Index for Breast Cancer

Cracking Cancer Toolkit Jeffrey Dach MDINDEX for Breast Cancer in the book, Cracking Cancer Toolkit by Jeffrey Dach MD

P 27  DIM
Thomson, Cynthia A., Emily Ho, and Meghan B.
Strom. “Chemopreventive properties of 3, 3′-diindolylmethane
in breast cancer: evidence from experimental
and human studies.” Nutrition reviews 74.7
(2016): 432-443.

P 38  Detecting CTC’s
Papasotiriou, Ioannis, et al. “Detection of circulating
tumor cells in patients with breast, prostate,
pancreatic, colon and melanoma cancer: A blinded
comparative study using healthy donors.” Journal of
Cancer Therapy 6.07 (2015): 543.

Ntanovasilis, Dimitrios-Athanasios, Panagiotis
Apostolou, and Ioannis Papasotiriou. “Flow Cytometric
Detection of Circulating Tumor Cells in Breast Cancer
Patients: A Blinded Study.” Journal of Cancer Therapy
10.08 (2019): 708.

PTEROSTILBENE

P 41
Dr. Dora Moon
et al. (2013) studied in vitro breast cancer cells
showing that pterostilbene induces apoptosis
via the mitochondrial pathway in breast cancer
cell lines. (13)

A 2015 study by Julie A. Alosi et al. on pterostilbene in breast cancer showed similar findings
with apoptosis induced by mitochondrial pathways. (15)

A 2012 study by Yanshang Wang et al. showed pterostilbene induces apoptosis and
cell-cycle arrest in breast cancer cells. (16)

P 45
13) Moon, Dora, et al. “Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro.” Journal of Surgical Research 180.2 (2013): 208-215.

15) 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.

16) 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, no. 1 (2012): 44.

Chinese Skullcap

p 42

Chinese Skullcap (also called Oroxylin A and Baicalin) is extracted from a medicinal
plant called Scutellaria baicalensis Georgi. Researchers found that Chinese Skullcap inhibits
GLYCOLYSIS and induces disassociation of HK2 from the mitochondria in human breast
carcinoma cell lines, thus inducing apoptosis. Chinese Skullcap blocked the translocation of
the anti-apoptotic protein Bcl-2 to the mitochondria, keeping the VDAC pores open, allowing
apoptosis to take place. (24–25)

24) Wei, L., et al. “Oroxylin A induces dissociation of hexokinase II from the mitochondria and inhibits glycolysis by SIRT3-mediated deacetylation of cyclophilin D in breast carcinoma.” Cell death & disease 4.4 (2013): e601.

Quercetin

p 43

In 2018, Dr. Lijun Jia et al. studied the anti-cancer effects of QUE in a breast cancer
model using in vitro and in vivo xenografts. Dr. Jia found that QUE blocked cancer-cell
GLYCOLYSIS, inhibited glucose uptake, decreased lactic acid, and decreased glycolysis-
related proteins such as pyruvate kinase, glucose transporter1 (GLUT1), and lactate
dehydrogenase A (LDHA). As expected, inhibition of mTOR resulted in autophagy induction

in this model. Dr. Jia’s group writes:
Quercetin suppressed the progression of breast cancer by inhibiting cell mobility
and glycolysis through Akt-mTOR pathway mediated autophagy induction and may
provide a potential therapeutic target for breast cancer treatment. (43)

p 46

43) Jia, Lijun, et al. “Quercetin suppresses the mobility of breast cancer by suppressing glycolysis through Akt-mTOR pathway mediated autophagy induction.” Life sciences 208 (2018): 123-130

DCA

p 51
In 2013, Dr. G. Sutendra et al. used animal xenografts of non-small-cell lung cancer
and breast cancer to study DCA inhibition of HIF-1 and angiogenesis. They observed that
DCA increases pyruvate dehydrogenase (PDH) activity, inhibiting GLYCOLYSIS, which inhibited cancer cell proliferation and induced apoptosis. In addition, DCA treatment suppressed new tumor vessel formation (angiogenesis), and
also inhibited HIF1α. They write:

Effective inhibition of HIF1α is shown by a decrease in the expression of several HIF1α
regulated gene products as well as inhibition of angiogenesis in vitro in matrigel
assays…. DCA increases pyruvate dehydrogenase activity … pro-apoptotic and
antiproliferative effects and suppresses angiogenesis as well, normalizing the pseudo-
hypoxic signals that lead to normoxic HIF1α activation in solid tumors. (32)

p 64

32) Sutendra, G., et al. “Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer.” Oncogene 32.13 (2013): 1638.

p 57 Breast Cancer Stem Cells have Pro-Glycolytic Phenotype !!!!

In 2014, Dr. Weiguo Feng et al. studied the anti-CSC effects of DCA in a breast cancer
model, finding that the CSCs (called TICs, for tumor-initiating cells) have a “pro-glycolytic
phenotype” with fewer, less active mitochondria. Dr. Feng found that breast CSCs rely on
GLYCOLYSIS and are eliminated in vitro and in vivo by metabolic reprogramming with a glycolysis inhibitor, such as DCA. Dr. Feng’s group concludes:

Transcriptome profiling using RNA-Seq revealed TICs [tumor-initiating cells, cancer
stem cells] under-express genes involved in mitochondrial biology and mitochondrial
oxidative phosphorylation and metabolic analyses revealed TICs preferentially
perform glycolysis over oxidative phosphorylation compared to NTCs [non-tumor-
initiating cells]. Mechanistic analyses demonstrated that decreased expression
and activity of pyruvate dehydrogenase [PDH], a key regulator of oxidative phosphorylation,
play a critical role in promoting  the pro-glycolytic phenotype of TICs.
Metabolic reprogramming [with DCA] via forced activation of PDH preferentially
eliminates TICs both in vitro and in vivo.(94)

p 67
94) Feng, Weiguo, et al. “Targeting unique metabolic properties of breast tumor initiating cells.” Stem cells 32.7 (2014): 1734-1745.

DCA Enhances effect of DOXORubicin p 59

In 2017, Dr. Minghao Wang
et al. studied the effect of DCA in combination with Doxorubicin (DOX) in a breast cancer cell
model, finding DCA significantly inhibits the autophagy induced by DOX, markedly enhancing cancer cell death in vitro and in vivo with prolongation of mouse survival time. Dr. Wang’s group writes:
DCA inhibit[s] doxorubicin-inducing autophagy and provide[s] a novel strategy
for improving the anti-cancer efficacy of chemotherapy. (152)

p 68

114) Sun, Ramon C., et al. “Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo.” Breast cancer research and treatment 120.1 (2010): 253-260.

Solomons Seal p 63

7) Ouyang, Liang, et al. “Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFRmediated Ras-Raf-MEK-ERK pathway in human MCF-7
breast cancer cells.” Phytomedicine 21.12 (2014): 1658-1665.

Methyl Jasmonate p 65

57) Yehia, Rania, et al. “Anti-tumor efficacy of an integrated methyl dihydrojasmonate transdermal microemulsion system targeting breast cancer cells:in vitro and in vivo studies.” Colloids and Surfaces B:Biointerfaces 155 (2017): 512-521.

p 66
78) Haugrud, Allison B., et al. “Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells.” Breast cancer research and treatment 147.3 (2014): 539-550.

p 69

131) Wang, Minghao, et al. “Sensitization of breast cancer cells to paclitaxel by dichloroacetate through inhibiting autophagy.” Biochemical and biophysical
research communications 489.2 (2017): 103-108.

Propranolol Beta Blocker p. 72

Similarly, in 2010, Dr. Erica Sloan et al. studied the effect of stress-induced neuroendocrine
activation in a mouse model of breast cancer using in vivo bioluminescence imaging
to track metastatic spread of disease. This study revealed that stress-increased beta-adrenergic signaling, which increased macrophage infiltration into the tumor, induced a
pro-metastatic gene expression and induced a 30-fold increase in distant metastatic lesions.
This effect was blocked by treating the animals with the beta blocker propranolol. Dr. Sloan et al. conclude:

These findings identify activation of the sympathetic nervous system as a novel
neural regulator of breast cancer metastasis and suggest new strategies for
antimetastatic therapies that target the βeta-adrenergic induction of pro-metastatic
gene expression in primary breast cancers. (10)

p 81
10) Sloan, Erica K., et al. “The sympathetic nervous system induces a metastatic switch in primary breast cancer.” Cancer research 70.18 (2010): 7042-7052.

p 73
According to Dr. Alexa Montoya et al. (2019), retrospective studies using propranolol
(beta-adrenergic antagonists) in breast cancer patients showed reduced tumor proliferation,
decreased mortality, decreased metastases, longer survival, and reduced cancer recurrence.
Most significantly, propranolol downregulates hexokinase 2 and inhibits glucose metabolism.
Propranolol has anti-angiogenic effects, as well. (19–20)

p 82

19) Montoya, Alexa, et al. “The beta adrenergic receptor antagonist propranolol alters mitogenic and apoptotic signaling in late stage breast cancer.” Biomedical Journal 42.3 (2019): 155.

20) Osawa, H., et al. “Regulation of hexokinase II gene transcription and glucose phosphorylation by catecholamines, cyclic AMP, and insulin.” Diabetes 44.12 (1995): 1426.

p 74

In 2016, Dr. Pan Pantziarka et al. reviewed the nonselective beta blocker propranolol as
a repurposed anti-cancer drug. Propanolol anti-cancer activities include:

• Abrogates norepinephrine-induced increased migratory activity in breast
cancer cells (blocks metastatic spread).
• Exerts an anti-angiogenic effect by downregulating VEGF in cancers and
infantile hemangiomas. (29)

p 82

29) Pantziarka, Pan, et al. “Propranolol and breast cancer—A work in progress.” ecancermedicalscience 12 (2018).

p 74

Propranolol for Breast Cancer Patients

In 2013, Dr. Edoardo Botteri et al. identified 800 post-menopausal women who underwent
surgery to remove a triple-negative breast cancer (negative for hormone receptors). Of the
800, 74 women were coincidentally on beta blockers.

Beta blocker (BB) intake was associated with a significantly decreased risk of recurrence,
metastasis, and death from breast cancer.

The hazard ratio (HR) for women on beta blockers was 0.32 for metastases and 0.42 for
death from breast cancer, compared to controls not on beta blockers. This is remarkable. (34)   In 2011, Dr. Amal Melhem-Bertrandt et al. found improved relapse-free survival in the cohort of women using beta blocker drugs. (HR= 0.32) (35)

In 2015, Dr. Kurtis Childers et al. did a  meta-analysis, finding risk of death from breast
cancer reduced in half: Breast cancer death results were contained in 4 studies, which also suggested a significant reduction in risk (HR, 0.50; 95% CI,
0.32–0.80). (36)

In 2011, Dr. Thomas Barron et al. analyzed women with breast cancer in Ireland over a
5-year period from 2001 to 2006. Compared to matched controls, women taking propranolol
had less locally invasive or metastatic cancers at diagnosis, with marked reduction in
metastases (OR, 0.20; 95% CI, 0.04 to 0.88) and striking reduction in breast cancer mortality
(HR, 0.19; 95% CI, 0.06 to 0.60). Propranolol has been suggested as an adjunct to breast
cancer treatment. Additional studies describe beta blockers conferring similar reduction in
cancer mortality in prostate cancer, melanoma, colon, ovarian, prostate, non-small-cell lung,
and hepatocellular, multiple myeloma. (37)

p 82

34) Botteri, Edoardo, et al. “Therapeutic effect of β-blockers in triple-negative breast cancer postmenopausal women.” Breast cancer research and treatment 140.3 (2013): 567-575

35) Melhem-Bertrandt, Amal, et al. “Beta-blocker use is associated with improved relapse-free survival in patients with triple-negative breast cancer.” Journal of clinical oncology 29.19 (2011): 2645.

36) Childers, W. Kurtis, Christopher S. Hollenbeak, and Pramil Cheriyath. “β-blockers reduce breast cancer recurrence and breast cancer death: a meta-analysis.” Clinical breast cancer 15.6 (2015): 426-431.

37) Barron, Thomas I., et al. “Beta blockers and breast cancer mortality: a population-based study.” J Clin Oncol 29.19 (2011): 2635-2644.

p 75 Evaluation of Cancer Markers after Propranolol

In 2019, Dr. Alexa Montoya et al. studied late-stage breast cancer in a 44-year-old female
with a 6.5 cm. invasive lobular breast cancer. After initial biopsy, the patient was treated
with propranolol, 80 mg per day for 25 days and then underwent definitive radical mastectomy .

The histology on microscopic slides of both biopsy sample and mastectomy sample
were compared. The use of propranolol decreased the Ki-67 proliferation marker, the
BCL-2 anti-apoptosis protein, and increased pro-apoptosis p53 expression on the surgical
specimen histology slides. (19)

19) Montoya, Alexa, et al. “The beta adrenergic receptor antagonist propranolol alters mitogenic and apoptotic signaling in late stage breast cancer.” Biomedical Journal 42.3 (2019): 155.

p 76 Propranolol Reduces HK2 and PET Uptake in Breast Cancer

In 2014, Dr. Fei Kang et al. studied breast cancer using in vitro and in vivo mouse xenografts. Their studies showed that beta-adrenergic receptors were overexpressed in
breast cancer cell lines. Propranolol significantly decreased HK2 protein levels through
post-transcription mechanisms. In vivo studies using propranolol in this animal model of breast cancer showed reduced expression of HK2. In addition, PET scans of the propranolol-treated mice showed reduced 18-FDG uptake in tumor and brown adipose tissue. Dr. Kang et al. suggested the use of propranolol in humans might reduce true-positive PET scan uptake in small or low-uptake lesions, concluding:
The expression of HK-2 was regulated by the activation of ADRB2 [Adrenergic Receptor Beta2] in 4T1 [mouse] breast cancer cells primarily at the post-transcriptional
level. Additionally, propranolol prevented glucose metabolism and 18FFDG
PET imaging of 4T1 breast cancer tumors. (40)

40) Kang, Fei, et al. “Propranolol inhibits glucose metabolism and 18F-FDG uptake of breast cancer through posttranscriptional downregulation of hexokinase-2.” Journal of Nuclear Medicine 55.3 (2014):439-445.

p 76 Synergy of Propranolol with Metformin in Breast Cancer Model

In Oncotarget (2017), Dr. Maria Rico et al. studied the combination of metformin and propranolol in a breast cancer cell model, finding reduction in cancer progression, metastasis, and reduced migratory and invasive behavior of the cancer cells. The combination of metformin and propranolol showed a “strong inhibition of
mitochondrial bioenergetics” and “drastically activated glycolysis…abolished mitochondrial
respiratory activity…with a noticeable increase in glycolysis.” The metabolic pathway of the
cancer cell was skewed away from mitochondrial OXPHOS and toward GLYCOLYSIS. Dr. Rico et al. theorize that this metabolic derangement starved the cancer cells of glucose, causing cell death:
By enhancing glycolysis to an extraordinarily high rate, the combination probably
leads to glucose deprivation in the tumor cell micro-environment… ultimately produce
synthetic lethality, as glucose levels decrease. (41)

41) Rico, María, et al. “Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models.” Oncotarget 8.2 (2017): 2874.

p 54

69) Hiller, Jonathan G., et al. “Pre-operative β-blockade with propranolol reduces biomarkers of metastasis in breast cancer: a Phase II randomized trial.” Clinical Cancer Research (2019).

70) Pasquier, Eddy, et al. “Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: implication in breast cancer treatment.” Oncotarget 2.10 (2011): 797.

71) Powe, Desmond G., et al. “Beta-blocker drug therapy reduces secondary cancer formation in breast cancer and improves cancer specific survival.” Oncotarget
1.7 (2010): 628.

72) Powe, D. G., et al. “Alpha-and beta-adrenergic receptor (AR) protein expression is associated with poor clinical outcome in breast cancer: an immunohistochemical
study.” Breast cancer research and treatment 130.2 (2011): 457-463

73) Spini, A., et al. “Abstract P3-14-08: Preclinical and clinical evidence about the use of betablockers for the treatment of triple negative breast cancer: A systematic
review.” (2019): P3-14.

83) Dezong, Gao, et al. “Carvedilol suppresses migration and invasion of malignant breast cells by inactivating Src involving cAMP/PKA and PKCδ signaling
pathway.” Journal of cancer research and therapeutics 10.4 (2014): 991.

p 79 Propranolol in Breast Cancer Epidemiology Study

In 2010, Dr. Desmond Powe et al. reviewed the medical records of 466 women with operable
breast cancer with more than 10 years follow-up. Women on beta blockers (propranolol)
had a 57% reduction in metastatic disease and recurrence, and a 71% reduction in mortality
compared to controls not on propranolol. Dr.Powe writes:

We performed an epidemiological study of breast cancer patients with long-term
clinical follow-up [>10 years] and showed that patients receiving antihypertensive
beta-blocker drugs [propranolol] significantly benefit by a 57% reduction in distant
metastasis formation and a 71% reduced risk of dying from breast cancer compared
to control patients…. . (71–72)

71) Powe, Desmond G., et al. “Beta-blocker drug therapy reduces secondary cancer formation in breast cancer and improves cancer specific survival.” Oncotarget
1.7 (2010): 628.

72) Powe, D. G., et al. “Alpha-and beta-adrenergic receptor (AR) protein expression is associated with poor clinical outcome in breast cancer: an immunohistochemical
study.” Breast cancer research and treatment 130.2 (2011): 457-463

p 79 and 80

In 2019, Dr. A. Spini et al. reviewed the preclinical and clinical evidence for use of beta
blockers (propranolol) for TNBC, finding 616 published studies, including 62 preclinical (in
vitro and in vivo). The clinical studies included four retrospective observational cohort studies
showing TNBC patients using beta blockers (propranolol) had a 65% reduction in mortality,
and a 68% reduction in metastatic disease. Dr. Spini and colleagues write:

The in vitro studies showed a high expression of Beta-2 adrenoreceptors in TNBC cell
lines. Propranolol (and carvedilol) significantly decreased proliferation, migration
and invasion of TNBC cells…. In vivo studies reported a reduction of metastasis,
angiogenesis and tumor growth in animals exposed to propranolol. (73)

73) Spini, A., et al. “Abstract P3-14-08: Preclinical and clinical evidence about the use of betablockers for the treatment of triple negative breast cancer: A systematic review.” (2019): P3-14

Alpha Lipoic Acid p 92

42) Tripathy, Joytirmay, et al. “α-Lipoic acid inhibits the migration and invasion of breast cancer cells through inhibition of TGFβ signaling.” Life sciences 207 (2018): 15-22.

43) Na, Mi Hee, Eun Young Seo, and Woo Kyoung Kim. “Effects of α-lipoic acid on cell proliferation and apoptosis in MDA-MB-231 human breast cells.” Nutrition research and practice 3.4 (2009): 265-271.

44) Kuban-Jankowska, Alicja, Magdalena Gorska-Ponikowska, and Michal Wozniak. “Lipoic acid decreases the viability of breast cancer cells and activity of PTP1B and SHP2.” Anticancer research 37.6 (2017): 2893-2898.

45) Nur, Gökhan, Mustafa Nazıroğlu, and Haci Ahmet Deveci. “Synergic prooxidant, apoptotic and TRPV1 channel activator effects of alpha-lipoic acid and cisplatin
in MCF-7 breast cancer cells.” Journal of Receptors and Signal Transduction 37.6 (2017): 569-577.

46) Li, B. J., et al. “Effect of lipoic acid combined with paclitaxel on breast cancer cells.” Genet Mol Res 14 (2015): 17934-17940.

48) Dozio E, Ruscica M, Passafaro L, et al. The natural antioxidant alpha-lipoic acid induces p27(Kip1)-dependent cell cycle arrest and apoptosis in MCF-7 human breast cancer cells. Eur J Pharmacol. 2010 Sep 1;641(1):29-34

49) Lee HS, Na MH, Kim WK. alpha-Lipoic acid reduces matrix metalloproteinase activity in
MDA-MB-231 human breast cancer cells. Nutr Res. 2010 Jun;30(6):403-9

Thiamine for Breast Cancer p 93

In 2018, Dr. Xiaowen Liu studied the effect of thiamine on breast cancer cells in vitro, finding
an anti-cancer mechanism similar to ALA: The treatment of MCF7 breast cancer cells
with 1–2 microgram/mL of thiamine for 24 hours significantly reduced their proliferation.
This reduction is associated with a reduction in glycolysis and activation of
the PDH complex in breast cancer cells. (39)

39) Liu, Xiaowen, et al. “The effects of thiamine on breast cancer cells.” Molecules 23.6 (2018): 1464.

Melatonin p 94

Melatonin levels rise at night during darkness and fall during daylight. In mouse breast
cancer xenograft and in human breast cancer studies, high nighttime circulating melatonin
switches the breast cancer cells from GLYCOLYSIS to OXPHOS. Dr. Russel Reiter et al.
speculate this is done by inhibition of PDK, the same target as DCA. In this way, the anti-cancer mechanism of melatonin is similar to that of DCA.

p 95

In 2016, Dr. Naiane do Nascimento Goncalves et al. studied melatonin in breast CSCs in human and canine lines, finding melatonin effective for EMT markers and reducing invasive properties. Melatonin inhibits EMT by degrading Beta-Catenin via activation of GSK3-Beta. This prevents the Beta-Catenin from translocating to the nucleus, and complexing with TCF/LEF to induce transcription of Wnt target genes. Melatonin promotes apoptosis and inhibits invasiveness of CSCs, effective at the 1 millimolar concentration. (72–78)

72) do Nascimento Goncalves, Naiane, et al. “Effect of melatonin in epithelial mesenchymal transition markers and invasive properties of breast cancer stem cells of canine and human cell lines.” PloS one 11.3 (2016).

Melatonin has synergy with ATRA (all-trans retinoic acid, a vitamin A analog) and berberine. (79–80)
Melatonin has synergy with most chemotherapies and oncology drugs, while reducing the
toxicity of chemotherapy. (81–84)

79) Margheri M, et al. (2012) Combined effects of melatonin and all-trans retinoic acid and somatostatin on breast cancer cell proliferation and death: molecular basis for the anticancer effect of these molecules.

89) Amin, Negin, et al. “Melatonin is an appropriate candidate for breast cancer treatment: Based on known molecular mechanisms.” Journal of cellular biochemistry
120.8 (2019): 12208-12215

Vitamin C improves chemotherapy p 105

In 1996, Dr. Christian Kubacher et al. reported that vitamin C improves the antineoplastic
activity of doxorubicin, cisplatin, and paclitaxel in breast cancer cells in vitro. (1)

1) Kurbacher, Christian M., et al. “Ascorbic acid (vitamin C) improves the antineoplastic activity of doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells in vitro.” Cancer letters 103.2 (1996): 183-189.

p 107

Vitamin C Depletes Glutathione,Inhibits GLYCOLYSIS, and Targets Cancer Stem Cells

In 2017, Dr. Bonuccelli et al. studied the effect of vitamin C (ascorbate) on breast cancer
cell cultures and stated that ascorbate depletes the glutathione pool, inhibits GLYCOLYSIS, and targets CSCs: Vitamin C has two mechanisms of action. First, it is a potent pro-oxidant, that actively depletes the reduced glutathione pool, leading to cellular oxidative stress
and apoptosis in cancer cells. Moreover, it also behaves as an inhibitor of glycolysis,
by targeting the activity of GAPDH, a key glycolytic enzyme… Here, we show that
Vitamin C can also be used to target the CSC Cancer Stem Cell population, as it is
an inhibitor of energy metabolism that feeds into the mitochondrial TCA cycle
[TriCarboxylic Acid Cycle] and OXPHOS….
A breast cancer based clinical study has already shown that the use of Vitamin C,
concurrent with or within 6 months of chemotherapy, significantly reduces both
tumor recurrence and patient mortality. (12)

12) Bonuccelli G, De Francesco EM, de Boer R, Tanowitz HB, Lisanti MP. NADH autofluorescence, a new metabolic biomarker for cancer stem cells: Identification
of Vitamin C and CAPE as natural products targeting “stemness.” Oncotarget. 2017;8(13):20667-20678.

p 109

The combination of IV ascorbate (vitamin C) and auranofin was found to be “strongly”
synergistic in vitro for malignant B cells (lymphoma) and in a mouse xenograft breast cancer
model as reported by Drs. Agnieszka Graczyk et al. (2019) and Dr. Elie Hatem et al. (2018).
(31–32)

32) Hatem, Elie, et al. “Auranofin/vitamin C: a novel drug combination targeting triple-negative breast Cancer.” JNCI: Journal of the National Cancer Institute 111.6 (2018): 597-608.

p 110

In 2015, Dr. Lisanti’s group published a study using an in vitro breast cancer cell model showing that doxycycline and high-dose IV Vitamin C induced “synthetic lethality,” leading to eradication of CSCs. (35)(81)

p 114
70) Gonzalez, Michael J., Miguel J. Berdiel, and Amanda V. Cintrón. “High Dose IV Vitamin C and Metastatic Breast Cancer: A Case Report.” Journal of Orthomolecular Medicine 32.6 (2017).

K2 p 115

97) Kiely, Maeve, et al. “Real-time cell analysis of the inhibitory effect of vitamin K2 on adhesion and proliferation of breast cancer cells.” Nutrition Research 35.8 (2015): 736-743.

Doxycycline and Vit C p 116

Working with MCF7 breast cancer-cell cultures, the Lisanti Group showed that the combined use of doxycycline and vitamin C was a “lethal metabolic strategy for eradicating cancer stem cells.” (9)

9) De Francesco, E. M., Michael Lisanti et al. “Vitamin C and Doxycycline: a synthetic lethal combination therapy targeting metabolic flexibility in cancer stem
cells (CSCs).” Oncotarget (2017).

p 120 Estrogen and Exemestane—Aromatase Inhibitor for Breast Cancer

Exemestane combined with Statin for breast cancer P 121

Using a breast cancer cell line in 2015, Dr. Yuanyuan Shen studied the synergy of combining
exemestane with simvastatin (a common statin drug) showing that it:

markedly increased the efficacy, as compared with the single-agent treatment,
suggesting that combination treatment could become a highly effective approach
for breast cancer…. co-administration of exemestane and simvastatin was shown
to result in marked inhibition of tumor cell proliferation, significant cell cycle arrest at
G0/G1 phase and induction of apoptosis, as compared with that of the control and
individual drug-treated cells. (32)

32) Shen, Yuanyuan, et al. “Synergistic effects of combined treatment with simvastatin and exemestane on MCF‑7 human breast cancer cells.” Molecular medicine reports 12.1 (2015): 456-462.

p121 Bone Metastasis on Zoledronic Acid

In those with bone metastasis from breast cancer already on Zoledronic acid, the addition
of doxycycline to the Zoledronic acid might be synergistic and more effective than each single agent. (33–34)

p 122 Exemestane and Anti-Cancer Metabolites

In the drug development for exemestane, researchers may have accidentally stumbled
upon a highly effective anti-cancer drug by virtue of the metabolites of exemestane, which
seem to have a potent biological effect, inducing “cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation.” Dr. Cristina Amaral et al. (2015) write:

Our results indicate that metabolites induced, in sensitive breast cancer cells,
cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8
activation… It was also concluded that….the biological effects of [exemestane]
metabolites are different from the ones of exemestane, which suggests that exemestane
efficacy in breast cancer treatment may also be dependent on its metabolites.
Note: caspase 8 activation initiates apoptosis.(35)

p 123 Doxycycline for Breast Cancer Stem Cells

Doxycycline preclinical studies have shown favorable results for breast CSCs by inhibiting
the cancer stem phenotype epithelial to mesenchymal transition (EMT). In 2016, Dr. Le Zhang et al. studied breast cancer cells in vitro, building on the 2015 work by Dr. Rebecca Lamb’s group, finding doxycycline:

inhibition of the viability and proliferation of breast cancer cells and BCSCs [stem
cells], decrease mammosphere forming efficiency, migration and invasion, and EMT
[epithelial to mesenchymal transition] of breast cancer cells. Expression of stem
cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after
doxycycline treatment. (45)

Doxycycline Autophagy Inhibitor p 123

Dr. Zhang further writes that doxycycline treatment of breast cancer cells induced a
decrease in LC3B protein marker, indicating inhibition of autophagy. In addition, inhibition
of autophagy specifically targets CSCs:

LC3B-II, is one of the most specific biomarkers of autophagy…. a growing number of
studies suggest a link between autophagy and BCSC s [Breast cancer stem cells]….
Guan et al. has reported that BCSCs have a higher autophagic flux than non-CSC
cells… and Cufí et al. has demonstrated that autophagy positively regulates the
CD44+CD24− breast cancer stem-like phenotype… and Maycotte et al. reported that
autophagy supports BCSC (stem cell) maintenance by modulating IL6 secretion, and
that inhibition of autophagy decreases cell survival, as well as mammosphere forming
efficiency…. Here, we report that doxycycline downregulates the autophagy-related
protein levels of LC-3BI and LC-3BII, suggesting a role for autophagy in the doxycycline-
induced suppression of proliferation, invasion, and self-renewal of breast cancer
cells … Moreover, doxycycline could down regulate the expression of the autophagy
marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible
in part for the observed effects on proliferation, EMT and stem cell markers … The
potent inhibition of EMT and cancer stemlike characteristics in breast cancer cells by
doxycycline treatment suggests that this drug can be repurposed as an anti-cancer
drug in the treatment of breast cancer patients in the clinic. (45)

Doxycycline Inhibits NF-kB and IL-6 p 124

In 2017, Dr. Xiaoyun Tang et al. studied doxycycline in a mouse model of breast cancer,
showing potent anti-inflammatory effects, with a 50% reduction in NF-kB transcriptional
activity:

Under basal condition without stimulation, doxycycline was able to decrease the
transcriptional activity of nuclear NF-κB by ~50%, which explained the decreased
IL-6 mRNA and secretion of IL-6, CCL2 and CXCL2 by doxycycline when LPA and TNFα
were absent. (47)

47) Tang, Xiaoyun, et al. “Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB
activation.” Molecular cancer 16.1 (2017): 36.

48) Fife, Rose S., and George W. Sledge Jr. “Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells.” The Journal of laboratory and clinical medicine 125.3 (1995): 407-411.

Doxycycline and Cancer Stem Cells

6) Scatena, Cristian, et al. “Doxycycline, an inhibitor of mitochondrial biogenesis, effectively reduces cancer stem cells (CSCs) in early breast cancer patients: a clinical
pilot study.” Frontiers in oncology 8 (2018): 452.

7) Lin, Chang‑Ching, et al. “Doxycycline targets aldehyde dehydrogenase‑positive breast cancer stem cells.” Oncology reports 39.6 (2018): 3041-3047.

67) Lin, Chang‑Ching, et al. “Doxycycline targets aldehyde dehydrogenase‑positive breast cancer stem cells.” Oncology reports 39.6 (2018): 3041-3047.

68) Scatena, Cristian, et al. “Doxycycline, an inhibitor of mitochondrial biogenesis, effectively reduces cancer stem cells (CSCs) in early breast cancer patients: a clinical pilot study.” Frontiers in oncology 8 (2018): 452.

75) Zhong, Weilong, et al. “Doxycycline inhibits breast cancer EMT and metastasis through PAR-1/NF-κB/ miR-17/E-cadherin pathway.” Oncotarget 8.62 (2017):
104855.

76) Jiang, Xianpeng, et al. “Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection.”
(2019): 3600-3600.

Exemestane

16) Amaral, Cristina, et al. “Apoptosis and autophagy in breast cancer cells following exemestane treatment.” PLoS One 7.8 (2012): e42398.

mTOR Inhibitor

26) Awada, Ahmad, et al. “The oral mTOR inhibitor RAD001 (everolimus) in combination with letrozole in patients with advanced breast cancer: results of a phase I study with pharmacokinetics.” European journal of cancer 44.1 (2008): 84-91.

27) Rudloff, Joëlle, et al. “The mTOR pathway in estrogen response: A potential for combining the rapamycin derivative RAD001 with the aromatase inhibitor Letrozole (Femara®) in breast carcinoma.” (2004): 1298-1298.

28) Boulay, Anne, et al. “Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer.” Clinical Cancer Research
11.14 (2005): 5319-5328.

29) Lee, Joycelyn JX, Kiley Loh, and Yoon-Sim Yap. “PI3K/Akt/mTOR inhibitors in breast cancer.” Cancer biology & medicine 12.4 (2015): 342.

Synergy of Exemestane and Simvastatin

32) Shen, Yuanyuan, et al. “Synergistic effects of combined treatment with simvastatin and exemestane on MCF‑7 human breast cancer cells.” Molecular medicine reports 12.1 (2015): 456-462.

33) Duivenvoorden, W. C. M., et al. “Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer.” British journal of cancer 96.10 (2007): 1526.

35) Amaral, Cristina, et al. “Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane.” The international journal of biochemistry & cell biology 69 (2015):
183-195.

Doxycycline

45) Zhang, Le, et al. “Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer.” Cell Cycle 16.8 (2017):
737-745.

47) Tang, Xiaoyun, et al. “Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.” Molecular cancer 16.1 (2017): 36.

48) Fife, Rose S., and George W. Sledge Jr. “Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells.” The Journal of laboratory and clinical medicine 125.3 (1995): 407-411.

67) Lin, Chang‑Ching, et al. “Doxycycline targets aldehyde dehydrogenase‑positive breast cancer stem cells.” Oncology reports 39.6 (2018): 3041-3047.

68) Scatena, Cristian, et al. “Doxycycline, an inhibitor of mitochondrial biogenesis, effectively reduces cancer stem cells (CSCs) in early breast cancer patients: a clinical pilot study.” Frontiers in oncology 8 (2018):452.

75) Zhong, Weilong, et al. “Doxycycline inhibits breast cancer EMT and metastasis through PAR-1/NF-κB/ miR-17/E-cadherin pathway.” Oncotarget 8.62 (2017):
104855.

76) Jiang, Xianpeng, et al. “Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection.”
(2019): 3600-3600.

Curcumin and EGCG for Breast Cancer Stem Cells p132

The combination of curcumin and epigallocatechin gallate from green tea (EGCG) inhibit CSCs via downregulation of STAT3-NF-kB signaling.

13) Chung, Seyung S., and Jaydutt V. Vadgama. “Curcumin and Epigallocatechin Gallate Inhibit the Cancer Stem Cell Phenotype via Down-regulation
of STAT3–NFκB Signaling.” Anticancer research 35.1 (2015): 39-46.

Sulforaphane Eliminates Breast Cancer Stem Cells in Vivo p 136

In 2010, and 2013, Drs. Y. Li and T. Zhang studied sulforaphane in a breast cancer mouse
xenograft model in vivo, finding that sulforaphane is “highly potent” for elimination of
breast CSCs. Sulforaphane inhibited CSCs at a low concentration of (0.5 to 5 MicroMolar)
without affecting the bulk (non-stem cell) cancer cells. Elimination of CSCs in primary xenografts was confirmed by failure of implanted tumors to grow in secondary mice. Βeta-
Catenin and Cyclin D1 levels were decreased by 80–85%

63) Li, Yanyan, et al. “Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells.” Clinical Cancer Research 16.9 (2010): 2580-2590.

64) Li, Y., and T. Zhang. “Targeting cancer stem cells with sulforaphane, a dietary component from broccoli and broccoli sprouts.” Future Oncology (London, England) 9.8 (2013): 1097-1103.

Sulforaphane Reaches Therapeutic Levels p 136

In 2007, Dr. Cornblatt did a pilot study of eight healthy women undergoing reduction
mammoplasty who were given a single dose of a broccoli sprout preparation containing 200
μmol (MicroMol) of sulforaphane one hour before surgery. When the breast tissue in the
removed surgical specimen was examined, sulforaphane metabolites were readily measurable at concentrations effective against breast CSCs, based on the concentrations used in Dr. Li’s study above. (65)

65) Cornblatt, Brian S., et al. “Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast.” Carcinogenesis 28.7 (2007): 1485-1490.

Triple-negative breast cancer stem cells (68–69) p 137

68) Castro, Nadia P., et al. “Sulforaphane suppresses the growth of triple-negative breast cancer stem-like cells in vitro and in vivo.” Cancer Prevention Research 12.3 (2019): 147-158.

69) Burnett, Joseph P., et al. “Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells.” Cancer Letters 394 (2017): 52-64

p 138 Autphagy Inhibition Synergy with Sulfora[hane

In 2010, Dr. Kanematsu showed that autophagy inhibition “significantly enhanced” apoptosis
(programmed cell death) when combined with sulforaphane in a breast cancer model,
writing:

These results indicate a cytoprotective role of autophagy against [sulforaphane]
SFN-induced apoptosis and that the combination of SFN treatment with autophagy
inhibition may be a promising strategy for breast cancer control. (88)

88) Kanematsu, Sayaka, et al. “Autophagy inhibition enhances sulforaphane-induced apoptosis in human breast cancer cells.” Anticancer Research 30.9 (2010):
3381-3390.

Sulforaphane Synergy with Chemotherapy Agents p 139

In 2017 and 2018, studies by Drs. Milczarek and Lee showed sulforaphane synergizes with
chemotherapy agents 5-FU in triple-negative breast cancer and cisplatin in mesothelioma.
(92–93)

92) Milczarek, Małgorzata, et al. “Autophagic cell death and premature senescence: New mechanism of 5-fluorouracil and sulforaphane synergistic anticancer effect in MDA-MB-231 triple negative breast cancer cell line.” Food and Chemical Toxicology 111 (2018): 1-8.

93) Lee, Yoon‑Jin, and Sang‑Han Lee. “Pro-oxidant activity of sulforaphane and cisplatin potentiates apoptosis and simultaneously promotes autophagy in malignant mesothelioma cells.” Molecular Medicine Reports 16.2 (2017): 2133-2141

Also Sulforaphane synergy with TAXANES

69) Burnett, Joseph P., et al. “Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells.” Cancer
Letters 394 (2017): 52-64.

Pterostilbene p 139

In 2015, Dr. Chi-Hao Wu studied the effect of pterostilbene targeted against breast CSCs
(MCF-7 cells) in vitro. His study showed that pterostilbene selectively killed breast CSCs
which express the CD44 surface antigen. In addition, pterostilbene increased the sensitivity
of breast CSCs to the killing effects of chemotherapy. The underlying mechanism of
pterostilbene is degradation of Beta-Catenin via inhibition of Hedgehog/Akt/GSK3β signaling,
thus inhibiting downstream expression of cancer growth factors C-Myc and Cyclin D1.(104)

104) Wu, Chi-Hao, et al. “Targeting cancer stem cells in breast cancer: potential anticancer properties of 6-shogaol and pterostilbene.” Journal of Agricultural and Food Chemistry 63.9 (2015): 2432-2441.

Feverfew • Breast cancer stem cells (118–119) p141

118) Carlisi, D., et al. “Parthenolide and DMAPT exert cytotoxic effects on breast cancer stem-like cells by inducing oxidative stress, mitochondrial dysfunction and necrosis.” Cell Death & Disease 7.4 (2016): e2194-e2194.

119) Araújo, Thaise Gonçalves, et al. “Parthenolide and Its Analogues: A New Potential Strategy for the Treatment of Triple-Negative Breast Tumors.” Current Medicinal Chemistry (2019).

==============================

7) Gu, Hao-Feng, Xue-Ying Mao, and Min Du. “Prevention of breast cancer by dietary polyphenols—role of cancer stem cells.” Critical Reviews inFood Science and Nutrition (2019): 1-16.

9) Mbese, Zintle, Vuyolwethu Khwaza, and Blessing Atim Aderibigbe. “Curcumin and Its Derivatives as Potential Therapeutic Agents in Prostate, Colon and Breast Cancers.” Molecules 24.23 (2019): 4386.

28) Li, Xiaoting, et al. “Sonic hedgehog and Wnt/β-catenin pathways mediate curcumin inhibition of breast cancer stem cells.” Anti-cancer drugs 29.3 (2018): 208-215.

Berberine p 144

43) Lin, Yen‐Shu, et al. “Different mechanisms involved in the berberine‐induced antiproliferation effects in triple‐negative breast cancer cell lines.” Journal of
Cellular Biochemistry 120.8 (2019): 13531-13544.

44) Kaboli, Parham Jabbarzadeh, et al. “Antitumor effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study.” Pharmacological Reports 71.1 (2019): 13-23.

45) El Khalki, Lamyae, et al. “Berberine Impairs the Survival of Triple Negative Breast Cancer Cells: Cellular and Molecular Analyses.” Molecules 25.3 (2020): 506.

50) Kaboli, Parham Jabbarzadeh, et al. “Targets and mechanisms of berberine, a natural drug with potential to treat cancer with special focus on breast cancer.” European Journal of Pharmacology 740 (2014): 584-595.

56) Wang, Kai, et al. “Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death.” Scientific Reports 6 (2016): 26064.

Diallyl Trisulfide (Garlic Allicin)
p 149

153) Li, Xiaoting, et al. “Diallyl Trisulfide inhibits breast cancer stem cells via suppression of Wnt/β‐catenin pathway.” Journal of Cellular Biochemistry 119.5 (2018): 4134-4141

154) Xie, Xinhua, et al. “Diallyl disulfide inhibits breast cancer stem cell progression and glucose metabolism by targeting CD44/PKM2/AMPK signaling.” Current
Cancer Drug Targets 18.6 (2018): 592-599.

Sulfasalazine Breast cancer (15) p 152

15) Narang, Vishal S., et al. “Suppression of cystine uptake by sulfasalazine inhibits proliferation of human mammary carcinoma cells.” Anticancer research 23.6C
(2002): 4571-4579

Chloroquine p 155

In 2014, Dr. Dong Soon Choi et al. studied the effects of chloroquine in triple-negative breast
cancer cells, finding inhibition of breast CSCs. (51)

51) Choi, Dong Soon, et al. “Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.” Stem cells 32.9 (2014): 2309-2323.

• Triple-negative breast cancer (51)(54)

54) Liang, Diana H., et al. “The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair.” Cancer letters 376.2 (2016): 249-258.

CQ, an autophagy inhibitor with anti-CSC effects, may be an effective adjunct to current TNBC chemotherapy regimens with carboplatin.

ATRA p 158

Curative for Pro-Myelocytic Leukemia— Targets Cancer Stem Cells

The vitamin A derivative, all-trans retinoic acid (ATRA), also known as tretinoin
(Vesinoid®) is curative for pro-myelocytic leukemia, and has been found to target CSCs in
glioblastoma, breast cancer, and head and neck cancer cell lines in vitro. (91–99)

92) Zeng, W. G., et al. “All-trans retinoic acid effectively inhibits breast cancer stem cells growth in vitro.” Zhonghua zhong liu za zhi [Chinese journal of oncology] 35.2 (2013): 89-93.

98) Huang, Lu, et al. “All-trans-retinoic acid (ATRA) markedly augments anti-tumor immunity.” (2019): 3279-3279.

99) Moreb, Jan S., Deniz A. Ucar-Bilyeu, and Abdullah Khan. “Use of retinoic acid/aldehyde dehydrogenase pathway as potential targeted therapy against cancer stem cells.” Cancer chemotherapy and pharmacology 79.2 (2017): 295-301.

p 159
Dr. Lima and colleagues studied the ATRA induced miRNA expression in cancer cells,
finding:

ATRA… was able to modulate the expression of more than 300 miRNAs, and inhibit
invasive behavior and deregulated growthof cancer cells, resulting in total tumor
remission in some cases. ATRA may thus be broadly effective for neoplasm treatment
and prevention…. ATRA has also been shown to function as an anti-cancer
agent in several neoplasms, such as gastric cancer, breast cancer, leukemia, nephroblastoma, melanoma, lung cancer, and neuroblastoma (103)

103) Lima, Lara, et al. “Modulation of all-trans retinoic acid-induced MiRNA expression in neoplastic cell lines: a systematic review.” BMC cancer 19.1 (2019): 866.

Doxycycline and High-Dose IV Vitamin C – A Lethal Combination for Cancer Stem Cells p 159

Dr. Michael Lisanti’s group published a study in Oncotarget (June 2017) using an in vitro
breast cancer cell model showing that doxycycline and high-dose IV vitamin C are a lethal
combination for eradication of CSCs. Indeed, this is a major advance in our understanding
of CSC eradication. (107–109)

107) Lamb, Rebecca, et al. “Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: treating cancer like an infectious disease.” Oncotarget 6.7 (2015): 4569.

108) Bonuccelli, Gloria, et al. “NADH autofluorescence,
a new metabolic biomarker for cancer stem
cells: Identification of Vitamin C and CAPE as natural
products targeting “stemness”.” Oncotarget 8.13
(2017): 20667.

109) De Francesco, Ernestina Marianna, et al. “Vitamin
C and Doxycycline: A synthetic lethal combination
therapy targeting metabolic flexibility in cancer stem
cells (CSCs).” Oncotarget 8.40 (2017): 67269.

p 160

8) Yu, Haochen, et al. “Sulfasalazine‑induced ferroptosis
in breast cancer cells is reduced by the inhibitory
effect of estrogen receptor on the transferrin receptor.”
Oncology reports 42.2 (2019): 826-838.

p 161

29) Narang, Vishal S., et al. “Sulfasalazine-induced
reduction of glutathione levels in breast cancer cells:
enhancement of growth-inhibitory activity of doxorubicin.”
Chemotherapy 53.3 (2007): 210-217.

30) Wei, Chyou-Wei, et al. “Anti-Cancer Effects of
Sulfasalazine and Vitamin E Succinate in MDA-MB
231 Triple-Negative Breast Cancer Cells.” International
journal of medical sciences 16.4 (2019): 494.

Pyrvinium p 165

Pyrvinium exhibits potent anti-cancer activity as a Wnt pathway inhibitor against the following
CSC lines:

Pyrvinium Inhibits Wnt Pathway and Cancer Stem Cells p 166

In 2016, Drs. Liang and W. Xu et al. studied pyrvinium in a breast cancer cell model, finding
pyrvinium inhibited proliferation of breast CSCs in vitro and in vivo related to Wnt pathway
inhibition, with measurable reduction of CSC markers. (6–7)

6) Xu, Liang, et al. “WNT pathway inhibitor pyrvinium pamoate inhibits the self-renewal and metastasis of breast cancer stem cells.” International journal of
oncology 48.3 (2016): 1175-1186.

7) Xu, W., et al. “P1-04-01: The Mechanism of Anti-Breast Cancer TICs Effect of Pyrvinium Pamoate Is through WNT/beta-Catenin Signaling.” (2011): P1-04.

Pyrvinium Synergy with Chloroquine p 169

For example, in 2013, Dr. Longfei Deng studied pyrvinium (10mg/kg i.g.) by oral gavage
once daily in a mouse xenograft 4T1 breast cancer model, with good reduction in tumor
volume and enhanced activity (synergy) with the autophagy inhibitor, chloroquine. (30)

Synergy with the autophagy inhibitor chloroquine is not unexpected, since pyrvinium is
an OXPHOS inhibitor that stimulates protective autophagy

30) Deng, Longfei, et al. “Pyrvinium targets autophagy addiction to promote cancer cell death.” Cell death & disease 4.5 (2013): e614-e614.

Aspirin as Anti-Cancer Stem Cell Agent in Breast Cancer—Breast Cancer Stem Cells are Glycolytic!! p 174

As discussed in chapter 5 on DCA, in 2014, Dr. Weiguo Feng et al. did transcriptome profiling using RNA sequencing, which revealed that breast cancer stem cells—also called tumor-initiating cells (TICs)—preferentially perform GLYCOLYSIS over mitochondrial OXPHOS compared to non-stem cancer cells. This is due to increased expression and activity of pyruvate dehydrogenase kinase (PDK), which promotes
pro-glycolytic phenotype by inhibiting pyruvate dehydrogenase (PDH). They write:

Glycolysis-associated events/processes, such as glucose uptake, glycolytic enzyme
expression, lactate production and ATP levels, are significantly elevated in CSCs,
[cancer stem cells] which is also linked to a decrease in mitochondrial oxidative metabolism.
Conversely, inhibition of glycolysis reversely suppresses the CSC maintenance.
(4)

The key point here is: Metabolic reprogramming of PDH to force OXPHOS eliminated breast
CSCs.
Dr. Feng et al. write: “Metabolic reprogramming via forced activation of PDH preferentially
eliminated TICs [tumor initiating cells, also called cancer stem cells] both in vitro and
in vivo”. (4)

4) Feng, Weiguo, et al. “Targeting unique metabolic properties of breast tumor initiating cells.” Stem Cells 32.7 (2014): 1734-1745.

Aspirin is a GLYCOLYSIS Inhibitor p 175

In 2018, Dr. F. Peng et al. studied the effect of aspirin on breast CSCs, finding aspirin restricted tumor GLYCOLYSIS and stemness in vitro and in vivo by significantly decreasing lncRNA H19 and pyruvate dehydrogenase kinase (PDK1). H19 is a long, noncoding RNA oncogene that targets, and increases, PDK1, the master controller
for switching between mitochondrial OXPHOS and GLYCOLYSIS. For example, as discussed in chapter 5, DCA (dichloroacetate) inhibits PDK, which then upregulates PDH,
which then switches metabolic pathway from GLYCOLYSIS to mitochondrial OXPHOS. Dr. Peng and colleagues summarize their findings:

Intriguingly, we also uncovered that aspirin can suppress glycolysis and BCSC [breast
cancer stem cell] maintenance through repressing H19 and PDK1. Taken together,
our studies identify a novel role and regulatory mechanism of PDK1 in BCSC reprogramming, which provides a promising strategy for breast cancer therapy. (5)

5) Peng, F., et al. “Glycolysis gatekeeper PDK1 reprograms breast cancer stem cells under hypoxia.” Oncogene 37.8 (2018): 1062.

Doxorubicin Increases Cancer Stem Cells p 176

Adding Aspirin Helps !!

Another unfavorable result of chemotherapy is the increased number of CSCs. In 2019,
Dr. Bee Luan Khoo et al. used patient-derived breast cancer cells from blood samples
of 68 patients, finding that low-dose aspirin enhanced the cancer-cell-killing effects of the
chemotherapy agent doxorubicin by inhibiting IL-6 secretion.

When doxorubicin alone was used, this treatment increased the number of
CSCs over 7 days. However, when the aspirin was added to the doxorubicin, there was a significant decrease in generation of CSCs. Dr. Khoo et al. write:

Our findings serve as a basis for optimism regarding… low-dose aspirin in combination
with anti-cancer drugs as an effective chemopreventive therapy against breast
cancer and potentially other cancer types…. our study is the first to connect
the anti-cancer effects of aspirin and anti-cancer drugs with the reduction of
CSCs [cancer stem cells]. (11)

11) Khoo, Bee Luan, et al. “Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.” British journal of cancer 120.4 (2019): 407.

Breast cancer stem cells (4–5)(16–17) p 177

16) Tu, L., et al. “Effect of aspirin on breast cancer stem cells and stemness of breast cancer.” Zhonghua yi xue za zhi 98.44 (2018): 3598-3602.

17) Saha, Shilpi, et al. “Aspirin suppresses the acquisition of chemoresistance in breast cancer by disrupting an NFκB–IL6 signaling axis responsible for the generation
of cancer stem cells.” Cancer Research 76.7 (2016): 2000-2012.

p 177

In 2019, Dr. Ling LI et al. studied EGFR in lung and breast cancer cell lines, in vitro and
in vivo, using the new EGFR drugs gefitinib and osimertinib. Initially the drugs work effectively. However, over time, the cancer cell lines gradually become resistant to the drugs.

Overcoming TKI Resistance with Aspirin

Dr. Li et al. searched a gene signature database to find a drug to overcome TKI drug resistance. The top-ranked candidate was aspirin, which suppresses activation of NF-kB, and suppresses cancer cell “stemness.” The concentration of aspirin in the mice was roughly
equivalent to a 500 mg dose in a 60 kg male, a commonly used dose considered safe in
humans. (23)

23) Li, Ling, et al. “Repositioning Aspirin to Treat Lung and Breast Cancers and Overcome Acquired Resistance to Targeted Therapy.” Frontiers in Oncology 9 (2019).

p 178

In 2018, Dr. Maria Amaral et al. also found synergy of aspirin with metformin in various
breast cancer cell types in vitro. This combination bound to the estrogen receptor and was
most effective in cell lines having upregulated COX-2 (cyclo-oxygenase 2 inflammatory pathway). (35)

35) Amaral, Maria Eduarda Azambuja, et al. “Preclinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes.” Investigational new drugs 36.5 (2018): 782-796.

Aspirin is an Antiplatelet Agent p 178

Additionally, in 2019, Dr. Kelly Johnson et al. found that aspirin inhibits release of platelet
factors that activate the Akt pathway. Activation of Akt stimulates breast cancer cells to secrete IL-8, which promotes an invasive, aggressive phenotype. Dr. Johnson and colleagues write:
Platelets treated with aspirin did not activate the Akt pathway, resulting in
reduced IL-8 secretion and impaired tumor cell invasion. Of note, patients with breast
cancer receiving aspirin had lower circulating IL-8, and their platelets did not increase
tumor cell invasion compared with patients not receiving aspirin. Our data suggest
platelets support breast tumor metastasis by inducing tumor cells to secrete IL-8. Our
data further support that aspirin acts as an anti-cancer agent by disrupting the communication between platelets and breast tumor cells. (38) error shuld be (39)

39) Johnson, Kelly E., et al. “Aspirin inhibits platelets from reprogramming breast tumor cells and promoting metastasis.” Blood advances 3.2 (2019): 198-211.

p 179 aspirin tumor micro environment

In 2018, Dr. Chia-Chien Hsieh studied the effect of aspirin on the tumor micro-environment
in a triple-negative breast cancer (4T1) cell model in vitro, using a culture medium
simulating the macrophage infiltration of the tumor micro-environment. Dr. Hsieh found
that aspirin treatment decreased inflammatory cytokines in the tumor microenvironment,
writing:

[the aspirin] interfered with crosstalk between cancer cells and macrophages…
and decreased angiogenic and inflammation- associated cytokine VEGF, PAI-1,
MCP-1, IL-6, IL-10, and TGF-β production…suggesting that aspirin is a promising agent
to prevent tumor progression. (44)

44) Hsieh, Chia-Chien, and Chih-Hsuan Wang. “Aspirin disrupts the crosstalk of angiogenic and inflammatory cytokines between 4T1 breast cancer cells and macrophages.” Mediators of Inflammation 2018 (2018).

Metformin Inhibits Hexokinase Function p 185

In 2013, Drs. Ceclia Marini and Barbara Salani et al., in a follow-up to a previous study,
examined the effect of metformin in a triple-negative breast cancer cell model (in vitro) and in
mouse xenografts (in vivo). This elegant study used both PET FDG scan imaging and immunofluorescent imaging. Dr. Marini et al. found reduced glucose consumption, which activated AMP-kinase in the cancer cells, thought to be related to inhibition of HK2 function. (11–12)

11) Salani, Barbara, et al. “Metformin impairs glucose consumption and survival in Calu-1 cells by direct inhibition of hexokinase-II.” Scientific reports 3 (2013).

[Metformin] actually inhibits HK function, resulting in an immediate cytotoxic effect
both in vitro and in vivo as well as in a significant reduction of cancer growth rate
under chronic treatment… Metformin strikingly impaired glucose consumption
of MDA-MB-231[breast cancer cells] in a dose- and time-dependent manner …
that progressively reduced FDG [PET Scan tracer] uptake down to its minimum
values after 48 hours…. More importantly, metformin treatment had a relevant
influence on cancer growth. In fact, weight of explanted lesions was almost halved in
“prolonged” mice treated with metformin for the whole month of study duration,
while it was obviously not affected by pulsed treatment… The present study
documents that metformin reduces cancer metabolism and growth at least partially
via a direct and selective inhibition of HK I and II enzymatic function … and its interaction
with mitochondria hampering a crucial aspect for tumor immortality. (12)

12) Marini, Cecilia, et al. “Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer.” Cell Cycle 12.22 (2013): 3490-3499

p 187
As mentioned above, metformin activates AMP-kinase (AMPK), which inhibits the mTOR
signal pathway, which then induces protective autophagy with perinuclear clustering of lysosomes. Similarly, dichloroacetate (DCA) and many other anti-cancer drugs activate protective autophagy, a survival mechanism for the cancer cell. (17)

Metformin Synergy with Autophagy Inhibitor chloroquine p 187

In 2011, Dr. Alelejandro Vasquez-Martin et al. made this same suggestion, proposing the combination  of both drugs to eradicate cancer stem cells (CSC) in premalignant lesions, specifically, ductal carcinoma of the breast in situ (DCIS).(19)

19) Vazquez-Martin, Alejandro, et al. “Repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions.” Drug Resistance Updates 14.4-5 (2011): 212-223.

Metformin as an Anti-Cancer Stem Cell Agent p 191

In 2013, Dr. Heather Hirsch et al. studied a breast cancer mouse xenograft model, finding
metformin “selectively kills” cancer stem cells by inhibiting NF-kB activation and STAT3 activation. Metformin is also effective as a stem cell agent in mouse xenograft studies involving inflammatory prostate cancer and melanoma cell lines. (9)

9) Hirsch, Heather A., Dimitrios Iliopoulos, and Kevin Struhl. “Metformin inhibits the inflammatory response associated with cellular transformation and cancer stem cell growth.” Proceedings of the National Academy of Sciences 110.3 (2013): 972-977

In 2017, Dr. Peiguo Shi et al. found metformin suppresses triple-negative breast cancer
stem cells. (38)

38) Shi, Peiguo, et al. “Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation.” Cell discovery 3.1 (2017): 1-13.

p 191 Metronomic Low Dose DOXO rubicin / Metformin

In 2019, Dr. Indranil Banerjee et al. studied the combination of metronomic low-dose
doxorubicin with metformin in a mouse xenograft breast cancer model, showing a “strong
anti-cancer effect,” with inhibition of the Wnt/Beta-Catenin pathway and inhibition of cancer
stem cells. (41)

41) Banerjee, Indranil, et al. “Combination of metformin and metronomic liposomal doxorubicin exerts a robust anticancer effect in triple negative breast cancer by inhibiting breast cancer stem cells & the Wnt/ beta-catenin pathway.” (2019): 3638-3638.

p 192 Metformin

In 2017, Dr. Cuyas Elisabet et al. found that metformin inhibits receptor activator of NF-kB
ligand (RANKL) and sensitizes breast cancer stem cells to the cancer drug denosumab, a
RANKL inhibitor originally developed for treating osteoporosis. (43)

43) Cuyas, Elisabet, et al. “Metformin inhibits RANKL and sensitizes cancer stem cells to denosumab.” Cell Cycle 16.11 (2017): 1022-1028

p 196

In 2016, Dr. Young Kwang Chae et al. reported in Oncotarget:

Metformin activates the T-cell-mediated immune response against cancer cells,
reduces KI-67 proliferation rate in endometrial cancer and breast cancer patients. (58)

58) Chae, Young Kwang, et al. “Repurposing metformin for cancer treatment: current clinical studies.” Oncotarget 7.26 (2016): 40767

Metformin Synergy with Chemotherapy p 196

In 2009, in a breast cancer xenograft mouse model, Dr. Hirsch et al. observed that metformin
acts synergistically with chemotherapy using doxorubicin to block tumor growth and
prolong remission in four genetically different breast cancers, concluding that:
The combination of metformin and chemotherapeutic drugs might improve treatment
of patients with breast [and possibly other] cancers. (9)

Metformin and propranolol combination p 197

In 2017, Dr. Maria Rico et al. found the combination of metformin and propranolol synergistic
in triple-negative breast cancer cell (TNBC) lines studied in vitro and in vivo mouse
xenografts. Combining metformin and propranolol strongly inhibited mitochondrial energy
production and:

achieve[d] a complete suppression of the mitochondrial bioenergetics, and drastically
activates glycolysis…. recent studies demonstrate that OXPHOS is indispensable
for tumor migration and metastasis …Disseminating breast cancer cells indeed
display increased levels of mitochondrial respiration. The ability of Met + Prop combination
to inhibit rapidly and efficiently the mitochondrial bioenergetics thus likely underlies the strong antimetastatic properties of the treatment. (70)

70) Rico, María, et al. “Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models.” Oncotarget 8.2 (2017): 2874

Metformin Synergy with Curcumin p 198

Curcumin, used for centuries as a spice and anti-inflammatory botanical, has been found
synergistic with metformin. In 2017, Dr. Rabah Rashad Falah et al. studied breast cancer in a mouse xenograft model, observing that the metformin plus curcumin combination inhibited
angiogenesis, modulated the immune system, and induced apoptosis independent of p53
status. (75)

75) Falah, Rabah Rashad, Wamidh H. Talib, and Seba Jamal Shbailat. “Combination of metformin and curcumin targets breast cancer in mice by angiogenesis
inhibition, immune system modulation and induction of p53 independent apoptosis.” Therapeutic advances in medical oncology 9.4 (2017): 235-252.

p 199 hyperthermia

In 2014, Dr. Hyemi Lee et al. found that hyperthermia treatment increased metformin
cytotoxicity against breast cancer cells as well as the CSCs by activating AMPK and inactivating mTOR. Dr. Lee’s group concludes that:
The effects of metformin against cancer cells including CSCs can be markedly
enhanced by hyperthermia. (83)

83) Lee, Hyemi, et al. “Response of breast cancer cells and cancer stem cells to metformin and hyperthermia alone or combined.” PloS one 9.2 (2014): e87979.

Metformin Use as Adjuvant for Cancer p 199

A number of authors have suggested metformin as an adjuvant for breast cancer and
other cancers as well. In view of the studies outlined above, it seems reasonable to offer
metformin to the cancer patient as an adjuvant treatment before, during, and after conventional treatment with chemotherapy, surgery, or radiation. (84–86)

84) Roshan, Mohsin HK, et al. “Metformin as an Adjuvant in Breast Cancer Treatment.” SAGE Open Medicine, Vol.7 (2019): 1 –16.

85) Coyle, C., et al. “Metformin as an adjuvant treatment for cancer: a systematic review and meta-analysis.” Annals of Oncology 27.12 (2016): 2184.

86) Heckman-Stoddard, Brandy M., et al. “Repurposing metformin for the prevention of cancer and cancer recurrence.” Diabetologia 60.9 (2017): 1639-1647.

61) Orecchioni, Stefania, et al. “The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both
neoplastic and microenvironment cells.” International Journal of Cancer 136.6 (2015): E534-E544.

68) Talarico, Giovanna, et al. “Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells.” Scientific reports 6 (2016): 18673.

69) Kang, Fei, et al. “Propranolol inhibits glucose metabolism and 18F-FDG uptake of breast cancer through posttranscriptional downregulation of hexokinase-2.” Journal of Nuclear Medicine 55.3 (2014):439-445.

70) Rico, María, et al. “Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models.” Oncotarget 8.2 (2017): 2874

75) Falah, Rabah Rashad, Wamidh H. Talib, and Seba Jamal Shbailat. “Combination of metformin and curcumin targets breast cancer in mice by angiogenesis inhibition, immune system modulation and induction of p53 independent apoptosis.” Therapeutic advances
in medical oncology 9.4 (2017): 235-252

====================================================================

p 211

5) Amaral, Cristina, et al. “Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane.” The international journal of biochemistry & cell biology 69 (2015):
183-195.

p 212

29) Hossain, F., et al. “Repurposing sulindac sulfide as a Notch inhibitor to target cancer stem-like cells in triple negative breast cancer.” CANCER RESEARCH. Vol. 79. No. 4. AMER ASSOC CANCER RESEARCH, 2019.

p 214
Pterostilbene

USDA Studies of Pterostilbene—Potent and Effective The USDA and the University of Mississippi have been studying resveratrol analogs for two decades. In 2002, Dr. Agnes M. Rimando et al. reported pterostilbene inhibited breast cancer in a mouse model. (8)

8) Rimando, Agnes M., et al. “Cancer chemopreventive and antioxidant activities of pterostilbene, a naturally occurring analogue of resveratrol.” Journal of agricultural
and food chemistry 50.12 (2002): 3453-3457.

p 216 Pterostilbene Anti-Cancer Effects

Breast Cancer p 217

Dr. Yanshang Wang et al. (2012), showed pterostilbene simultaneously induces apoptosis,
cell-cycle arrest and cyto-protective autophagy in breast cancer cells. (32)

Dr. D. Moon et al. (2012), showed that pterostilbene induces apoptosis in breast cancer cell
lines via the mitochondrial pathway. (33)

Dr. Julie A. Alosi et al. (2010) studied pterostilbene in breast cancer in vitro showing similar
findings with apoptosis induced by mitochondrial pathways. (35)

30) Wakimoto, Rei, et al. “Differential anticancer activity of pterostilbene against three subtypes of human breast cancer cells.” Anticancer research 37.11 (2017):
6153-6159.

31) Mak, Ka‐Kit, et al. “Pterostilbene, a bioactive component of blueberries, suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF‐κ B/microRNA 448 circuit.” Molecular nutrition & food research 57.7
(2013): 1123-1134.

32) 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.

33) Moon, D., et al. “Pterostilbene Induces Mitochondrially-Derived Apoptosis in Breast Cancer in Vitro Via Bax Activation and Cytosolic Calcium Overload.” Journal of Surgical Research 172.2 (2012):342.

34) Pan, Min-Hsiung, et al. “Suppression of heregulin-β1/HER2-modulated invasive and aggressive phenotype of breast carcinoma by pterostilbene via inhibition of matrix metalloproteinase-9, p38 kinase cascade and Akt activation.” Evidence-Based
Complementary and Alternative Medicine, vol. 2011,Article ID 562187.

35) 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.

p 224 Boswellia Chapter 17

High Thru-put Screen for Triple-Negative Breast Cancer- Boswellia

In 2017, Dr. Elizabeth Mazzio et al. performed a high thru-put screen of a library of 1,640
plant-derived chemicals and botanical herbs for their cytotoxicity to triple-negative breast
cancer (TNBC) cells, in vitro, at low micromolar concentrations. Frankincense (Boswellia serrata extract, or BSE) and 3-O-acetyl-β-boswellic acid (3-OAβBA) were identified as the leading compounds. Dr. Mazzio then did a “whole transcriptome data analysis of RNA” from the breast cancer cells after boswellia treatment, showing that the primary mode of cell death is:

ER [endoplasmic reticulum] stress leadingto a UPR [unfolded protein response] …
commonly associated with activated cell death. (6)

6) Mazzio, Elizabeth A., Charles A. Lewis, and Karam FA Soliman. “Transcriptomic profiling of MDA-MB-231 cells exposed to Boswellia serrata and 3-O-acetyl-BBoswellic acid; ER/UPR mediated programmed cell death.” Cancer Genomics-Proteomics 14.6 (2017): 409-425.

Silver Nanoparticles p 224

Similarly, in 2016, Drs. Jean-Christophe Simard et al. showed that silver nanoparticles
also induce “irremediable endoplasmic stress leading to unfolded protein response in breast
cancer cells,” thereby conferring anti-cancer activity. (10)

10) Simard, Jean-Christophe, Isabelle Durocher, and Denis Girard. “Silver nanoparticles induce irremediable endoplasmic reticulum stress leading to unfolded protein response dependent apoptosis in breast cancer cells.” Apoptosis 21.11 (2016): 1279-1290.

p 225 Boswellia Effective for Breast Cancer Using In Vivo Mouse Model

Drs. Hiva Alipanah and Parvin Zareian (2018) studied orally administered boswellia
extract in a mouse breast cancer xenograft model showing potent anti-cancer activity. The
cancer cells stopped dividing, growth of new blood vessels was halted (angiogenesis), and
metastatic spread was blocked. (23)

23) Alipanah, Hiva, and Parvin Zareian. “Anti-cancer properties of the methanol extract of Boswellia serrata gum resin: Cell proliferation arrest and inhibition of angiogenesis and metastasis in BALB/c mice breast cancer model.” Physiology and Pharmacology 22.3
(2018): 183-194.

Solomon’s Seal for Breast cancer (17–18) p 233

17) Tai, Yu, et al. “Effect of Polygonatum odoratum extract on human breast cancer MDA-MB-231 cell proliferation and apoptosis.” Experimental and therapeutic medicine 12.4 (2016): 2681-2687.

18) Ouyang, Liang, et al. “Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells.” Phytomedicine 21.12 (2014): 1658-1665.

Mistletoe Breast Cancer p 234

3) Marvibaigi M, Supriyanto E, Amini N, Abdul Majid FA, Jaganathan SK. Preclinical and clinical effects of mistletoe against breast cancer. Biomed Research International, 2014, 785479 (2014).

EGF p234

11) Jung, Kyung-Ho, et al. “EGF receptor stimulation shifts breast cancer cell glucose metabolism toward glycolytic flux through PI3 kinase signaling.” PloS one
14.9 (2019).

12) Lim, Seung-Oe, et al. “EGFR signaling enhances aerobic glycolysis in triple-negative breast cancer cells to promote tumor growth and immune escape.” Cancer research 76.5 (2016): 1284-1296.

17) Tai, Yu, et al. “Effect of Polygonatum odoratum extract on human breast cancer MDA-MB-231 cell proliferation and apoptosis.” Experimental and therapeutic
medicine 12.4 (2016): 2681-2687.

18) Ouyang, Liang, et al. “Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells.” Phytomedicine 21.12 (2014): 1658-1665.

CBD p 238

25) Sultan, Ahmed S., Mona A. Marie, and Salah A. Sheweita. “Novel mechanism of cannabidiol-induced apoptosis in breast cancer cell lines.” The Breast 41
(2018): 34-41.

26) McAllister, Sean D., et al. “Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells.” Molecular cancer therapeutics 6.11 (2007): 2921-2927.

27) McAllister, Sean D., et al. “Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis.” Breast
cancer research and treatment 129.1 (2011): 37-47.

28) Shrivastava, Ashutosh, et al. “Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy.”
Molecular cancer therapeutics 10.7 (2011): 1161-1172.

29) Elbaz, Mohamad, et al. “Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: Novel anti-tumor mechanisms of Cannabidiolin breast cancer.” Molecular oncology 9.4 (2015):906-919.

30) Sultan, Ahmed S., Mona A. Marie, and Salah A. Sheweita. “Novel mechanism of cannabidiol-induced apoptosis in breast cancer cell lines.” The Breast 41
(2018): 34-41.

31) Caffarel, María M., et al. “Δ9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation.” Cancer research 66.13
(2006): 6615-6621.

p 247 pyrvinium

Dr. Liang Xu reported in the 2016 International Journal of Oncology:
The Wnt pathway inhibitor pyrvinium pamoate inhibits the self-renewal and
metastasis of breast cancer stem cells. (38)

38) Xu, Liang, et al. “WNT pathway inhibitor pyrvinium pamoate inhibits the self-renewal and metastasis of breast cancer stem cells.” International journal of
oncology 48.3 (2016): 1175-1186.

40) Xu, Wei, et al. “The antihelmintic drug pyrvinium pamoate targets aggressive breast cancer.” PloS one 8.8 (2013): e71508.

86) Zhang, Le, et al. “Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer.” International Journal of Radiation Oncology, Biology,
Physics. 103.1 (2019): 195-207.

Artemisinin p 260

Perinuclear Clustering of Lysosomes p 263

In 2010, studying MCF-7 breast cancer cells in vitro, Dr. Anne Hamacher-Brady et al.
observed that artemisinin causes a peculiar re-arrangement of the lysosomes and mitochondria in a pattern typical for autophagy. (20)

20) Hamacher-Brady, Anne, et al. “Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production.” J. Biol. Chem 2011.286 (2010): 6587-6601.

139) Greenshields, Anna L., Wasundara Fernando, and David W. Hoskin. “The anti-malarial drug artesunate causes cell cycle arrest and apoptosis of triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells.” Experimental and molecular pathology
107 (2019): 10-22

p 267 Sulforaphane

In 2010, Dr. Yanyan Li et al. reported that sulforaphane eliminated breast cancer stem cells
in vivo in a mouse xenograft model. Tumorbearing primary mice were treated with sulforaphane. The tumor cells from the primary mice were then re-implanted into secondary
mice, showing no growth of tumor cells, indicating the cancer stem cells had been eradicated.

Dr. Li’s group writes:
Sulforaphane decreased the protein level of βeta-catenin by up to 85% in MCF7 and
SUM159 [breast cancer] cells; and the expression of cyclin D1, one of the Wnt/β-
catenin target genes, declined by up to 77% as well. (64–65)

64) Li, Yanyan, et al. “Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells.” Clinical Cancer Research 16.9 (2010):
2580-2590.

65) Li, Y., and T. Zhang. “Targeting cancer stem cells with sulforaphane, a dietary component from broccoli and broccoli sprouts.” Future oncology (London,England) 9.8 (2013): 1097-1103.

Mefloquine Superiority to Chloroquine p 269

In a 2012 breast cancer cell study, Dr. Natasha Sharma et al. found mefloquine superior
to chloroquine for causing cancer cell death. (88)

88) Sharma, Natasha, et al. “Inhibition of autophagy and induction of breast cancer cell death by mefloquine, an antimalarial agent.” Cancer letters 326.2 (2012): 143-154.

Ivermectin p 272

Dr. Dobrin Draganov et al. reported in 2015 that ivermectin kills mouse and human triple-negative breast cancer [TNBC] cells through augmented P2X7-dependent purinergic
signaling associated with caspase-1 and caspase-3 activation…. also involved is
the recruitment and activation of T cells, macrophages and dendritic cells, a form of
immunomodulation and cancer immunotherapy.(109)

109) Draganov, Dobrin, et al. “Modulation of P2X4/ P2X7/pannexin-1 sensitivity to extracellular ATP via ivermectin induces a non-apoptotic and inflammatory
form of cancer cell death.” Scientific reports 5 (2015).

p 280 Parthenolide feverfew

54) Araújo, Thaise Gonçalves, et al. “Parthenolide and Its Analogues: A New Potential Strategy for the Treatment of Triple-Negative Breast Tumors.” Current medicinal chemistry (2020).

p 281 Sulfasalazine

74) Narang, Vishal S., et al. “Sulfasalazine-induced reduction of glutathione levels in breast cancer cells: enhancement of growth-inhibitory activity of doxorubicin.” Chemotherapy 53.3 (2007): 210-217

Blocking Wnt Pathway p 287

In 2012, Dr. Robin Hallet et al. reported that blocking the Wnt /Beta-Catenin signaling pathway in a breast cancer model eradicated cancer stem cells. (11)

11) Hallett, Robin M., et al. “Small molecule antagonists of the Wnt/beta-catenin signaling pathway target breast tumor-initiating cells in a Her2/Neu mouse
model of breast cancer.” PloS one 7.3 (2012): e33976

Ivermectin Effective Against Triple-Negative Breast Cancer Cells (TNBC); p 287

In 2015, Dr. Dobrin Dragonov et al. reported in Scientific Reports on the mechanism of cancer- cell-killing by ivermectin. Dr. Dragonov’s lab was heavily involved in studying P2X receptors, a family of ATP-gated cation channels. These are cell-membrane pores that open in response to ATP in the micro-environment. Dr. Dragonov’s group makes the observation
that tumors have upregulated P2X7 receptors,which are involved with regulation of high
ATP concentrations in the tumor micro-environment, promoting tumor progression. The
application of ivermectin sensitizes the cation channels to ATP, further opening the channel
pores, allowing an influx of large cations causing cancer cell death. They write:

Ivermectin kills mouse and human triple-negative breast cancer [TNBC] cells
through augmented P2X7-dependent purinergic signaling associated with caspase-1
and caspase-3 activation. [Note: caspase activation means apoptotic programmed
cell death controlled by mitochondria]. (15–17)

15) Draganov, Dobrin, et al. “Modulation of P2X4/ P2X7/pannexin-1 sensitivity to extracellular ATP via ivermectin induces a non-apoptotic and inflammatory
form of cancer cell death.” Scientific reports 5 (2015).

16) Mackay, Laurent, et al. “Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.” PLoS computational biology 13.7 (2017): e1005643.

17) Latapiat, Verónica, et al. “P2X4 receptor in silico and electrophysiological approaches reveal insights of ivermectin and zinc allosteric modulation.” Frontiers in pharmacology 8 (2017): 918.

p 288 Ivermectin Effective at Low Concentration

Dr. Dragonov reports that mouse and human TNBC (triple negative breast cancer) cells are
sensitive to ivermectin, with IC50 values as low as 2 μM with 24 hour exposure time. In
addition, Dr. Dragonov hypothesized that ivermectin kills cancer cells by enhancing receptor
P2X7 sensitivity to extracellular ATP. In doing so, ivermectin induces both an apoptotic and a
non-apoptotic, inflammatory type of cell death . The inflammatory cell death stimulates the
immune system, an added beneficial effect, so that the patient’s own immune system will kill
new cancer cells in the future

Ivermectin Effective for Canine Breast Cancer p 289

In 2019, Dr. Hongxiu Diao et al. studied ivermectin’s inhibitory effect on canine breast
cancer cells involved in regulating cell-cycle progression, and Wnt signaling. Dr. Diao
reported cell-cycle arrest at G1 phase via downregulation of CDK4 and cyclin D1 expression.
However, unlike the 2019 study of HeLa cervical cancer cells by Dr. Zhang, there was
no significant induction of apoptosis. Dr. Diao et al. observed significantly reduced Beta-
Catenin nuclear translocation after treatment with ivermectin, resulting in the inactivation of
Wnt signaling. Significant suppression of tumor growth by ivermectin was observed in canine
mammary tumor xenografts. (21)

21) Diao, Hongxiu, et al. “Ivermectin inhibits canine mammary tumor growth by regulating cell cycle progression and WNT signaling.” BMC Veterinary Research 15.1 (2019).

Ivermectin Reverses Drug Resistance p 289

In 2019, using breast cancer and colorectal cancer cell lines, Dr. Lu Jiang et al. showed
that ivermectin reverses cancer cell drug resistance to chemotherapy by reducing expression of P-glycoprotein (P-gp). This is done by inhibiting the epidermal growth factor receptor (EGFR) and its downstream signaling cascade ERK/Akt/NF-kB, not by directly inhibiting P-gp activity. Dr. Jiang and colleagues write that ivermectin could be useful in drug-resistant cancers:

Thus, ivermectin, a FDA-approved antiparasitic drug, could potentially be used
in combination with chemotherapeutic agents to treat cancers and in particular,
the drug-resistant cancers. (23)

23) Jiang, Lu, et al. “Ivermectin reverses the drug resistance in cancer cells through EGFR/ERK/Akt/NF-KB pathway.” Journal of Experimental & Clinical Cancer Research 38.1 (2019): 265.

Ivermectin mTOR Inhibitor—Induces Autophagy p 290

Ivermectin degrades the PAK protein, which then inhibits mTOR, which activates autophagy.
Note: PAK1 is P21-activated kinase (see Glossary). In 2016, Dr. Kui Wang et al. studied
the effect of ivermectin in a breast cancer cell line in vitro and in vivo, writing:

Ivermectin markedly inhibits the growth of breast cancer cells by stimulating cytostatic
macroautophagy/autophagy in vitro and in vivo. (24)

24) Wang, Kui, et al. “Ivermectin induces PAK1-mediated cytostatic autophagy in breast cancer.”Autophagy 12.12 (2016): 2498-2499

Ivermectin degrades PAK1 protein, which then inhibits mTOR, which leads to increased
autophagic flux. Dr. Qianhui Dou et al. (2016) write:

Ivermectin-induced autophagy is cytostatic in breast cancer cells, and suppression of
autophagy may attenuate the anti-cancer effect of ivermectin (26)
Thus, the addition of autophagy inhibitor chloroquine failed to increase (attenuates) the
effectiveness of ivermectin in Dr. Dou’s breast cancer model. (24–26)
As mentioned above,

25) Liu, Jian, et al. “Progress in Understanding the Molecular Mechanisms Underlying the Antitumour Effects of Ivermectin.” Drug Design, Development and
Therapy 14 (2020): 285.

26) Dou, Qianhui, et al. “Ivermectin induces cytostatic autophagy by blocking the PAK1/Akt axis in breast cancer.” Cancer research 76.15 (2016): 4457-4469.

Niclosamide p 295

Degrades the LRP6 CoReceptor at One Micromolar

In 2011, Dr. Lu found that niclosamide inhibited the Wnt pathway by targeting the LRP6-
Wnt co-receptor on the cancer cell surface, degrading the LRP6 protein. This was found at
low concentrations of the drug with IC50 values less than 1 micromolar for prostate and
breast cancer cells. (5)

5) Lu, Wenyan, et al. “Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway.” PloS one 6.12 (2011): e29290.

Niclosamide for Breast Cancer Stem Cells p 301

In 2013, Dr. Yu-Chi Wang et al. screened the LOPAC chemical library of 1,258 compounds,
identifying niclosamide as the best inhibitor of breast cancer stem cells. (33) In 2014, Dr. Tinghong Ye et al. studied the effect of niclosamide on breast cancer cells in vitro,
and in vivo with a mouse breast cancer model, showing dramatic growth inhibition, and apoptosis in a dose-dependent manner…. Apoptosis was associated downregulation of Bcl-2, Mcl-1 and Survivin…. . Niclosamide blocked cancer cell migration and invasion [properties of cancer stem cells] … reduced STAT3 [a cancer stem cell pathway], … without
detectable toxicity.(34)

33) Wang, Yu-Chi, et al. “Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells.” PloS one 8.9 (2013): e74538.

Dr. Ye et al. report on histological and immunohistochemical analyses in the mouse breast
cancer model. After niclosamide treatment, Dr. Ye observed:

decrease in Ki67-positive cells, VEGF-positive cells and microvessel density [MVD] and an increase in cleaved caspase-3-positive cells upon niclosamide…. .Notably, niclosamide
reduced the number of myeloid-derived suppressor cells [MDSCs] in tumor tissues
and blocked formation of pulmonary metastases. (34)

34) Ye, Tinghong, et al. “The anthelmintic drug niclosamide induces apoptosis, impairs metastasis and reduces immunosuppressive cells in breast cancer model.” PloS one 9.1 (2014): e85887.

Micro-Environment Study of Breast Cancer p 302

The tumor micro-environment (TME) is composed of cells and tissues around the cancer
mass, playing a supportive role in feeding the cancer mass with nutrients and growth factors.
According to Drs. Freja Venning et al. (2015), the tumor micro-environment includes:
immune cells, fibroblasts, pericytes, endothelial cells, adipocytes, and mesenchymal
stem cells, and also the interstitial fluids and the extracellular matrix [ECM]. (36)

36) Venning, Freja A., Lena Wullkopf, and Janine T. Erler. “Targeting ECM disrupts cancer progression.” Frontiers in oncology 5 (2015): 224.

EMT Transition in Breast Cancer p 302

Reversed by Niclosamide

A 2019 study by Dr. Jones Gyamfi et al. examined the micro-environment of breast cancer,
composed predominantly of adipocytes, or fat cells. Dr. Gyamfi’s group says these adipocytes surrounding the cancer mass induce “epithelial mesenchymal transition (EMT) of breast cancer cells through paracrine IL-6/Stat3 signaling.” (37)

37) Gyamfi, Jones, et al. “Niclosamide reverses adipocyte induced epithelial-mesenchymal transition in breast cancer cells via suppression of the interleukin- 6/STAT3 signalling axis.” Scientific reports 9.1 (2019): 1-14.

Dr. Gyamfi’s breast cancer cell study found that the Niclosamide inhibition of IL-6/
STAT3 reversed adipocyte-induced EMT. They state:

Niclosamide inhibited adipocyte-induced effects and significantly decreased cell
motility in a dose-dependent manner….. niclosamide reversed adipocyte-induced
EMT with a correlated inhibition of IL-6/Stat3 activation and downregulation
of EMT-TFs TWIST and SNAIL. Moreover, niclosamide markedly impaired
MDA-MB-468 and MCF-7 [breast cancer cells] migration and invasion. (37)

p 307 Niclosamide Prevents Conversion of Non-Stem cells to Stem Cells

In 2013, Dr. Seog-Young Kin et al. studied how IL-6/STAT3 signaling transforms breast
cancer cells into breast cancer stem cells, finding that niclosamide effectively prevents this
conversion by blocking OCT-4 gene expression. Data from cytokine array assay show that
IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment
plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-
JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by
treatment with anti-IL-6 antibody (1 μg/ml) or niclosamide (0.5–2 μM)/LLL12 (5–10 μM)
effectively prevented OCT-4 gene expression. (87)

87) Kim, Seog-Young, et al. “Role of the IL-6-JAK1- STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells.” Cellular signalling 25.4 (2013): 961-969.

33) Wang, Yu-Chi, et al. “Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells.” PloS one 8.9 (2013): e74538.

p 316 Mebendazole Synergy with oncology drugs and radiation:

18) Zhang, Le, et al. “Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer.” International Journal of Radiation Oncology* Biology*
Physics 103.1 (2019): 195-207

25) Alam, Syed Mahboob, Farah Asad, and Hina Shams. “Mebendazole Inexplicably Reducing the Breast Cancer Cells Viability Preclinically by Incitement Effects
with Methotrexate.” RADS Journal of Pharmacy and Pharmaceutical Sciences 6.2 (2018): 101-106.

p 320 Fenbendazole is effective in other cell types, including: • Breast cancer (63)

63) Duan, Qiwen, Yanfeng Liu, and Sara Rockwell. “Fenbendazole as a potential anticancer drug.” Anticancer research 33.2 (2013): 355-362.

p 320 Flubendazole is effective in the following:
• Breast cancer stem cells (70–72)

70) Hou, Zhi-Jie, et al. “Flubendazole, FDA-approved anthelmintic, targets breast cancer stem-like cells.” Oncotarget 6.8 (2015): 6326.

71) Oh, Eunhye, et al. “Flubendazole elicits anti‐metastatic effects in triple‐negative breast cancer via STAT3 inhibition.” International journal of cancer 143.8 (2018): 1978-1993.

72) Kim, Yoon-Jae, et al. “Flubendazole overcomes trastuzumab resistance by targeting cancer stem-like properties and HER2 signaling in HER2-positive breast
cancer.” Cancer Letters 412 (2018): 118-130.

p 331

21) Mahmoud, Abeer M. “Cancer testis antigens as immunogenic and oncogenic targets in breast cancer.” Immunotherapy 10.9 (2018): 769-778.

p 335 Mifepristone Induces Apoptosis in Cancer Cells

As mentioned above, mifepristone has a second anti-cancer mechanism directly inducing
apoptosis in cancer cells at appropriate concentrations. In 2013, Dr. Ji Hoon Jang et al. studied the effect of mifepristone on (U937) lymphoma cells, finding reduction in mitochondrial potential,  activation of p38 MAPK, and induction of mitochondrial apoptosis. Overexpression of BCL-2 (anti-apoptotic protein) blocked this effect. Induction of apoptosis was also found for breast, lung, and colon cancer cells. (18)

18) Jang, Ji Hoon, et al. “RU486, a glucocorticoid receptor antagonist, induces apoptosis in U937 human lymphoma cells through reduction in mitochondrial membrane potential and activation of p38 MAPK.” Oncology reports 30.1 (2013): 506-512.

p 338 Mifepristone Breast Cancer Studies

A number of studies show the efficacy of mifepristone for breast cancer, suppressing
growth, preventing metastatic disease, and suppressing cancer stem cells. (30–33)

30) Liu, Rong, et al. “Mifepristone Derivative FZU-00,003 Suppresses Triple-negative Breast Cancer Cell Growth partially via miR-153-KLF5 axis.” Int J Biol Sci 16.4 (2020): 611-619.

31) Yu, Suhong, et al. “Pharmacoproteomic analysis reveals that metapristone (RU486 metabolite) intervenes E-cadherin and vimentin to realize cancer metastasis chemoprevention.” Scientific reports 6 (2016): 22388.

32) Liu, Rong, et al. “Mifepristone suppresses basal triple-negative breast cancer stem cells by down-regulating KLF5 expression.” Theranostics 6.4 (2016): 533.

33) Rubin, Ayelen, et al. “Effect of the combined treatment with mifepristone and chemotherapy on breast cancer brain metastases.” (2017): 4912-4912.

p 340 8) Bhuvanalakshmi, G., et al. “Breast cancer stem-like cells are inhibited by diosgenin, a steroidal saponin, by the attenuation of the Wnt β-catenin signaling via the Wnt antagonist secreted frizzled related protein-4.” Frontiers in pharmacology 8 (2017): 124.

p 348

18) Cserni, Gábor, et al. “Spontaneous pathological complete regression of high-grade triple-negative breast cancer with axillary metastasis.” Polish Journal of Pathology 70.2 (2019): 139-143.

p 358 Cancer Stem Cell Crosstalk with T-Regulatory Cells

In 2019, Dr. A. Dutta et al. achieved a new insight in the mechanism of how cancer cells
evade immune surveillance. Dr. Dutta studied breast cancer stem cells and breast tumor tissues, finding that the breast cancer stem cells actually generated their own immunosuppressive T-reg cells. This was done by secretion of exosomes (small vesicles related to macropinocytosis) into the micro-environment. The exosomes contain the FOXP3 protein, which binds to a receptor on the T cell, which then converts the T cell into an immunosuppressive T-reg cell. Thus, Dr. Dutta and colleagues linked together immune evasion of cancer stem cells to the lysosmal/autophagy pathway.

Dr. Dutta et al. write:
Collectively our data demonstrates that bCSC [breast cancer stem cell]-shed exosomal
FOXP3 [protein] plays an important role in procreation of T-reg cells within the
tumor micro-environment thus leading to tumor-induced immune suppression. (8–9)

8) Dutta, A., et al. “338P A new insight into tumour immune-evasion: Crosstalk between cancer stem cells and T regulatory cells.” Annals of Oncology 30.Supplement_9 (2019): mdz438-020.

9) Wee, Ian, et al. “Role of tumor-derived exosomes in cancer metastasis.” Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1871.1 (2019): 12-19.

p 361 Beta glucans benefits in various cancer cell lines:

• Breast cancer, stem cells (27–28)

27) Graham, Émilie A., et al. “MicroRNA signature in the chemoprevention of functionally-enriched stem and progenitor pools (FESPP) by active hexose correlated compound (AHCC).” Cancer biology & therapy 18.10 (2017): 765-774.

28) Matsushita, Kazuhiro, et al. “Combination therapy of active hexose correlated compound plus UFT significantly reduces the metastasis of rat mammary adenocarcinoma.”
Anti-cancer drugs 9.4 (1998): 343-350.

p 370
92) El-Din, Nariman K. Badr, et al. “Biobran/MGN-3, arabinoxylan from rice bran, sensitizes breast adenocarcinoma tumor cells to paclitaxol in mice.” (2015): 5312-5312.

p 374 Molecular Iodine as Treatment for Breast Cancer

Dr. Gerson had no way of knowing that medical research would eventually show he was
quite correct to use iodine for cancer patients. Over the years, a number of studies have
accumulated evidence that molecular iodine is effective treatment for breast cancer. The
mechanism of action (MOA) involves activation of peroxisome proliferator-activated receptors (PPAR) and production of iodolactones, which mediate apoptotic effects. (25–29)

22) Mansel, Robert E., et al. “A Randomized Controlled Multicenter Trial of an Investigational Liquid Nutritional Formula in Women with Cyclic Breast Pain Associated with Fibrocystic Breast Changes.” Journal of Women’s Health 27.3 (2018): 333-340.

23) Rappaport, Jay. “Changes in dietary iodine explains increasing incidence of breast Cancer with distant involvement in young women.” Journal of Cancer 8.2 (2017): 174.

24) Gerson Therapy, by Charlotte Gerson and Morton Walker, DPM (2001) NY: Kensington Publishing Corp. ISBN 1-57566-628-6 (Trade paperback, 371 pages, plus
appendixes and index.)

25) Aceves, Carmen, et al. “Antineoplastic effect of iodine in mammary cancer: participation of 6-iodolactone (6-IL) and peroxisome proliferator-activated receptors (PPAR).” Molecular Cancer 8.1 (2009): 33.

26) Arroyo-Helguera, O., et al. “Signaling pathways involved in the antiproliferative effect of molecular iodine in normal and tumoral breast cells: evidence that 6-iodolactone mediates apoptotic effects.” Endocrine-related cancer 15.4 (2008): 1003-1011.

27) Nava-Villalba, Mario, and Carmen Aceves. “6-Iodolactone, key mediator of antitumoral properties of iodine.” Prostaglandins & other lipid mediators 112 (2014): 27-33.

28) Nava-Villalba, Mario, et al. “Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone.” Molecular cancer 14.1 (2015): 168.

29) Xu, Zack, et al. “Elucidating the mechanism of action of molecular iodine on breast cancer cells.” (2017): 2243-2243.

p 374 Clinical Study: Iodine as Adjuvant for Breast Cancer

Iodine treatment has a dual anti-cancer effect. It has a direct anti-cancer effect by
inducing apoptosis of the cancer cells, and secondarily, iodine “activates the anti-tumor
immune response.” In 2019, Dr. Aura Morena- Vega et al. studied the use of molecular iodine in thirty breast cancer patients, either alone or as adjuvant with chemotherapy, in two groups— early and advanced. In the early group, thirty women were treated with either 5 mg per day of molecular iodine (I2) or placebo for 7 to 35 days before surgery. For the advanced group, all patients received chemotherapy (5-fluorouracil/ epirubicin/cyclophosphamide or
taxotere/epirubicin (FEC/TE) and were randomized to receive either molecular iodine 5
mg/day or placebo before and 170 days after surgery. Five-year disease-free survival was
significantly higher for patients treated with molecular iodine (I2) before and after surgery
compared to placebo—82 per cent vs. 46 per cent. Examination of histology of tissue samples showed Iodine treated patients had “activation of the anti-tumoral immune response.”

Dr. Moreno-Vega et al. write:
I2 supplementation showed a significant attenuation of the [chemotherapy] side
effects and an absence of tumor chemoresistance … I2-treated tumors exhibit
less invasive potential, and significant increases in apoptosis, estrogen receptor
expression, and immune cell infiltration…. Transcriptomic analysis indicated activation
of the antitumoral immune response. (34)

34) Moreno-Vega, Aura, et al. “Adjuvant Effect of Molecular Iodine in Conventional Chemotherapy for Breast Cancer. Randomized Pilot Study.” Nutrients 11.7
(2019): 1623

p 375 Dr. Mendieta proposes iodine supplementation as a possible adjuvant in breast cancer
therapy, writing:

I2 [Iodine] decreases the invasive potential of a triple-negative basal cancer cell
line, and under in vivo conditions the oral supplement of this halogen [iodine] activates
the anti-tumor 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. (35)

35) 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 19.1 (2019): 261.

p 375 In 2018, Dr. Xóchitl Zambrano-Estrada et al. studied canine mammary cancer using the combination of Iodine and chemotherapy, writing:
The [combination Doxorubicin chemotherapy plus iodine] mDOX+I2 scheme
improves the therapeutic outcome, diminishes the invasive capacity, attenuates the
adverse events and increases disease-free survival. These data led us to propose
mDOX+I2 as an effective treatment for canine mammary cancer. (41)

41) Zambrano-Estrada, Xóchitl, et al. “Molecular iodine/doxorubicin neoadjuvant treatment impair invasive capacity and attenuate side effect in canine mammary cancer.” BMC veterinary research 14.1 (2018): 87.

p 376 DIM, Selenium D3
In addition to iodine, a typical breast cancer prevention program includes supplementation
with DIM (di-indole-methane) and calcium-D-glucarate, while avoiding xeno-estrogens
in food and plastics. It is also useful to test for selenium and vitamin D3 and supplement
if found low. (163-165) (169)

163) Short, Sarah P., and Christopher S. Williams. “Selenoproteins in tumorigenesis and cancer progression.” Advances in cancer research. Vol. 136. Academic Press, 2017. 49-83.

164) Sanmartín, Carmen, et al. “Selenium compounds, apoptosis and other types of cell death: an overview for cancer therapy.” International journal of molecular sciences 13.8 (2012): 9649-9672.

165) Misra, Sougat, et al. “Redox-active selenium compounds— From toxicity and cell death to cancer treatment.” Nutrients 7.5 (2015): 3536-3556.

169) Thomson, Cynthia A., Emily Ho, and Meghan B. Strom. “Chemopreventive properties of 3, 3′-diindolylmethane in breast cancer: evidence from experimental and human studies.” Nutrition reviews 74.7 (2016): 432-443.

==============================

p 380
In 2002, Dr. Je-Ruei Liu et al. studied the anti-tumor effect of milk kefir in mouse xenografts
inoculated with human sarcoma cells, finding 64% tumor inhibition compared to controls
in mice fed the kefir. Other in vitro and in vivo studies show efficacy in colorectal cancer,
malignant T lymphocytes, breast cancer, and lung carcinoma. (113–114)
Dr. Mohammadreza Sharifi et al. (2017),write:

Kefir is one of the best therapeutic natural ingredients, applying its anti-cancer effect
through different cellular and molecular pathways. Kefir is likely to be recognized
for effective treatment of malignancies and as an anti-cancer agent in the near future.
(114)

113) Liu, Je-Ruei, et al. “Antitumor activity of milk kefir and soy milk kefir in tumor-bearing mice.” Nutrition and cancer 44.2 (2002): 183-187.

114) Sharifi, Mohammadreza, et al. “Kefir: a powerful probiotics with anticancer properties.” Medical Oncology 34.11 (2017): 183.

p 390 Mefloquine Most Potent

In 2012, Dr. Natasha Sharma et al. studied the effect of mefloquine on breast cancer cells
in vitro, finding mefloquine the most potent of the three antimalarial drugs, causing expansion and disruption of lysosomes. (23)

23) Sharma, Natasha, et al. “Inhibition of autophagy and induction of breast cancer cell death by mefloquine, an antimalarial agent.” Cancer Letters 326.2 (2012): 143-154

Vitamin D3 p 404
Dr. Yu and colleagues write:
Calcitriol causes antiproliferative effects through multiple mechanisms, including
the induction of cell cycle arrest, apoptosis and differentiation in vitro and in vivo in
a variety of cancer cell types including prostate, breast, colon, skin and leukemic
cells. (19)

19) Yu, Wei-Dong, et al. “Calcitriol enhances gemcitabine antitumor activity in vitro and in vivo by promoting apoptosis in a human pancreatic carcinoma model system.” Cell cycle 9.15 (2010): 3094-3101.

=============================

p 406 Triple-Negative Breast Cancer Cell Model: Combining OXPHOS and Autophagy Inhibition

In 2019, Dr. Etna Abad et al. studied in vitro and in vivo mouse xenografts of a triple-negative breast cancer cell line, using a combination of antibiotic to target mitochondrial ribosomes, along with an autophagy inhibitor. Dr. Abad’s group first created in their lab parent CSCs, as well as chemotherapy resistant cancer cells, finding the latter had increased mitochondrial metabolic activity and higher metastatic ability compared to the parent cancer cells. They theorized that an antibiotic to induce mitochondrial dysfunction would inhibit OXPHOS and suppress cancer growth. The antibiotic selected was linezolid, which inhibits mitochondrial ribosomal protein production, similar to the mechanism of doxycycline and clarithromycin. The authors found the antibiotic increased
reactive oxygen species (ROS) and activated “protective autophagy.” The authors then added the autophagy inhibitor hydroxychloroquine, finding this combination reduced metastatic capacity and delayed tumor growth for both CSCs and drug-resistant cancer cells. One might speculate that Dr. Abad’s results would have been even better if a glycolysis Inhibitor (DCA) had been added, targeting all three major metabolic
pathways. Dr. Abad et al. write:

we propose that antibiotics serving as MDF-inducers [MDF= mitochondrial
dysfunction] can suppress cancer cell proliferation and decrease tumor growth.
In combination with autophagy blockers, such drugs can be repurposed as part of
the multitarget anti-cancer therapy. (42)

p 407 Preventing the Switch from Dormant to Aggressive

In 2018, Dr. Laura Vera-Ramirez et al. studied autophagy inhibition in “dormant” breast
cancer using in vitro and in vivo mouse xenograft models. “Dormant” breast cancer cells
are synonymous with the CSC phenotype. As we discussed in previous chapters, CSCs have metabolic plasticity and can switch back and forth from a dormant to an aggressive proliferative state. Autophagy inhibition prevented the “wake-up” of dormant breast CSCs, preventing the switch from the dormant to the proliferative state. Dr. Vera-Ramirez et al. write:

Autophagy inhibition effectively reduced the metastatic burden in the lungs of
transplanted mice and it was proposed that autophagy is required for the switch
from dormancy to tumor cell growth, as autophagy inhibition specifically depleted
dormant cells from tumors, leaving the proliferative tumor cells intact … Inhibition of autophagy may therefore be a potential mechanism to eliminate dormant tumour
cells and prevent recurrence of BC [breast cancer]. (43)

p 408 Chloroquine Targets Cancer Stem Cells and Decreases Ability to Metastasize

In 2016, Dr. Diana Liang et al. studied the effect of chloroquine on triple-negative CSCs in
vitro and in vivo xenografts, finding that: CQ [chloroquine] effectively targets CSCs
via autophagy inhibition, mitochondrial structural damage, and impairment of
double-stranded DNA break repair…. CQ effectively diminishes the TNBC [triple
negative breast cancer] cells’ ability to metastasize in vitro and in a TNBC xenograft
model (55)

55) Liang, Diana H., et al. “The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair.” Cancer letters 376.2 (2016): 249-258

p 412 D3 Metformin Synergy

22) Guo, Li-Shu, et al. “Synergistic antitumor activity of vitamin D3 combined with metformin in human breast carcinoma MDA-MB-231 cells involves m-TOR related signaling pathways.” Die Pharmazie-An International Journal of Pharmaceutical Sciences 70.2 (2015):
117-122.

43) Vera-Ramirez, Laura, et al. “Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence.” Nature communications 9.1 (2018): 1-12

58) Maycotte, Paola, et al. “Autophagy supports breast cancer stem cell maintenance by regulating IL6 secretion.” Molecular cancer research 13.4 (2015): 651-658.

60) Han, Yanyan, et al. “Role of autophagy in breast cancer and breast cancer stem cells.” International journal of oncology 52.4 (2018): 1057-1070.

62) Bousquet, Guilhem, et al. “Targeting autophagic cancer stem-cells to reverse chemoresistance in human triple negative breast cancer.” Oncotarget 8.21
(2017): 35205.

63) Cufí, Sílvia, et al. “Autophagy positively regulates the CD44+ CD24-/low breast cancer stem-like phenotype.” Cell cycle 10.22 (2011): 3871-3885

p 421 In Vivo Studies PPI Drugs Enhance Chemotherapy

In 2015, Dr. Tan et al. studied the PPI drug pantoprazole, added to the chemotherapy
agent docetaxol, against three cancer cell lines (breast, prostate, and epidermoid skin cancer) as mouse xenografts, finding dramatic enhancement of docetaxol activity by the PPI
drug. Note: docetaxol is a taxane microtubule agent. This was further studied and found to
be due to autophagy inhibition by the PPI drug. Similar to many other chemotherapy drugs,
docetaxol upregulates “protective autophagy” which promotes survival in cancer cells.
Inhibition of “protective autophagy” with a PPI drug is confirmed by finding increased protein
markers LC3-II and p62. Dr. Tan and colleagues write:

Our results suggest that pantoprazole inhibits autophagy by raising lysosomal
pH and/or by inhibiting fusion of autophagosomes with lysosomes, leading to
the accumulation of autophagosomes….pantoprazole increased the accumulation
of both LC3-II and p62. (30–32)

30) Tan, Q., et al. “Effect of pantoprazole to enhance activity of docetaxel against human tumour xenografts by inhibiting autophagy.” British journal of cancer 112.5 (2015): 832-840.

31) Baquero, Pablo, et al. “Targeting quiescent leukemic stem cells using second generation autophagy inhibitors.” Leukemia 33.4 (2019): 981-994.

32) Tan, Qian, et al. “Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity.”
Cancer chemotherapy and pharmacology 79.5 (2017): 959-969.

p 430  PPIs plus FASN inhibitors

40) Wang, Chao. “Utilization of Proton Pump Inhibitors in Combination Regimen for Breast Cancer Treatment by Targeting Fatty Acid Synthase.” (2019).

p 422 Results of PPIs in Clinical Trials

As described by Dr. Elisabetta lessi et al. in 2018, a number of clinical trials showing adjuvant PPI use increases efficacy of chemotherapy in osteosarcoma and metastatic breast
cancer, metastatic colorectal cancer and other solid tumors. PPI use increased survival in
head-and-neck squamous cell carcinoma.

Dr. Iessi and colleague writes:
All these studies provided the first clinical evidence that PPIs pretreatment could be
easily included into the standard protocols in clinical oncology with a clear benefit for
patients having the less favorable prognostic factors. Indeed, pretreatment with
PPIs, by inhibiting proton pumps, induced a decrease of the protonation of extracellular
tumor environment, in turn allowing the chemotherapeutics to be fully effective,
improving the effectiveness of either chemical and biological drugs against cancer.
Thus, tumor alkalinization could improve the outcome of patients by counteracting
tumor chemoresistance. (54–55)

54) Iessi, Elisabetta, et al. “Rethinking the combination of proton exchanger inhibitors in cancer therapy.” Metabolites 8.1 (2018): 2.

55) Papagerakis, Silvana, et al. “Proton pump inhibitors and histamine 2 blockers are associated with improved overall survival in patients with head and neck squamous
carcinoma.” Cancer Prevention Research 7.12 (2014): 1258-1269.

p 426 Loratidine More Cohort Studies

Additional cohort studies have been done for melanoma, ovarian cancer, and breast cancer,
finding that loratadine users have improved survival. Melanoma patients using loratadine
had striking reduction in mortality with HR of 0.50 (hazard ratio reduced by half). (63–66)

65) Olsson, Håkan Lars, Ildiko Fritz, and Philippe Wagner. “Abstract P5-06-07: Desloratadine and loratadine increase breast cancer survival.” (2020): P5-06.

66) Bens, Annet, et al. “The role of H1 antihistamines in contralateral breast cancer: a Danish nationwide cohort study.” British Journal of Cancer (2020): 1-7.

p 436 Thymoquinone

Immune System Activation by TQ

In 2018, Dr. Lena Odeh et al. found synergy of TQ with melatonin in a breast cancer
mouse xenograft model. In addition, there was a beneficial effect on the immune system with
activation of T-helper-1 anti-cancer immune response. (27)

Breast cancer (25–27)(48–54)(59)

25) Aumeeruddy, M. Zakariyyah, and M. Fawzi Mahomoodally. “Combating breast cancer using combination therapy with 3 phytochemicals: Piperine, sulforaphane, and thymoquinone.” Cancer 125.10 (2019): 1600-1611.\

26) Alobaedi, Omar H., Wamidh H. Talib, and Iman A. Basheti. “Antitumor effect of thymoquinone combined with resveratrol on mice transplanted with breast cancer.”
Asian Pacific journal of tropical medicine 10.4 (2017): 400-408.

27) Odeh, Lena Hisham, Wamidh H. Talib, and Iman A. Basheti. “Synergistic effect of thymoquinone and melatonin against breast cancer implanted in mice.”
Journal of cancer research and therapeutics 14.9 (2018): 324.

48) Sutton, Kimberly M., Anna L. Greenshields, and David W. Hoskin. “Thymoquinone, a bioactive component of black caraway seeds, causes G1 phase cell cycle arrest and apoptosis in triple-negative breast cancer cells with mutant p53.” Nutrition and cancer
66.3 (2014): 408-418.

49) Yegin, Z., T. Duran, and I. H. Yildirim. “Thymoquinone Down-regulates VEGFA and Up-regulates FLT1 Transcriptional Levels in Human Breast Cancer Cells.”
Int J Hum Genet 20.1 (2020): 19-24.

50) Yıldırım, İbrahim Halil, Ali Ahmed Azzawri, and Tuğçe Duran. “Thymoquinone induces apoptosis via targeting the Bax/BAD and Bcl-2 pathway in breast cancer cells.” Dicle Tıp Dergisi 46.3 (2019): 411-417.

51) Talib, Wamidh H. “Regressions of breast carcinoma syngraft following treatment with piperine in combination with thymoquinone.” Scientia pharmaceutica 85.3 (2017): 27.

52) Barkat, Md A., et al. “Insights into the targeting potential of thymoquinone for therapeutic intervention against triple-negative breast cancer.” Current drug targets 19.1 (2018): 70-80

53) Shanmugam, Muthu K., et al. “Thymoquinone inhibits bone metastasis of breast cancer cells through abrogation of the CXCR4 signaling axis.” Frontiers in pharmacology 9 (2018): 1294.

54) Kabil, Nashwa, et al. “Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer.” Breast cancer research and treatment 171.3 (2018): 593-605.

59) Ng, Wei Keat, et al. “Thymoquinone-loaded nanostructured lipid carrier exhibited cytotoxicity towards breast cancer cell lines (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa).” BioMed research international 2015 (2015).

p 437

In 2016, Dr. Mehdi Nikbakht Dastjerdi et al. studied TQ in a MCF-7 breast cancer cell line,
finding induction of apoptosis associated with dramatic upregulation of the P53 gene. (33)

33) Dastjerdi, Mehdi Nikbakht, et al. “Effect of thymoquinone on P53 gene expression and consequence apoptosis in breast cancer cell line.” International journal of preventive medicine 7 (2016).

p 446 Dual Inhibition Targets Cancer Stem Cells

In 2016, in Breast Cancer Research, Dr. Lisanti’s group studied breast CSCs. Because
of the metabolic plasticity of the CSC, they suggested that a dual blockade of both OXPHOS
and the GLYCOLYSIS metabolic pathways would be a better way to eradicate CSCs.
[Dual Blockade] may represent a better way to eradicate CSC [Cancer Stem Cell]
heterogeneity than focusing exclusively on glycolysis inhibition or suppression of
mitochondrial respiration [OXPHOS]. (5)

5) Peiris-Pagès, Maria, et al. “Cancer stem cell metabolism.” Breast Cancer Research 18.1 (2016): 55.

p 455 Itraconazole

Retrospective studies of patients with ovarian cancer and recurrent triple-negative breast
cancer taking itraconazole revealed significant increases in overall survival, thought to be due to the anti-angiogenic effects of itraconazole. Other angiogenesis-dependent diseases such as macular degeneration and diabetic retinopathy may benefit from itraconazole, as well. (3–7)

5) Tsubamoto, Hiroshi, et al. “Repurposing itraconazole as an anticancer agent.” Oncology Letters 14.2 (2017): 1240-1246.

p 461 Itraconazole for Breast Cancer Inhibits Hedgehog (Hh) Pathway – Autophagic Cell Death

In 2017, Dr. Xiaoya Wang et al. reported in Cancer Letters that itraconazole has antiproliferative effects on breast cancer cell lines and the xenograft mouse model by inhibiting the Hedgehog pathway and inducing apoptosis and autophagic cell death. Dr. Wang and colleagues write:

In breast cancer cell lines, itraconazole-induced apoptosis by altering mitochondria
membrane potential, reducing BCL-2 expression and elevating caspase-3 activity.
Itraconazole also induced autophagic cell death via LC3-II expression upregulation …
Itraconazole treatment inhibited hedgehog pathway key molecular expression, such
as SHH and Gli1, resulting in promotion of apoptosis and autophagy. The anti-proliferation
effect of itraconazole-induced apoptosis and autophagy via hedgehog pathway
inhibition was confirmed … A human xenograft nude mouse model corroborated the
anti-breast cancer activity as evidenced by reduced tumor size, and increased tumor
tissue apoptosis and autophagy. (36)

36) Wang, Xiaoya, et al. “Anti-proliferation of breast cancer cells with itraconazole: Hedgehog pathway inhibition induces apoptosis and autophagic cell death.”
Cancer letters 385 (2017): 128-136.

p 462
Upregulation of Hh in Breast Cancer In 2017, Dr. Helen Oladapo et al. studied triple-
negative inflammatory breast cancer cells, reporting in Cancer Letter upregulation of the
Hedgehog (Hh/Gli) pathway: Activation of the Hedgehog [Hh] pathway
effector GLi1 is linked to tumorigenesis and invasiveness in a number of cancers, and
pharmacological targeting of GLi1 inhibits proliferation, tumor emboli formation
and in vivo tumor growth of inflammatory breast cancer cells. (39)

39) Oladapo, Helen O., et al. “Pharmacological targeting of GLI1 inhibits proliferation, tumor emboli formation and in vivo tumor growth of inflammatory breast cancer cells.” Cancer letters 411 (2017): 136-149.

In 2014, Drs. Hiroshi Tsubamoto et al. reported on their study of 13 patients with
Triple-Negative Breast Cancer with relapsed/ refractory metastatic disease who were treated
by adding itraconazole to conventional chemotherapy. Median overall survival was prolonged by 20 months when compared to similar studies using chemotherapy alone. (40)

40) Tsubamoto, Hiroshi, Takashi Sonoda, and Kayo Inoue. “Impact of itraconazole on the survival of heavily pre-treated patients with triple-negative breast cancer.” Anticancer research 34.7 (2014): 3839-3844.

p 462 Anti-Cancer Concentrations Similar to Antifungal

In Cell (2019), Dr. Riobo-Del Galdo et al. reported on itraconazole’s role as a hedgehog
inhibitor in breast cancer, writing that anticancer activity occurs at concentrations similar to
those used for anti-fungal treatment:

its inhibitory effect on Hh signaling is distinct and appears to be through prevention
of ciliary accumulation of SMO [smoothened protein]. When administered
systemically in mice, itraconazole suppressed the growth of medulloblastoma
and reduced Hh activation markers at similar concentrations than required for its
antifungal activity. (41)

41) Galdo, Riobo-Del, et al. “Role of Hedgehog Signaling in Breast Cancer: Pathogenesis and Therapeutics.” Cells 8.4 (2019): 375.

p 463 Itraconazole as Adjuvant for Breast Cancer

Dr. Galdo’s group also reports itraconazole-induced cell death in in vitro breast-cancer
cell lines, reducing angiogenesis both in vitro and in vivo, and synergizing with 5-FU, a
common chemotherapy agent. In concluding, Dr. Galdo et al. suggest itraconazole is a drug
of choice for use as adjuvant in breast cancer treatment:

The low cost of itraconazole and wellknown safety profile makes it a possible
drug of choice for use as an adjuvant in cancer treatment in developing countries
or areas of socioeconomic disadvantage. (41)

41) Galdo, Riobo-Del, et al. “Role of Hedgehog Signaling in Breast Cancer: Pathogenesis and Therapeutics.” Cells 8.4 (2019): 375.

p 480 Fenofibrate Breast cancer (50–52)

50) Li, Ting, et al. “Fenofibrate induces apoptosis of triple-negative breast cancer cells via activation of NF-KappaB pathway.” BMC cancer 14.1 (2014): 96.

51) Sun, Jianguo, et al. “Fenofibrate potentiates chemosensitivity to human breast cancer cells by modulating apoptosis via aKT/NF-κB pathway.” OncoTargets and therapy 12 (2019): 773.

52) Nguyen, Chi Huu, et al. “Fenofibrate inhibits tumour intravasation by several independent mechanisms in a 3-dimensional co-culture model.” International journal
of oncology 50.5 (2017): 1879-1888

p 485 Quercetin Inhibits FASN

99) Sultan, Ahmed S., et al. “Quercetin induces apoptosis in triple-negative breast cancer cells via inhibiting fatty acid synthase and β-catenin.” Int. J. Clin. Exp.
Pathol 10.1 (2017): 156-172.

p 490 Celecoxib Inhibits Wnt Pathway

In 2017, Dr. Chaolin Huang et al. studied the effect of celecoxib on breast CSCs, finding downregulation of the Wnt pathway. Dr. Huang’s group found that celecoxib decreases both
Beta-Catenin, the main Wnt pathway protein, as well as messenger RNA (mRNA) expression levels of Wnt pathway target genes, which produce the downstream proteins cyclin-D1 and C-Myc. Their excellent study demonstrated the potent anti-CSC properties of celecoxib. (54)

54) Huang, Chaolin, et al. “Celecoxib targets breast cancer stem cells by inhibiting the synthesis of prostaglandin E2 and down-regulating the Wnt pathway activity.” Oncotarget 8.70 (2017): 115254.

p 491 Celecoxib – Anti-Cancer Stem Cell Agent

In 2019, Dr. Jerry Harb et al. discussed cancer treatment with Wnt pathway signaling inhibitors. Celecoxib and pyrvinium (discussed in its own chapter 12) were prominently mentioned.
Dr. Harb’s group found celecoxib suppressed breast CSCs and synergized with chemotherapy agents with “dramatically increased sensitivity.” They summarized the anti-CSC activity of celecoxib in breast and colon cancer cell studies, writing:

Celecoxib has been shown to suppress mammary cancer stem cell renewal,
enhance responsiveness to chemotherapeutic agents, impede epithelial
to mesenchymal transition [EMT], and mitigate metastatic potential in MCF-7 and
MDA-MB-231 [breast cancer] cells by inhibiting prostaglandin E2 [the COX-2 pathway]
and promoting Β-catenin degradation [the Wnt pathway] … combination use of both
celecoxib and conventional chemotherapeutic drugs dramatically increased the
chemo-sensitivity of breast cancer cells … (56)

56) Harb, Jerry, Pen-Jen Lin, and Jijun Hao. “Recent development of Wnt signaling pathway inhibitors for cancer therapeutics.” Current oncology reports 21.2 (2019): 12.

p 492 Tumor Micro-Environment (TME) COX-2 in TAMS

In a 2015, Dr. Hongzhong Li studied breast cancer cells and the TME, finding tumor-associated macrophages (TAMS) express COX-2 protein. This stimulates the cancer cells in a positive-feedback loop between TAMs and cancer cells. The secreted COX-2 protein stimulates cancer proliferation and invasive behavior. In addition, COX-2 protein increases the anti-apoptotic protein BCL-2 and the drug-resistant protein P-gp. Dr. Li writes:

65) Li, Hongzhong, et al. “Cyclooxygenase-2 in tumor-associated macrophages promotes breast cancer cell survival by triggering a positive-feedback loop between macrophages and cancer cells.” Oncotarget 6.30 (2015): 29637.

p 497 Celecoxib
• Breast cancer (23)(37)(65)(76)(113–
116)(127–135)

23) Kundu, Namita, and Amy M. Fulton. “Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease in a murine model of breast cancer.” Cancer research 62.8 (2002): 2343-2346.

37) Glynn, Sharon A., et al. “COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer.” BMC cancer
10.1 (2010): 626.

65) Li, Hongzhong, et al. “Cyclooxygenase-2 in tumor-associated macrophages promotes breast cancer cell survival by triggering a positive-feedback loop between macrophages and cancer cells.” Oncotarget 6.30 (2015): 29637.

76) Zatelli, Maria Chiara, et al. “Cyclooxygenase-2 inhibitors prevent the development of chemoresistance phenotype in a breast cancer cell line by inhibiting glycoprotein p-170 expression.” Endocrine-related cancer 14.4 (2007): 1029-1038.

113) Wang, Guanying, et al. “Celecoxib induced apoptosis against different breast cancer cell lines by down-regulated NF-κB pathway.” Biochemical and
biophysical research communications 490.3 (2017):
969-976.
114) Regulski, Miłosz, et al. “COX-2 inhibitors: a novel strategy in the management of breast cancer.” Drug discovery today 21.4 (2016): 598-615.

115) Friedrich, Michael, et al. “Effects of combined treatment with vitamin D and COX2 inhibitors on breast cancer cell lines.” Anticancer research 38.2
(2018): 1201-1207.

116) Li, Jieqing, et al. “Celecoxib in breast cancer prevention and therapy.” Cancer management and research 10 (2018): 4653.

127) Shaashua, Lee, et al. “Perioperative COX-2 and β-adrenergic blockade improves metastatic biomarkers in breast cancer patients in a phase-II randomized trial.” Clinical Cancer Research 23.16 (2017): 4651-4661.

128) Haldar, Rita, et al. “Perioperative inhibition of β-adrenergic and COX2 signaling in a clinical trial in breast cancer patients improves tumor Ki-67 expression, serum cytokine levels, and PBMCs transcriptome.” Brain, behavior, and immunity 73 (2018):
294-309.

129) Xie, WanYing, et al. “βblockers inhibit the viability of breast cancer cells by regulating the ERK/COX2 signaling pathway and the drug response is affected
by ADRB2 singlenucleotide polymorphisms.” Oncology reports 41.1 (2019): 341-350.

130) Regulski, Miłosz, et al. “COX-2 inhibitors: a novel strategy in the management of breast cancer.” Drug discovery today 21.4 (2016): 598-615.

131) Majumder, Mousumi, et al. “COX‐2 induces breast cancer stem cells via EP4/PI3K/AKT/NOTCH/WNT axis.” Stem Cells 34.9 (2016): 2290-2305.

132) Xu, Han, et al. “CXCR2 promotes breast cancer metastasis and chemoresistance via suppression of AKT1 and activation of COX2.” Cancer letters 412 (2018): 69-80.

133) Li, Bailong, et al. “miR-221/222 promote cancer stem-like cell properties and tumor growth of breast cancer via targeting PTEN and sustained Akt/NF-κB/
COX-2 activation.” Chemico-biological interactions 277 (2017): 33-42.

134) Krishnamachary, Balaji, et al. “Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.” Oncotarget 8.11 (2017):17981.

135) Xu, Feng, et al. “Clinicopathological and prognostic significance of COX-2 immunohistochemical expression in breast cancer: a meta-analysis.” Oncotarget 8.4
(2017): 6003.

Quercetin

66) Duo, Jian, et al. “Quercetin inhibits human breast cancer cell proliferation and induces apoptosis via Bcl-2 and Bax regulation.” Molecular medicine reports 5.6 (2012): 1453-1456.

DIM

69) Hong, Chibo, Gary L. Firestone, and Leonard F. Bjeldanes. “Bcl-2 family-mediated apoptotic effects of 3, 3′-diindolylmethane (DIM) in human breast cancer cells.” Biochemical pharmacology 63.6 (2002): 1085-1097.

p 510 Sulindac as Cancer Stem Cell Agent

In 2019, Dr. F. Hossain et al. found sulindac targets breast CSCs as a Notch inhibitor (a
CSC pathway). (61) Sulindac also serves as an immune modulator in breast cancer models.
(62–65) In addition, Sulindac suppresses Βeta-Catenin expression, a major signaling protein
for the Wnt pathway, a CSC pathway. (66–68)

62) Hossain, Fokhrul, et al. “Abstract P5-04-19:Sulindac sulfide as a non-immune suppressive gamma secretase modifier to target triple negative breast cancer.”
(2020): P5-04.

63) Yin, Tao, et al. “Sulindac, a non-steroidal anti-inflammatory drug, mediates breast cancer inhibition as an immune modulator.” Scientific reports 6 (2016):
19534.

64) McDonell, Shannon B., et al. “Sulindac reverses an immunosuppressive tumor microenvironment associated with obesity-driven metastatic mammary tumors
in mice.” (2019): 2815-2815.

65) Sui, He‑Huan, et al. “Effects of sulindac sulfide on proliferation and apoptosis of human breast cancer cell.” Oncology letters 15.5 (2018): 7981-7986

Dipyridamole p 516 Breast Cancer Patients

A 2013 study by Dr. Daniela Spano et al. showed that DP prevents progression of triple-
negative breast cancer in a mouse xenograft model.

Low dose dipyridamole significantly reduced primary tumor growth and metas
tasis … while high dose resulted in an almost a total reduction in primary tumor
… Dipyridamole had significant effects on Wnt, ERK1/2-MAPK and NF-kB pathways in
both animal models. Moreover, dipyridamole significantly decreased the infiltration
of tumor-associated macrophages and myeloid-derived suppressor cells in primary
tumors (p < 0.005), and the inflammatory cytokines. (15)

15) Spano, Daniela, et al. “Dipyridamole prevents triple- negative breast-cancer progression.” Clinical & experimental metastasis 30.1 (2013): 47-68.

In 2013, Dr. Chunmei Wang used a transgenic mouse model to study breast cancer,
showing dipyridamole was preventive for primary tumors and metastatic lesions, such as
bone metastases. (16)

16) Wang, Chunmei, et al. “Chemoprevention activity of dipyridamole in the MMTV-PyMT transgenic mouse model of breast cancer.” Cancer Prevention Research 6.5 (2013): 437-447.

p 518 Histamine as Autocrine Growth Factor

Histamine, an intracellular messenger that promotes platelet aggregation, is also an autocrine growth factor for cancer, and histamine antagonists have anti-cancer effects. (23–27)
In 1995, Dr. Cricco studied histamine as a growth factor for breast cancer in a mouse
model, concluding:

In an animal model of experimental breast carcinomas, endogenous histamine was
critical for cell proliferation…. A major effect of histamine is stimulation of cancer
cell growth by activating H2 membrane receptors on the cancer cell. (28)
Histamine is discussed in more detail in chapter 28 on cimetidine as anti-cancer, histamine-
blocking drug. Loratidine is also an antihistamine, discussed in chapter 33.

p 524
Zolendronic Acid for Breast Cancer Bone Metastasis

In 2013, Drs. C. Riganti and M. Massaia noted that the bisphosphonate family of
drugs for osteoporosis (such as Zoledronic acid) also inhibit the mevalonate pathway,
similar to simvastatin, causing depletion of intracellular pools of isoprenoids. The bisphosphonates: cause the deprivation of intracellular
isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate
(GGPP)… Isoprenoids are critical for the post-translational modification of proteins
that are essential for both cell proliferation and differentiation. (114)

Zolendronic Acid and Doxycycline Synergy

Drs. Reganti and Massaia think the bisphosphonate drugs are superior to the statins for
this purpose because, in addition to blocking mevalonate, they override multidrug resistance
(MDR) and restore immunogenic cell death (ICD) mechanisms. This is fortuitous because
Zoledronic acid is commonly given IV monthly as a bone agent for breast cancer bone involvement (bone metastasis). Without realizing it, oncologists using Zolendronic acid have been blocking the mevalonate pathway, as statins do, thus having an anti-CSC effect. (114)

114) Riganti, C., and M. Massaia. “Inhibition of the mevalonate pathway to override chemoresistance and promote immunogenic cell death in cancer cells: hitting two birds with one stone. ONCOIMMUNOLOGY (2013) 2.” (2019).

In this case, these patients benefit by adding doxycycline, which has synergy with Zoledronic
acid. There may be a combined benefit with use of all three—statins, Zoledronic acid, and doxycycline—in breast cancer. (115–116)

115) Duivenvoorden, W. C. M., et al. “Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer.” British journal of cancer 96.10 (2007): 1526.

116) Göbel, Andy, et al. “Combined inhibition of the mevalonate pathway with statins and zoledronic acid potentiates their anti-tumor effects in human breast cancer cells.” Cancer letters 375.1 (2016): 162-171.

119) Wang, Tingting, et al. “Simvastatin-induced breast cancer cell death and deactivation of PI3K/Akt and MAPK/ERK signalling are reversed by metabolic products of the mevalonate pathway.” Oncotarget 7.3 (2016): 2532.

120) Sethunath, Vidyalakshmi, et al. “Targeting the mevalonate pathway to overcome acquired anti-HER2 treatment resistance in breast cancer.” Molecular Cancer Research 17.11 (2019): 2318-2330.

Tocotrienol p 535 • Breast Cancer (23-32) (42-43) (74)

23) Algayadh, Ibrahim G., and Paul William Sylvester.  “Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells.” (2017): 1053-1053.

24) Sylvester, Paul William, and Venkateshwara Dronamraju. “γ-Tocotrienol reversal of the Warburg effect in breast cancer cells is associated with 5′-AMPactivated
kinase activation.” Molecular Nutrition.  Academic Press, 2020. 387-407.

25) Dronamraju, Venkateshwara, et al. “γ-Tocotrienol suppression of the Warburg effect is mediated by AMPK activation in human breast cancer cells.”Nutrition and cancer 71.7 (2019): 1214-1228.

26) Parajuli, Parash, Roshan Vijay Tiwari, and Paul William Sylvester. “Anticancer effects of γ-Tocotrienol are associated with a suppression in aerobic glycolysis.” Biological and Pharmaceutical Bulletin 38.9 (2015): 1352-1360.

27) Parajuli, P., R. V. Tiwari, and P. W. Sylvester. “Antiproliferative effects of γ‐tocotrienol are associated with suppression of c‐Myc expression in mammary tumour cells.” Cell proliferation 48.4 (2015): 421-435.

28) Ahmed, Rayan, and Paul W. Sylvester. “γ-Tocotrienol Reversal of Epithelial-to-Mesenchymal Transition in Human Breast Cancer Cells is Mediated through
a Suppression of Canonical Wnt and Hedgehog Signaling.” Vitamin E in Health and Disease (2018): 83.

29) Ahmed, R. A., O. A. Alawin, and P. W. Sylvester. “γ‐Tocotrienol reversal of epithelial‐to‐mesenchymal transition in human breast cancer cells is associated with inhibition of canonical Wnt signalling.” Cell proliferation 49.4 (2016): 460-470.

30) Alawin, Osama A., et al. “Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.” The Journal of nutritional biochemistry 27 (2016): 266-277.

31) Alawin, Osama A., et al. “γ-Tocotrienol-induced disruption of lipid rafts in human breast cancer cells is associated with a reduction in exosome heregulin
content.” The Journal of Nutritional Biochemistry 48 (2017): 83-93.

32) Sylvester, Paul W. “The Role of Lipid Rafts in Mediating the Anticancer Effects of γ-Tocotrienol.” Vitamin E in Human Health. Humana Press, Cham,
2019. 125-140.

42) Comitato, Raffaella, et al. “Tocotrienols activity in MCF‐7 breast cancer cells: Involvement of ERβ signal transduction.” Molecular nutrition & food research
54.5 (2010): 669-678.

43) Comitato, Raffaella, et al. “A novel mechanism
of natural vitamin E tocotrienol activity: involvement
of ERβ signal transduction.” American Journal
of Physiology-Endocrinology and Metabolism 297.2
(2009): E427-E437.

74) Loganathan, R., et al. “Tocotrienols promote
apoptosis in human breast cancer cells by inducing
poly (ADP‐ribose) polymerase cleavage and inhibiting
nuclear factor kappa‐B activity.” Cell proliferation 46.2
(2013): 203-213.

p 535 Synergy with Pterostilbene in Breast Cancer (23) (38)

23) Algayadh, Ibrahim G., and Paul William Sylvester. “Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells.” (2017): 1053-1053.

p 536

In 2020, Dr. Paul Sylvester studied anticancer
effects of Gamma Tocotrienol on breast cancer
cell models finding reversal of Warburg Effect
(inhibition of GLYCOLYSIS) with cancer cell
reduction in glucose consumption, and reduction
in expression of glycolytic enzymes. Dr.
Sylvester writes:

these findings indicate that gamma-tocotrienol-
induced reversal of the Warburg
effect in breast cancer cells is directly associated
with an increase in AMPK activation.
(24-26)
Note: AMPK activation inhibits the PI3K/Akt/
mTOR pathway, thus suppressing EGF signaling
(as seen below).

24) Sylvester, Paul William, and Venkateshwara
Dronamraju. “γ-Tocotrienol reversal of the Warburg
effect in breast cancer cells is associated with 5′-AMPactivated
kinase activation.” Molecular Nutrition.
Academic Press, 2020. 387-407.

25) Dronamraju, Venkateshwara, et al. “γ-Tocotrienol
suppression of the Warburg effect is mediated
by AMPK activation in human breast cancer cells.”
Nutrition and cancer 71.7 (2019): 1214-1228.

26) Parajuli, Parash, Roshan Vijay Tiwari, and Paul
William Sylvester. “Anticancer effects of γ-Tocotrienol
are associated with a suppression in aerobic glycolysis.”
Biological and Pharmaceutical Bulletin 38.9
(2015): 1352-1360.

p 536 Tocotrienol as Wnt, Hedgehog Pathway Inhibitors

In 2018, Dr. Rayan Ahmed studied effects of
tocotrienol in a breast cancer cell model finding
reversal of EMT was caused by inhibition of
Wnt and Hedgehog pathways (CSC pathways).

Dr. Ahmed writes:
Specifically, tocotrienols have been found
to suppress EGF-[Epithelial Growth Factor]
dependent mitogenic [Pro-Cancer] signaling…….
by significantly inhibiting activity
of the…[PI3K/Akt] pathway. (28-29)

28) Ahmed, Rayan, and Paul W. Sylvester. “γ-Tocotrienol
Reversal of Epithelial-to-Mesenchymal Transition
in Human Breast Cancer Cells is Mediated through
a Suppression of Canonical Wnt and Hedgehog
Signaling.” Vitamin E in Health and Disease (2018): 83.

29) Ahmed, R. A., O. A. Alawin, and P. W. Sylvester.
“γ‐Tocotrienol reversal of epithelial‐to‐mesenchymal
transition in human breast cancer cells is associated
with inhibition of canonical Wnt signalling.” Cell proliferation
49.4 (2016): 460-470.

p 536 Tocotrienols Disrupt Lipid Rafts

Lipid rafts are involved in various cell functions
such as receptor signaling, endocytosis
(membrane transport), and the actin cytoskeleton.
In 2019, Dr. Paul Sylvester studied anticancer
effects of Gamma Tocotrienols in a breast
cancer cell model finding anti-cancer effects
are associated with accumulation in lipid rafts
in the cancer cell outer membrane with interference
with the Tyrosine Kinase receptor,
HERS2 (human epithelial growth factor receptor
2). These are key receptors for transmitting
growth signals into the cancer cell. (30-33)

of γ-tocotrienol are associated with lipid raft disruption
in HER2-positive human breast cancer cells.” The
Journal of nutritional biochemistry 27 (2016): 266-277.
31) Alawin, Osama A., et al. “γ-Tocotrienol-induced
disruption of lipid rafts in human breast cancer cells
is associated with a reduction in exosome heregulin
content.” The Journal of Nutritional Biochemistry 48
(2017): 83-93.
32) Sylvester, Paul W. “The Role of Lipid Rafts in
Mediating the Anticancer Effects of γ-Tocotrienol.”
Vitamin E in Human Health. Humana Press, Cham,
2019. 125-140.

p 537

In 2010, Dr. Comitato studied tocotrienols
in breast cancer cell models proposing
the anti-cancer (apoptotic) activity is
due to ER-Beta signaling. Dr. Comitato found
Tocotrienol (TTRF= Tocotrienol rich fraction)
treatment of the breast cancer cells increased
ER-Beta nuclear translocation, and profoundly
inhibited ER-Alpha expression (459 fold) with
complete disappearance of ER-Alpha from the
cancer cell nucleus. Note: TTRF contains both
Delta and Gamma Isoforms. (42-43)

42) Comitato, Raffaella, et al. “Tocotrienols activity in
MCF‐7 breast cancer cells: Involvement of ERβ signal
transduction.” Molecular nutrition & food research
54.5 (2010): 669-678.

43) Comitato, Raffaella, et al. “A novel mechanism
of natural vitamin E tocotrienol activity: involvement
of ERβ signal transduction.” American Journal
of Physiology-Endocrinology and Metabolism 297.2
(2009): E427-E437.

p 555 Auranofin AF

Recent
studies show triple combination of AF, Vitamin C
(ascorbate) and Vitamin K (Menadione) synergistic
against TNBC (triple negative breast cancer) in
vitro and in vivo. Similarly, the combination of
Auranofin and anti-PD-L1 checkpoint inhibitor drug
is synergistically effective for TNBC.

27) Raninga, Prahlad V., et al. “Therapeutic cooperation
between auranofin, a thioredoxin reductase
inhibitor and anti‐PD‐L1 antibody for treatment of
triple‐negative breast cancer.” International Journal
of Cancer 146.1 (2020): 123-136.

29) Bajor, Malgorzata, et al. “Triple Combination
of Ascorbate, Menadione and the Inhibition of
Peroxiredoxin-1 Produces Synergistic Cytotoxic Effects
in Triple-Negative Breast Cancer Cells.” Antioxidants
9.4 (2020): 320.

36) Jiao, Lin, et al. “Betulinic acid suppresses breast
cancer aerobic glycolysis via caveolin-1/NF-κB/c-Myc
pathway.” Biochemical pharmacology 161 (2019):
149-162

54) Stenkvist, B. J. Ö. R. N. “Is digitalis a therapy for
breast carcinoma?” Oncology reports 6.3 (1999):
493-499.

63) Coelho, Raquel Guimarães, et al. “Clotrimazole
disrupts glycolysis in human breast cancer without
affecting non-tumoral tissues.” Molecular genetics and
metabolism 103.4 (2011): 394-398.
64) Furtado, Cristiane M., et al. “Clotrimazole preferentially
inhibits human breast cancer cell proliferation,
viability and glycolysis.” PloS one 7.2 (2012).
65) Meira, Débora Dummer, et al. “Clotrimazole
decreases human breast cancer cells viability through
alterations in cytoskeleton-associated glycolytic
enzymes.” Molecular genetics and metabolism 84.4
(2005): 354-362.

82) Wei, Ran, et al. “Suppressing glucose metabolism
with epigallocatechin-3-gallate (EGCG) reduces breast
cancer cell growth in preclinical models.” Food & function
9.11 (2018): 5682-5696

85) Ahmed, R. A., O. A. Alawin, and P. W. Sylvester.
“γ‐Tocotrienol reversal of epithelial‐to‐mesenchymal
transition in human breast cancer cells is associated
with inhibition of canonical Wnt signalling.” Cell
Proliferation 49.4 (2016): 460-470.

107) Shrivastava, Ashutosh, et al. “Molecular iodine
induces caspase-independent apoptosis in human
breast carcinoma cells involving the mitochondria-mediated
pathway.” Journal of Biological Chemistry
281.28 (2006): 19762-19771.
108) Aceves, Carmen, et al. “Antineoplastic effect of
iodine in mammary cancer: participation of 6-iodolactone
(6-IL) and peroxisome proliferator-activated
receptors (PPAR).” Molecular Cancer 8.1 (2009): 33.
109) Ayre, S. G., D. Perez Garcia y Bellon, and D. Perez
Garcia Jr. “Insulin potentiation therapy: a new concept
in the management of chronic degenerative disease.”
Medical Hypotheses 20.2 (1986): 199-210.

140) Jia, Lijun, et al. “Quercetin suppresses the mobility
of breast cancer by suppressing glycolysis through
Akt-mTOR pathway mediated autophagy induction.”
Life sciences 208 (2018): 123-130.

157) Dronamraju, Venkateshwara, et al. “γ-Tocotrienol
suppression of the Warburg effect is mediated
by AMPK activation in human breast cancer cells.”
Nutrition and cancer 71.7 (2019): 1214-1228.

196) Caffa, Irene, et al. “Fasting-mimicking diet and
hormone therapy induce breast cancer regression.”
Nature 583.7817 (2020): 620-624.

Tai, Yu, et al. “Effect of Polygonatum odoratum extract on human breast cancer MDA-MB-231 cell proliferation and apoptosis.” Experimental and therapeutic medicine 12.4 (2016): 2681-2687.

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Polygonatum odoratum

Tai, Yu, et al. “Effect of Polygonatum odoratum extract on human breast cancer MDA-MB-231 cell proliferation and apoptosis.” Experimental and therapeutic medicine 12.4 (2016): 2681-2687.

 

Last updated on by Jeffrey Dach MD

About Jeffrey Dach MD

Medical Director of TrueMedMD, a Clinic in Davie Florida specializing in Bioidentical Hormones and Natural thyroid. Office address 7450 Griffin Road Suite 190, Davie, Florida 33314 telephone 954-792-4663