women in the lowest quartile of serum selenium, compared to 86.7% for women in the highest quartile (p < 0.001 for difference). Further studies are needed to confirm the protective effect of selenium on breast cancer survival. If confirmed this may lead to an investigation of selenium supplementation on survival of breast cancer patients.

Lubinski, J., et al. “Serum selenium levels predict survival after breast cancer.” Breast cancer research and treatment 167.2 (2018): 591-598.

Harris, Holly R., Leif Bergkvist, and Alicja Wolk. Selenium intake and breast cancer mortality in a cohort of Swedish women .” Breast cancer research and treatment 134 (2012): 1269-1277.

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Lower Iodine Excretion in Breast Cancer Cases

Kurniawati, Yulia, et al. “Analysis of Urinary Iodine Concentration in Differentiated Thyroid Cancer and Breast Cancer Cases.” Asian Pacific journal of cancer prevention: APJCP 25.6 (2024): 1869-1873.

In BC patients, regardless of subtypes, breast cancer subjects showed a significantly lower iodine excretion level. The median of UIC patients and controls were 80.05 ± 38.24 µg/L and 144.25 ± 36.79 µg/L, respectively, p=0.000.

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Sorrenti, Salvatore, et al. “Iodine: Its role in thyroid hormone biosynthesis and beyond.” Nutrients 13.12 (2021): 4469.

iodine oxidation to hypoiodite (IO−) has been shown to possess strong bactericidal as well as antiviral and antifungal activity. Finally, and importantly, iodine has been demonstrated to exert antineoplastic effects in human cancer cell lines. Thus, iodine, through the action of different tissue-specific peroxidases, may serve different evolutionarily conserved physiological functions that, beyond TH biosynthesis, encompass antioxidant activity and defense against pathogens and cancer progression.

iodine oxidation to hypoiodite (IO−) possesses strong bactericidal as well as antiviral and antifungal activity [6,7,8,9]. Finally, iodine has been demonstrated to exert antineoplastic effects in breast cancer, and in human melanoma- and lung cancer-derived cell lines

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2023

Ibrahim, Raihan Syah, and Aisyah Elliyanti. “The Potential of Iodine as A Treatment for Breast Cancer: A Narrative Review.” Jurnal Kesehatan Manarang 9.3 (2023): 159-165.

In-Vitro Cancer cell Lines, Breast, Lung, pancreatic, melanoma

Rösner, Harald, et al. “Antiproliferative/cytotoxic effects of molecular iodine, povidone-iodine and Lugol’s solution in different human carcinoma cell lines.” Oncology letters 12.3 (2016): 2159-2162.

Materials The following human cell lines were used in the present study:
MCF-7 (breast carcinoma);  HS-24, H1299 and A549 (lung carcinoma);
Capan-2 and PaTu 8902 (pancreatic carcinoma), and the IPC-298 (melanoma) cell line,

Iodine for prostate, lung carcinoma, pancreas carcinoma, melanoma, glioblastoma, and neuroblastoma cells

Aceves, Carmen, et al. “Molecular iodine has extrathyroidal effects as an antioxidant, differentiator, and immunomodulator.” International journal of molecular sciences 22.3 (2021): 1228.

Various groups have described apoptosis effects of iodine in several cancer cell lines and proposed different mechanisms and pathways (Figure 3). The most studied effects include a direct action, where the oxidized iodine dissipates the mitochondrial membrane potential, thereby triggering mitochondrion-mediated apoptosis [64], and an indirect effect through iodolipids formation and the activation of peroxisome proliferator-activated receptors type gamma (PPARγ) [65].

Moreover, recent reports have shown that antineoplastic effects of iodine or iodolipids are exerted on different types of cells that can take up I2 and exhibit apoptotic induction by PPAR agonists. Such cells include prostate, lung carcinoma, pancreas carcinoma, melanoma, glioblastoma, and neuroblastoma cells [79].

We propose that molecular iodine intake be increased in adults to at least 1 mg/day in specific pathologies to obtain the potential extrathyroidal benefits described in the present review.

64. Shrivastava A., Tiwari M., Sinha R.A., Kumar A., Balapure A.K., Bajpai V.K., Sharma R., Mitra K., Tandon A., Godbole M.M. Molecular Iodine Induces Caspase-Independent Apoptosis in Human Breast Carcinoma Cells Involving the Mitochondria-mediated Pathway. J. Biol. Chem. 2006;281:19762–19771. doi: 10.1074/jbc.M600746200. [PubMed] [CrossRef] [Google Scholar]

65. Aceves C., García-Solís P., Omar A.-H., Vega-Riveroll L., Delgado G., Anguiano B. Antineoplastic effect of iodine in mammary cancer: Participation of 6-iodolactone (6-IL) and peroxisome proliferator-activated receptors (PPAR) Mol. Cancer. 2009;8:33. doi: 10.1186/1476-4598-8-33. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

Published Studies of the Effects of Iodine Administration on Breast Cancer

Mendieta, Irasema, et al. “Molecular iodine exerts antineoplastic effects by diminishing proliferation and invasive potential and activating the immune response in mammary cancer xenografts.” BMC cancer 19 (2019): 1-12.

The present work analyzed the effect of I2 in human breast cancer cell lines with low (MCF-7) and high (MDA-MB231) metastatic potential under both in vitro (cell proliferation and invasion assay) and in vivo (xenografts of athymic nude mice) conditions.
Results

In vitro analysis showed that the 200 μM I2 supplement decreases the proliferation rate in both cell lines and diminishes the epithelial-mesenchymal transition (EMT) profile and the invasive capacity in MDA-MB231. In immunosuppressed mice, the I2 supplement impairs implantation (incidence), tumoral growth, and proliferation of both types of cells. Xenografts of the animals treated with I2 decrease the expression of invasion markers like CD44, vimentin, urokinase plasminogen activator and its receptor, and vascular endothelial growth factor; and increase peroxisome proliferator-activated receptor gamma. Moreover, in mice with xenografts, the I2 supplement increases the circulating level of leukocytes and the number of intratumoral infiltrating lymphocytes, some of them activated as CD8+, suggesting the activation of antitumor immune responses.
Conclusions: I2 decreases the invasive potential of a triple negative basal cancer cell line, and under in vivo conditions the oral supplement of this halogen activates the antitumor immune response, preventing progression of xenografts from laminal and basal mammary cancer cells. These effects allow us to propose iodine supplementation as a possible adjuvant in breast cancer therapy.
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Prostate

Anguiano, Brenda, et al. “Protective effects of iodine on rat prostate inflammation induced by sex hormones and on the DU145 prostate cancer cell line treated with TNF.” Molecular and Cellular Endocrinology 572 (2023): 111957.

In-vivo xenograft study mice

Aranda, Nuri, et al. “Uptake and antitumoral effects of iodine and 6‐iodolactone in differentiated and undifferentiated human prostate cancer cell lines.” The Prostate 73.1 (2013): 31-41.

METHODS. Non-cancerous (RWPE-1) and cancerous (LNCaP, DU-145) cells, as well as
nude mice xenotransplanted with DU-145 cells were used as cancer models. Iodine uptake was analyzed with radioactive tracers, transporter expression by qRT-PCR, cell proliferation by blue trypan, apoptosis by enzyme immunoassay or fluorescence, BAX and BCL-2 by western-blot, and caspsase 3 by enzymatic assay.

RESULTS. All three cell lines take up both forms of iodine. In RWPE-1 cells, I uptake depends on the Na/I symporter (NIS), whereas it was independent of NIS in LNCaP and DU-145 cells. Antiproliferative effects of iodine and 6-IL were dose and time dependent; RWPE-1 was most sensitive to I and 6-IL, whereas LNCaP was more sensitive to I2. In the three cell lines both forms of iodine activated the intrinsic apoptotic pathway (increasing the BAX/BCL-2 index and caspases). Iodine supplementation impaired growth of the DU-145 tumor in nude mice.
CONCLUSION. Normal and cancerous prostate cells can take up iodine, and depending on
the chemical form, it exerts antiproliferative and apoptotic effects both in vitro and in vivo.

Neuroblastoma

Mendieta, Irasema, et al. “Molecular iodine synergized and sensitized neuroblastoma cells to the antineoplastic effect of ATRA.” Endocrine-Related Cancer 27.12 (2020): 699-710.

Falkenberg, Torkel, et al. “Iodine loaded nanoparticles with commercial applicability increase survival in mice cancer models with low degree of side effects.” Cancer Reports 6.8 (2023): e1843.

The syngeneic colon cancer model cells (CT26), the xenograft breast cancer model cells and the orthotopic, syngeneic lung metastasis model cells (LL/2)…NO Anti Tumor Effect !!!

Thomasz, Lisa, et al. “6 Iodo-δ-lactone: A derivative of arachidonic acid with antitumor effects in HT-29 colon cancer cells.” Prostaglandins, Leukotrienes and Essential Fatty Acids 88.4 (2013): 273-280.

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references from: Fork Clinic Estrogen Detox Protocol

Calcium-D-glucarate. (2002). Alternative medicine review : a journal of clinical therapeutic, 7(4), 336–339.

Bastos, P., Gomes, T., & Ribeiro, L. (2017). Catechol-O-Methyltransferase (COMT): An Update on Its Role in Cancer, Neurological and Cardiovascular Diseases. Reviews of physiology, biochemistry and pharmacology, 173, 1–39.

Vanduchova, A., Anzenbacher, P., & Anzenbacherova, E. (2019). Isothiocyanate from Broccoli, Sulforaphane, and Its Properties. Journal of medicinal food, 22(2), 121–126.

Abdull Razis, A. F., Konsue, N., & Ioannides, C. (2018). Isothiocyanates and Xenobiotic Detoxification. Molecular nutrition & food research, 62(18), e1700916.

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Estrogen Metabolism

Nice Image Chart of Estrogen Metabolism

Duplicate with (67)

Fuhrman, Barbara J., et al. “Estrogen metabolism and risk of breast cancer in postmenopausal women.” Journal of the National Cancer Institute 104.4 (2012): 326-339.

More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

Above Image: Estrogen Metabolism Courtesy of Dr. Barbara Fuhrman and NIH, 2012 (67)

More recent chart (2023), Courtesy of: Al-Shami, Khayry, et al. “Estrogens and the risk of breast cancer: A narrative review of literature.” Heliyon 9.9 (2023). (33)

Fig. 3: Estrogen metabolism pathway in humans. The diagram shows the metabolism of estradiol and other natural estrogens such as estrone and estriol. It demonstrates that conjugation (e.g., sulfation and glucuronidation) occurs in the case of estradiol and metabolites of estradiol that have one or more available hydroxyl (–OH) groups. Catechol and quinone formation from estrone is shown and how the derivatives are reacting with DNA to form depurination DNA adducts (adapted from Ref. [16]). Courtesy of: Al-Shami, Khayry, et al. “Estrogens and the risk of breast cancer: A narrative review of literature.” Heliyon 9.9 (2023). (33)

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Al-Shami, Khayry, et al. “Estrogens and the risk of breast cancer: A narrative review of literature.” Heliyon 9.9 (2023).

Fig. 3 Estrogen metabolism pathway in humans. The diagram shows the metabolism of estradiol and other natural estrogens such as estrone and estriol. It demonstrates that conjugation (e.g., sulfation and glucuronidation) occurs in the case of estradiol and metabolites of estradiol that have one or more available hydroxyl (–OH) groups. Catechol and quinone formation from estrone is shown and how the derivatives are reacting with DNA to form depurination DNA adducts (adapted from Ref. [16]).

Shouman, Samia, Mohamed Wagih, and Marwa Kamel. “Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells.” Cancer Biology & Medicine 13.4 (2016): 505.

Fig 1. Estradiol is metabolized into 2-OHE2 and 4-OHE2 by CYP1A1 and CYP1B1, respectively. These catechols undergo further oxidation into semiquinones and quinones that react with DNA to form depurinating adducts leading to mutations associated with breast cancer. NQO1 Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1 (NQO1) reduces these quinones back to catechols which are detoxified into methoxy derivatives by the action of COMT which protects the cells against DNA adducts formation and lowers the potential for mutagenic damage. Used with permission from Ref.9.

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Sulforaphane

Kuran, Dominika, Anna Pogorzelska, and Katarzyna Wiktorska. “Breast cancer prevention-is there a future for sulforaphane and its analogs?.” Nutrients 12.6 (2020): 1559.

Naujokat, Cord, and Dwight L. McKee. “The “Big Five” phytochemicals targeting cancer stem cells: Curcumin, EGCG, sulforaphane, resveratrol and genistein.” Current medicinal chemistry 28.22 (2021): 4321-4342.

Saavedra-Leos, María Zenaida, et al. “Molecular pathways related to sulforaphane as adjuvant treatment: A nanomedicine perspective in breast cancer.” Medicina 58.10 (2022): 1377.

Pogorzelska, A., et al. “Antitumor and antimetastatic effects of dietary sulforaphane in a triple-negative breast cancer models.” Scientific Reports 14.1 (2024): 16016.

Asif Ali, Muhammad, et al. “Anticancer properties of sulforaphane: current insights at the molecular level.” Frontiers in oncology 13 (2023): 1168321.

Li, Shizhao, Huixin Wu, and Trygve O. Tollefsbol. “Combined broccoli sprouts and green tea polyphenols contribute to the prevention of estrogen receptor–negative mammary cancer via cell cycle arrest and inducing apoptosis in HER2/neu mice.” The Journal of Nutrition 151.1 (2021): 73-84.

Santana-Gálvez, Jesús, et al. “Synergistic combinations of curcumin, sulforaphane, and dihydrocaffeic acid against human colon cancer cells.” International journal of molecular sciences 21.9 (2020): 3108.

Wang, Yuan, et al. “Sulforaphane induces S-phase arrest and apoptosis via p53-dependent manner in gastric cancer cells.” Scientific reports 11.1 (2021): 2504.

Rutz, Jochen, et al. “Sulforaphane reduces prostate cancer cell growth and proliferation in vitro by modulating the Cdk-Cyclin axis and expression of the CD44 variants 4, 5, and 7.” International journal of molecular sciences 21.22 (2020): 8724.

Zhang, Fangxi, et al. “Sulforaphane inhibits the growth of prostate cancer by regulating the microRNA-3919/DJ-1 axis.” Frontiers in Oncology 14 (2024): 1361152.

Pterostilbene

Kumar, Viney, et al. “Pterostilbene-isothiocyanate inhibits breast cancer metastasis by selectively blocking IKK-β/NEMO interaction in cancer cells.” Biochemical Pharmacology 192 (2021): 114717.

Ma, Kang, et al. “Pterostilbene inhibits the metastasis of TNBC via suppression of β-catenin-mediated epithelial to mesenchymal transition and stemness.” Journal of Functional Foods 96 (2022): 105219.

Yang, Wenhui, et al. “Pterostilbene induces pyroptosis of breast cancer cells, inhibits the growth and invasion of transplanted tumor and reshapes its tumor microenvironment.” (2022).

Moon, Dora, et al. “Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro.” Journal of Surgical Research 180.2 (2013): 208-215.

Alosi, Julie A., et al. “Pterostilbene inhibits breast cancer in vitro through mitochondrial depolarization and induction of caspase-dependent apoptosis.” Journal of Surgical Research 161.2 (2010): 195-201.

Wang, Yanshang, et al. “Pterostilbene simultaneously induces apoptosis, cell cycle arrest and cyto-protective autophagy in breast cancer cells.” American journal of translational research 4.1 (2012): 44.

Elsherbini, Asmaa M., Salah A. Sheweita, and Ahmed S. Sultan. “Pterostilbene as a phytochemical compound induces signaling pathways involved in the apoptosis and death of mutant P53-breast cancer cell lines.” Nutrition and Cancer 73.10 (2021): 1976-1984.

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Estrogen metabolism 2024

https://www.researchsquare.com/article/rs-3944853/v1
Newman, Mark, and Jaclyn Smeaton. “Exploring the Impact of 3, 3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women.” (2024).
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Last updated on October 7th, 2024 by Jeffrey Dach MD