Targeting Cancer Stem Cells with NonToxic Therapies

Cancer Ste Cell Conference 2012 UK CambridgeTargeting Cancer Stem Cells with NonToxic Therapies

by Jeffrey Dach MD

When Jim was 36 year old, he was diagnosed with acute myeloblastic leukemia with symptoms of fatigue, anemia, and extremely high white count on his blood panel. Over three years, Jim received seven chemotherapy treatments, each one giving him a temporary remission shorter than the last one.  Eventually the chemotherapy was no longer effective, the cancer cells became resistant to chemotherapy.  After exhausting all of the usual treatments, the doctors offered Jim allogeneic hematopoetic stem cell transplantation, a procedure which obliterates the patient’s bone marrow with high dose chemotherapy.  The patient is then “rescued” with donor bone marrow stem cells from a family member or donor bank.  This procedure is associated with considerable morbidity and mortality from infection and graft vs. host disease.   The high dose chemotherapy produces permanent, irreversible infertility.  Jim had the procedure and is alive and doing well three years later.  Upper left image poster courtesy of Cancer Stem Cell Meeting 2012 Cambridge Cancer Stem Cell Symposium.

What can we learn from Jim’s story?

The first lesson is that hematologic malignancies, and all other cancers for that matter, tend to  come back (relapse) even after complete remission induced by cytotoxic chemotherapy.  For many years, the reason for this cancer recurrence was unknown.  However, in the past decade, the reason has been discovered.

Two Populations of Cancer Cells

There are actually two distinct populations of cancer cells in the cancer victim.  The bulky tumor masses are caused by rapidly replicating cancer cells, highly sensitive to killing effects of chemotherapy.  However, a  subset of these cancer cells lurk within the tumor mass, hiding in a dormant state, not actively replicating.  These are the cancer “stem cells” which are resistant to conventional cytotoxic chemotherapy, biding their time, waiting for a future opportunity to reactivate and seed metastatic cancer throughout the  body.

Cancer Stem Cell ConferenceAbove image courtesy of Case Western Reserve Cancer Stem Cell Conference.

This explains Jim’s futile experience with chemotherapy which kills off the rapidly replicating leukemia cells, while leaving the dormant leukemia “stem cells” unharmed.  This is also true for most other solid organ cancers.  The solution, of course, is to address the dormant cancer “stem cell”, at the same time as the rapidly replicating cancer cells.(1,1A)

Unfortunately,  modern day oncology/ hematology doctors are oblivious to this important lesson, and continue to mindlessly treat their patients with the same chemotherapy protocols as before.  Even though this information is in their own oncology/hematology journals,  they cannot bring themselves to acknowledge it.

Sadly, since their doctors will not help them, patients are left to fend for themselves.  In order to get off the chemotherapy merri-go-round, the patient must learn about non-toxic targeted therapies which kill cancer stem cells.  These are widely available.  As we will see below, some of these therapies are supplements at the health food store, and some are common drugs  requiring a doctor’s prescription.  With knowledge of nTargeting Cancer Stem Cells with NonToxic Therapieson-toxic targeted therapies,  a durable remission after the last chemotherapy treatment can be achieved.  Left Image Ovarian Cancer Stem Cells Courtesy of Yale News.

Many Natural Substance Target Cancer Stem Cells(12)

You might be surprised to know there are 3,000 plant species having anti-cancer activity.(1B)  In fact, there are hundreds of non-toxic natural substances that target cancer stem cells. (12)  There are so many to choose from, one must be selective, so we will discuss only a few of them below: (10-19)

curcumin jeffrey dach mdCurcumin

The food spice, Curcumin (tumeric) is widely available as a nutritional supplement in highly absorbance forms such as Curcumin Phytosome Meriva (r), CogumenLipospheric Curcumin. and the most bioavailable   Ultra-Cur .

Dr Huang reports in 2016 that “Curcumin Induces Apoptosis of Colorectal Cancer Stem Cells by Coupling with CD44 Marker”.

Dr Huang studied colorectal cancer stem cells, and found curcumin couples with the CD44 cancer stem cell membrane marker,  and “might have some blocking effect on the transport of glutamine into the cells, thus decreasing the glutamine content in the CD44+ cells and inducing apoptosis.”(2)

Dr Chung reported in 2015 that both “Curcumin and EGCG Epigallocatechin Gallate Inhibit the Cancer Stem Cells . (3) Dr Chung reported the cancer stem cell marker was denoted by the CD44+ protein.  The CD44+ cell population decreased following curcumin and EGCG (from Green Tea). (3)

Curcumin_Jeffrey Dach MDDr Hasanali in 2012 reported that Curcumin inhibits Nuclear Factor Kappa Beta activation and Downregulate Cyclin D1 in Mantle Cell Lymphoma, thereby inducing apoptosis. (4) Left image: molecular structure of curcumin courtesy of Wikimedia commons.

Dr Shishodia in 2005 reported that “Curcumin inhibits constitutive NF-κB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma.” (5)

The authors reported:

the expression of all NF-kappaB-regulated gene products were downregulated by Curcumin leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation.”

Dr Choudhuri reported in 2005 found that “Curcumin selectively induces apoptosis in cancer cells with deregulated cyclin D1 at G2 phase of cell cycle.”(6)  Normal cells were left unharmed. (6)

Drug inventors have been busy with new ideas to make curcumin more bioavailable and more effective, such as Curcumin nanodiscs. (7-9)   A massive body of basic science and medical studies on Curcumin shows the natural plant substance kills cancer stem cells while sparing normal cells as a non-toxic targeted therapy. (2-9)

Dr Peter Sordillo wrote an excellent summary of curcumin’s ability to kill cancer stem cells in ,” Curcumin and Cancer Stem Cells” AntiCancer Research 2015 (10)  Dr Sordillo reports that Curcumin inhibits release of cancer associated cytokines, IL6 and IL8, resulting in inhibition and suppression of cancer stem cells.   Both the WNT and Notch pathways are important for cancer stem cell survival.  Curcumin acts to inhibit and block multiple points along the Wnt pathway and Nctch pathways, downregulating β-catenin and the genes for VEGF, cyclin D1 and c-Myc.  Amazingly,  Curcumin has no deleterious effects on normal stem cells, and targets only the cancer stem cells.

The reason offered for this selectivity is:

1) Curcumin has much greater uptake by cancer cells compared to normal cells.

2) Curcumin affects the microenvironment around  Cancer Stem Cells by suppressing release of proinflammatory cytokines IL6 and IL8.

3) Curcumin induces Cancer Stem Cells to undergo cell differentiation into a mature cells which then undergo spontaneous or drug induced apoptosis.

4) Curcumin suppresses the Wnt pathway in Cancer Stem Cells with inhibition of β-catenin, yet has the opposite effect on neural stem cells as it stimulates neurogenesis. (10)(93)

Curcumin is highly lipophilic, and crosses the blood–brain barrier easily, and therefore suggested by Dr Sordillo as a “sensitizer” along  with conventional chemotherapy for treatment of Glioblastoma.(11B)

Curcumin and WNT/TCF Beta Cateninin Pathway Inhibition

Dr Park reported in 2005 curcumin’s mechanism to inhibit beta catenin/TCF signalling in various cancer cell lines.   Dr Park found no change in cytosolic or membranous Beta-Catenin in curcumin treated cancer cells.  Rather, Dr Park found decreased Beta-Catenin and TCF-4 in the nucleus of the cancer cells.(93)

Curcumin and Chemo Resistant AML Stem Cells

Dr Jia Rao studied the anti-cancer effects of  Curcumin in 9 separate acute myelogenous leukemia cell lines. (11C)   Dr Rao reports in 2011 that chemotherapy resistance in acute myelogenous leukemia may be due to upregulation of the anti-apoptotic protein Bcl-2 in CD34+ cancer stem cells.  Dr Rao found that treatment of the cancer stem cells with Curcumin reduces the Bcl-2 protein levels, leading to apoptosis (programmed cell death)  while showing no ill effects on normal cells.  “Curcumin demonstrated no major toxicities in phase I and II clinical studies at doses of up to 8 g/day”.(11C)

Berberine Synergy with CurcuOregon_Grape_Berberine_Jeffrey_Dach_MDmin

Berberine, also a WNT pathway inhibitor, was found synergistic when combined with Curcumin. (11A)

Berberine

Also called the Berberine (Oregon Grape)  Berberine is a natural WNT pathway inhibitor (48-51)  Berberine is actually patented as a targeted cancer stem cell therapy.(51)

See: Hsieh, Hsiu-Mei, et al. “Berberine for inhibiting cancer stem cell” U.S. Patent Application No. 14/790,154.

640px-Berberine_acsv.svgBerberine is a commonly used herbal extract with many health benefits.  Anti-cancer activity of Berberine is through inhibition of the WNT/B-Catenin pathway (48-50).  Berberine alters mitochondrial membrane potential, promotes release of cytochrome C, and triggers apoptosis in the cancer cell.(50)  Left image: chemical structure of Berberine courtesy of wikimedia commons.

In 2013, Dr Liu  studied anticancer effects of Berberine in a leukemia cell line lacking p53 finding Berberine induced apoptotic cell death via suppression of the anti-apoptotic protein (XIAP) which then inhibited MDM2 expression.  This also increased the sensitivity of leukemia cells to doxorubicin-induced apoptosis.(48A)

Milk Thistle

Milk Thistle (Silybin-phytosome – Siliphos) is a widely used herbal extract which inhibits the WNT/B-Catenin pathway. (52-54)  A number of clinical trials have shown benefits and promising effects against cancer stem cells. (52-54)

Sulforaphane_jeffrey dach _brocolliSulforaphane

Sulforaphane, a Brocolli Extract, down regulates glutathione. increases ROS, and Inhibits WNT signaling in the cancer cell (13-19)(95-96) Sulforaphane induced cell cycle arrest and apoptosis (cell death)  in ovarian cancer cell lines. (97)

Dr Kallifatidis et al studied Sulforaphane in a 2009 study of pancreatic cancer cells.(14)  The authors report sulforaphane “targets pancreatic tumour-initiating cells by NF-kappaB-induced antiapoptotic signalling”   The authors state:  “sulforaphane decreased the protein level of β-catenin by up to 85% in MCF7 and SUM159 cells; and the expression of cyclin D1, one of the Wnt/β-catenin target genes, declined by up to 77% as well.”(14)  “As a chemoprevention agent, sulforaphane possesses many advantages, such as high bioavailability and low toxicity.”

In a p53 depleted colon cancer cell  model, Dr Rudolph from the Czech Republic in 2011 found that sulforaphane induced lysosome and mitochondria-dependent cell death in colon cancer cells with deleted p53The effects were independent of P53 status.

SulforaphaneSulforaphane is a small molecule which easily crosses blood brain barrier producing anti-depressant and anti-anxiety effects in animal studies.(16)  Sulforaphane is neuroprotective and improves cognitive function after traumatic brain injury. The combination of Resveratrol(Pterostilbene) and Sulforaphane was effective against human glioblastoma in vitro cell cultures.  Sulforaphane is a HDAC (histone deacetylase inhibitor) and suppresses human prostate cancer in animal xenograft model.  Sulforaphane Anti-Cancer effects are independent of mutated P53 status.

Sulforaphane inhibited prostate cancer cells by down-regulating intracellular glutathione (GSH) and increasing ROS, causing mitochondrial apoptosis.(95-96)  In males followed after prostatectomy for prostate cancer, sulphoraphane reduced PSA doubling time in half, thus preventing “biochemical recurrence”. (98)

Pterostilbene_Source_naturalsPterostilbene Synergy With Chloroquin

Pterostilbene is a natural analog of Resveratrol found in blueberries, which is more bioavailable and more potent than resveratrol in activity against many different cancer cell types. (54-55)

Dr Chi-Hao Wu reported in 2015 that pterostilbene targets breast cancer stem cells.(54)  Using  breast cancer cells (MCF-7)  in vitro, his study showed that Pterotilbene selectively killed Breast Cancer Stem Cells which express the CD44 surface antigen.   In addition, Pterostilbene increased the sensitivity of Breast Cancer Stem Cells  to killing effects of chemotherapy.  The underlying mechanism of Pterostilbene was degradation of β-catenin,  thus inhibiting expression of cancer growth factors C-Myc and Cyclin D1.(54)

Dr Yang reported in 2013 that “Pterostilbene exerts antitumor activity”  in adenocarcinoma of the lung, both in vitro and in vivo with mouse tumor xenografts. (55)  Dr Yang showed pterostilbene significantly decreased the Mitochondrial Membrane Potential and increased Reactive Oxygen Species (ROS) with depletion of intracellular glutathione i the cancer cells.  Expression of apoptosis pathway proteins BAX and Cytochrome C were upregulated.(55)

Dr  Papandreou reported in 2015 on their study in which 1,726 small molecules were screened for activation of the Endoplamic Reticulum (ER) stress response Gene.   They said “plant stilbenes pterostilbene and piceatannol were the most potent inducers of ER stress from this group. (56)  Dr Papandreou then determined by molecular analysis that Pterostilbene blocks Wnt/B-Catenin pathway and also induces autophagy in acute lymphoblastic leukemia cells.   The authors found that:

combining pterostilbene (to induce ER stress) with chloroquine (to inhibit autophagy) leads to significant cellular toxicity in cells from aggressive acute lymphoblastic leukemia.“(56)

Parthenolide FeverfewParthenolide – Feverfew (41-43)

Parthenolide is the major active component in Feverfew, an herbal medicine used for centuries for migraine headache, and more recently for rheumatoid arthritis.   Left image : Feverew courtesy of wikimedia commons.

Dr Monica Guzman reported in 2005 Blood , that Parthenolide is a potent inhibitor of NFKB (Nuclear Factor Kappa Beta) and:

 “induces robust apoptosis in primary human acute melogenous leukemia (AML) cells while sparing normal hematopoietic cells. Furthermore, partheolide (PTL) also preferentially targets AML progenitor and stem cell populations. Notably, in comparison to the standard chemotherapy drug cytosine arabinoside (Ara-C), PTL is much more specific to leukemia cells. The molecular mechanism of PTL-mediated apoptosis is strongly associated with inhibition of nuclear factor κ B (NF-κB), proapoptotic activation of p53, and increased reactive oxygen species (ROS).” (41)

Dr Shanshan Pei reported in 2013 that Parthenolide targets the aberrant, upregulated glutathione metabolism in leukemia cancer stem cells by inducing almost complete glutatione depletion and severe cell death.(42)  At the same time, there was only limited and transient perturbation in normal hematopoetic cells.

parthenolide jeffrey dach mdDr Shanshan Pei noticed that Parthenolide contains an active α,β-unsaturated-γ-lactone group (see left image: From Fig. 3B, red ellipse denotes active area) that readily reacts with free thiol group of glutathione.(42)

In addition, the authors tested the combined effects of Pathenolide with commonly used chemotherapy drugs Ara-C and Idarubicin, finding augmented synergistic effects with anti-cancer activity substantially increased.

Repurposed Drugs Effective Against Cancer Stem Cells:

Azulfidine Pfizer Sulfasalazine Jeffrey Dach MDSulfasalazine (Azulfidine)

Sulfasalazine (Azulfidine) is an old rheumatology drug which targets cancer stem cells by inhibiting the xCT signal pathway for cystine uptake.  By starving the cancer stem cell of cystine,  intracellular glutathione is depleted, rendering the cancer cell defenseless to oxidative stress, ultimately resulting in cell death from oxidative damage.

Dr Shitara’s group reported at the 2014 ASCO Annual Meeting on the “effect of sulfasalazine (SSZ) on cancer stem-like cells (CSCs) via inhibiting the xCT signal pathway” .  Dr Shitera reported that the CD44 surface adhesion molecule is expressed in cancer stem-like cells.  They found cancer stem cells have a variant (CD44v) which interacts with xCT, the glutamate-cystine transporter and maintains high levels of the intracellular reduced glutathione (GSH). They found that “Sulfasalazine acts as an xCT inhibitor which suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in vivo study.”(20)

Dr Ishimoto in a 2011 study also found  Sulfasalazine inhibited the xCTmembrane transporter of cystine uptake, thereby suppresses CD44-Dependent Tumor Growth in cancer stem cells in vivo.(21)  Dr Ishimoto states, ” The activity of xCT-mediated cystine uptake in cancer cells is highly associated with cell proliferation, chemoresistance, and tumor growth.“(21)

Many studies show sulfasalazine effective for other cancers as well, including head and neck cancer, breast, prostate, lung, pancreatic cancer and lymphoma (20-38) .   Sulfasalazine was effective against Mantle Cell lymphoma cells in vitro.(23)  Although considered safe as a long term drug treatment in rheumatology patients, sulfasalazine crosses the blood brain barrier and can result in CNS toxicity, so caution is advised. (29-30)  Sufasalazine is poorly absorbed, (3-12%) with a 5-10 hour half life.  Sulfasalazine is cleaved by colonic bacteria into sulfapyridine and 5-ASA, thought to be active in treatment of inflammatory bowel disease. (Klotz 1985)

Mefloquin Lariam Jeffrey Dach mdMefloquin was second most active after Ivermectin

Chloroquin and Mefloquin (Lariam) are old Anti-Malaria drugs – They inhibit autophagy and serve as Lysosomal disruptors.

Dr Sukhai screened a library of drugs for greatest activity against AML (Acute myelogenous Leukemia stem cells.   Dr Sukhai found Mefloquin was second most active after Ivermectin with an EC50 of less than 8 microM.(39)  Mefloquin was less toxic to normal cells.  (39)  Mefloquine specifically targets lysosomal function and accumulates in lysosomes of the malarial parasite.  Dr. Sukhai found that Mefloquine directly disrupted lysosomes of Acute Myelogenous Leukemia cells (and progenitor stem cells)  in a dose-dependent manner, as measured by release of cathepsins into the cytosol. (39) Normal hematopoietic cells were unharmed. (39)

Serum concentrations of mefloquine up to 5 μM have been reported in individuals receiving 250 mg weekly for malaria prophylaxis . Thus, antileukemia concentrations of mefloquine may be pharmacologically achievable.(39)

Combination of Artemisinin with Mefloquin synergistic

The most synergistic combinations from Dr Sukhai’s screening efforts was Mefloquin with the anti-malarial drug, artemisinin.  The two in combination synergistically increased ROS (reactive oxygen sspecies) production.(39)

“Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC50 ~0.5-0.7 µM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors”.(59)   Mefloquin was effective agains prostate cancer cell lines(63)

The mechanism of mefloquin (64) :Mefloquine caused an expansion of the lysosomal apparatus, earliest seen by 24 h and lasting for some 7 days.

(1) mefloquine is a lysosomotropic drug that accumulates in lysosomes;
(2) mefloquine impairs lipid degradation with ensuing accumulation of lipids in lysosomes; and
(3) lysosomal trapping explains the high volume distribution of mefloquine.(64)

Dr Sachlos  screened 590 well-established annotated compounds from the NIH Clinical Collection and Canadian Compound Collection, finding that Mefloquin induced differentiation of neoplastic hematopoetic PSCs (human stem cells)  while not affecting normal hPSCs.(human stem cells). (69)  Within the top ten candidates, mefloquine were found to possess EC50 values lower than the 10 µM target threshold,  with no effect on normal hematopoiesis.  Mefloquin as a cancer treatment was patented by the University of California in 2002. (70)

Mefloquine Neuro-Toxicity

Caution is advised before using Mefloquine as well as other Quinolones.  Although originally marketed as safe for malaria prophylaxis, recent studies show an alarming incidence of neurologic toxicity, which may be “clinically occult , and in some cases, irreversible. (Remington Nevin 2014).   

A possible mechanism of Mefloquin toxicity was elucidated by Dr Anthony Mawson, in his 2013 article, “Mefloquine use, psychosis, and violence: a retinoid toxicity hypothesis,”    Dr Mawson says:

“The use of mefloquine in the prevention and treatment of malaria has been increasingly linked to a broad range of neuropsychiatric effects, including depression, psychosis, and violence.  The symptoms of mefloquine toxicity may result from the spillage of stored retinoids from the damaged liver into the circulation and their transport to the gut and brain, causing the adverse neuropsychiatric and gastrointestinal symptoms as a function of an endogenous form of hypervitaminosis A.”

Anopheles_stephensi_malaria_chloroquinChloroquin

Chloroquin, used for more than 80 years as an anti-malarial drug and more recently as a drug for auto-immune disease, increases pH in Lysosomal compartments and inhibits autophagy in cancer cells (57-58) “Daily uptake of clinically acceptable doses (less than 10mg/kg) of Chloroquine in addition to chemo-radio-therapy increases the survival of glioblastoma patients”  Patients reeived oral chloroquine at 150 mg/d for 12 months beginning on postoperative day 5 or placebo. Median survival after surgery was 24 months for chloroquine-treated patients and 11 months for controls.(66)  Upper left image: malaria mosquito engorged with blood.Courtesy wikimedia commons.

Dr Choi reports in 2014 that Chloroquin is an effective cancer stem cell agent.(57) See: “Chloroquine eliminates cancer stem cells.”  Chloroquin was effective against pancreatic cancer cells/(60) and melanoma(61)  and breast cancer cell lines(62) via inhibition of autophagy.

“Chloroquin (CQ) may be one of the most effective and safe sensitizers for cancer therapies. Taken together, it appears that the efficacy of conventional cancer therapies can be dramatically enhanced if used in combination with CQ and its analogs.”(65)

heartgard_Dog_canine_ivermectinIvermectin (Stromectol)

Ivermectin (Stromectol) is a potent blocker of the WNT pathway at low doses.  Ivermectin is an “astonishingly safe” anti-helminthic drug FDA approved in the US for treatment of pediatric scabies.  More than 200 million people take the drug globally for prevention or treatment of parasitic disease.  There is also extensive veterinary use in pets.(79-89)  Left image: Ivermectin is HeartGuard for Dogs.

Ivermectin Anti-Leukemic Activity

Dr Sharmeen and Sukhai at the University of Toronto screened a library of 100 drugs for activity against a leukemic cell line.(92) They reported Ivermectin as the top candidate inducing leukemic cell death at low micromolar concentrations, preferentially over normal blood cells.  The drug, Ivermectin also delayed leukemia tumor growth in mouse xenograft models.(92)

Ivermectin Patent for leukemia and lymphoma

Ivermectin was patented in 2012 as a treatment for hematological malignancy (including mantle cell lymphoma).(91)

Dr Simon Yu – Ivermectin Deficiency Syndrome

Dr Simon Yu author of Accidental Cure, suggested that Ivermectin should be handed out to all patients on the oncology wards.  Use could be justified by manufactured concerns for occult parasitic disease,  or as preventive of future risk of parasitic disease in immunocompromised patients after chemotherapy.  Thus, every oncology patient will soon be labeled with the diagnosis called,Ivermectin Deficiency Syndrome”. (90)

Dr Simon Yu discloses that, “Often, I will combine Ivermectin with pyrantel pamoate, praziquantel, or tinadazole for a variety of medically unexplainable symptoms.” End Quote (90)   This is available as a veterinary drug for dogs called Quadriguard.(99)

Tracking WNT-TCF Perfectly

Dr Alice Melotti published her study on “Ivermectin inhibition of  WNT‐TCF pathway in cancer. in 2914 EMBO molecular medicine. (79) Dr Melotti used a transcriptional reporter assay for TCF activity driven by ß-CATENIN to test a collection of 1,040 drugs and small molecules (Microsource 1040 library).  (See image below explaining TCF is the target gene for the WNT pathway)

Only one agent perfectly tracked the gene expression profile induced by blocking the TCF gene, and therefore blocks the WNT pathway.  This is Ivermectin, the anti-helmintic drug  derived from the bacteria strain Streptomyces avermitilis.(79)  This has profound significance for anti-cancer stem cell therapy, because blocking the WNT pathway is the key to killing cancer stem cells.

The WNT PATHWAY- Key to Cancer Stem Cell Destruction

Cancer Cells Upregulated WNT Pathway- WNT ON – This is BAD

The WNT pathway controls embryonical devlopment and cell proliferation, and  is massively upregulated in cancer stem cells.  Inhibiting the WNT pathways kills cancer stem cells.(45)

WNT ON Mantle Cell Lymphoma Jeffrey Dach MDLeft Image:

WNT PAthway ON – β-catenin is NOT degraded.

The accumulated β-catenin  enters the nucleus and activates the target genes such as LEF-1, c-myc and Cyclin D1.  Image

From : Chapter 3  Wnt/β-Catenin Signaling Pathway By Jae-Ik Han and Ki-Jeong Na. (44)

WNT Pathway OFF – This is GOOD – Beta Catenin Degraded

WNT_OFF_Jeffrey Dach MDLeft Image: 

WNT OFF- In the absence of Wnt signals, a cellular complex degrades β-catenin, so there is no entry into the nucleus, TCD/LEF is suppressed, and no nuclear transcription of Cyclin D1 or other growth signals. (44) Chapter 3  Wnt/β-Catenin Signaling Pathway  By Jae-Ik Han and Ki-Jeong Na.

100-Fold Elevated Expression of WNT Target Genes in Stem Cells

Dr Rohit Mathur from MD Anderson reports in 2015 cancer Stem Cells have elevated expression of Wnt target genes greater than 100-fold compared with Mantle Cell Lymphoma non-stem cells (MCL non-ICs).  The authors also report that blocking the WNT pathway kills cancer stem cells in  mantle cell lymphoma.  (45)   Dr Rohit Mathur says:

“The high rate of MCL relapse after initial apparent clinical remissions achieved with conventional chemotherapy suggests incomplete elimination of MCL cells and implicates a role for chemoresistant Mantle Cell Lymphoma – Initiating Cells (stem cells called MCL-ICs ) in relapse. Here we showed that MCL-ICs have functional properties of cancer stem cells: high expression of ALDH, antioxidant enzymes, chemoresistance-associated genes, and stem cell associated transcription factors, while still retaining t(11;14) (q13; q32) and overexpression of cyclin D1. Our analysis showed that MCL-ICs overexpress a subset of Wnt ligands and FZD receptors and that Wnt signaling is activated in MCL-ICs. Treatment of primary MCL cells with Wnt inhibitors preferentially eliminated MCL-ICs, which was not achieved with the current chemotherapy agents vincristine,  doxorubicin, or even with the recently FDA-approved agent ibrutinib [28]. Burton tyrosine kinase (BTK) has been shown to be a negative regulator of Wnt signaling [29]. Therefore, it is not surprising that ibrutinib (a BTK inhibitor) probably resulted in inducing Wnt signaling rather than inhibiting it and thereby could not eliminate MCL-ICs. Our results suggest that the inability of conventional chemotherapy to kill MCL-ICs can be overcome by adding inhibitors of Wnt signaling”

The Glutamate-Cystine Antiporter -depleting glutathione in the cancer cell

Cancer stem cells have upregulated the production of glutathione which protects them from oxidative damage and explains resistance to chemotherpy. Blocking the cystine uptake transport (X ct) with Azulfidine (sulfasalazine)  depletes the cancer stem cell of glutathione and kills the cancer stem cell.

Inhibiting Autophagy- Targeting the Lysosome -Chlorquin Mefloquin, PPI inhibitors

The anti-malaria drug, Chloroquin, accumulates in lysosomes and inhibits autophagy, a necessary function in cancer stem cells.  This kills the cancer stem cell.

Inducing Autophagy

Resveratrol(Pterostilbene), Curcumin and Berberine induce autophagy cancer cell death (1B)

Salinomycin

Salinomycin, FDA approved in veterinary use in animals was found to have striking activity against cancer stem cells. (43)  However toxicity to normal cells may represent a problem in human use.(43)

Common Antibiotics

Doxycyline and other commonly used antibiotics inhibit mitochondrial biogenesis in cancer stem cells and may ultimately find their place in routine use as anti-cancer stem cell agents. (73-78)

Conclusion: Targeting Cancer Stem Cells with numerous non-toxic therapies and re-purposed drugs is now available.  Although not currently accepted by mainstream oncology,  I predict many of these non-toxic modalities, such as Ivermectin FDA approved  for treatment of parasitic disease, will be incorporated into routine oncology protocols.

For Part Two of this series click Here:
Natural Substances Which Kill Cancer Stem Cells Part Two

Articles with related interest:

25 Cancer Stem Cell Killing Foods by Sayer Ji from GreenMedInfo

Artemisinin our Ultmate Anti-Cancer Weapon and other AntiCancer Botanicals

Cancer as a Metabolic Disease

Jeffrey Dach MD

Links and References

CD44 CANCER STEM CELLS  2015

1) Yan, Yongmin, Xiangsheng Zuo, and Daoyan Wei. “Concise review: emerging role of CD44 in cancer stem cells: a promising biomarker and therapeutic target.” Stem cells translational medicine 4.9 (2015): 1033-1043. Emerging role of CD44 in cancer stem cells promising biomarker therapeutic target Yan Yongmin Stem cells 2015

Cancer Stem Cell Reviews

1A)  Yu, Zuoren, et al. “Cancer stem cells.” The international journal of biochemistry & cell biology 44.12 (2012): 2144-2151.

Excellent Review Article 2015

1B) Gali-Muhtasib, H., et al. “Cell death mechanisms of plant-derived anticancer drugs: beyond apoptosis.” Apoptosis: an international journal on programmed cell death 20.12 (2015): 1531-1562. Cell Death Mechanisms Plant Anticancer Drugs Apoptosis Gali Muhtasib 2015

More than 3000 plant species have been reported to treat cancer and
about thirty plant-derived compounds have been isolated so far and have been tested in cancer clinical trials.

Recent studies have focused on the effects of plant-derived compounds on cell
cycle regulatory and apoptotic pathways [14, 15], yet little is known about their effects on non-apoptotic pathways e.g. autophagy, mitotic catastrophe, senescence leading to cell death, and programmed necrosis or ‘‘necroptosis’’

The naturally occurring structural analogue to resveratrol, pterostilbene, has also been reported to induce autophagy, cell cycle arrest, and apoptosis in different
cancer types including bladder, breast, and leukemic cancer

1C) Scarpa, E. S., and P. Ninfali. “Phytochemicals as Innovative Therapeutic Tools against Cancer Stem Cells.” International journal of molecular sciences 16.7 (2014): 15727-15742.

2016 Curcumin – Cancer Stem Cells

2) Huang, Yu-Ting, et al. “Curcumin Induces Apoptosis of Colorectal Cancer Stem Cells by Coupling with CD44 Marker.” Journal of agricultural and food chemistry (2016).
This study investigated the effect of curcumin on colorectal cancer stem cells (CCSCs) and its possible mechanism. Comparison of the metabolic profiles of human adenomatous polyp (N = 61) and colorectal cancer (CRC) (N = 57) tissue found statistically significant differences (p < 0.05) in their composition of adenosine monophosphate (AMP), adenine, 5′-methythioadenosine, 3-hydroxybutyric acid, prostaglandin E2, threonine, and glutamine. Our cell culture model study found that curcumin treatment (50 μM for 48 h) did indeed increase apoptosis of CRC cells as well as of CCSCs, but at a significant level only in CD44+ cells. Further metabolic profile studies of the CRC, CD44+, and CD44– cells indicated that curcumin treatment increased glyceraldehyde and hydroxypropionic acid in CD44– cells but decreased glutamine content in both curcumin-treated CRC and CD44+ cells. Based on our comparison of the metabolic profiles of human tissues and cancer cells, we suggest that curcumin might couple with CD44 and that curcumin-CD44+ coupling at the cell membrane might have some blocking effect on the transport of glutamine into the cells, thus decreasing the glutamine content in the CD44+ cells and inducing apoptosis.

2015 Curcumin  and EGCG  !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

3) 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.

The CD44+ cell population was also decreased following curcumin and EGCG treatments. Our results suggest that the final destination of STAT3 and NFκB signaling may be the CD44 expression and accompanied by a cancer stem cell phenotype.

Conclusion: This study suggests that curcumin and EGCG function as antitumor agents for suppressing breast CSCs. STAT3 and NFκB signaling pathways could serve as targets for reducing CSCs leading to novel targeted-therapy for treating breast cancer.

Curcumin – Mantle Cell Anti-cancer Effects

4) Hasanali, Zainul, Kamal Sharma, and Elliot Epner. “Flipping the cyclin D1 switch in mantle cell lymphoma.” Best Practice & Research Clinical Haematology 25.2 (2012): 143-152.

Curcumin, a plant flavonoid that is available naturally in turmeric and as an herbal supplement, has been shown in vitro to downregulate cyclins D1 and D3 at both the transcriptional and post-transcriptional levels in MCL and MM cell lines [50] . Curcumin is known to inhibit the COP9 signalosome, a multiprotein complex similar to the proteasome [51], [52], and [53]. Our laboratory has demonstrated that curcumin and bortezomib synergize in downregulating protein levels of cyclins D1 and D3 in MM and MCL cells [35] . Clinical trials of this agent in combination in lymphoid malignancies alone or in combination with bortezomib are ongoing.

Curcumin Effective Against Four MCL cell lines

5) Shishodia, Shishir, et al. “Curcumin (diferuloylmethane) inhibits constitutive NF-κB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma.” Biochemical pharmacology 70.5 (2005): 700-713. Biochem Pharmacol. 2005 Sep 1;70(5):700-13.  Shishodia S1, Amin HM, Lai R, Aggarwal BB.

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin’s lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL’s resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-kappaB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-kappaB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-kappaB and IkappaB kinase and phosphorylated forms of IkappaBalpha and p65. This correlated with expression of TNF, IkappaBalpha, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-kappaB. On treatment of cells with curcumin, however, downregulated constitutive active NF-kappaB and inhibited the consitutively active IkappaBalpha kinase (IKK), and phosphorylation of IkappaBalpha and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-kappaB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-kappaB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-kappaB complex and inhibited the proliferation of MCL cells. Constitutive NF-kappaB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-kappaB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-kappaB and IKK leading to suppression of expression of NF-kappaB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.

6) Choudhuri, Tathagata, et al. “Curcumin selectively induces apoptosis in deregulated cyclin D1-expressed cells at G2 phase of cell cycle in a p53-dependent manner.” Journal of Biological Chemistry 280.20 (2005): 20059-20068.   All of these data suggest that curcumin can exert its apoptogenic effect in those cells whose cyclin D1 expression is deregulated due to genetic manipulations.

7) Singh, Amareshwar TK, et al. “Curcumin nanodisk-induced apoptosis in mantle cell lymphoma.” Leukemia & lymphoma 52.8 (2011): 1537-1543.

8) Tadmor, Tamar, and Aaron Polliack. “Mantle cell lymphoma: curcumin nanodisks and possible new concepts on drug delivery for an incurable lymphoma.” Leukemia & lymphoma 52.8 (2011): 1418.

9) Yallapu, Murali M., Meena Jaggi, and Subhash C. Chauhan. “Curcumin nanomedicine: a road to cancer therapeutics.” Current pharmaceutical design 19.11 (2013): 1994.

Nice Review Article on Curcumin As Targeting Cancer Stem Cells

10) Curcumin and Cancer Stem Cells Asymmetrical Effects AntiCancer Peter SORDILLO 2015  SORDILLO, PETER P., and LAWRENCE HELSON. “Curcumin and Cancer Stem Cells: Curcumin Ηas Asymmetrical Effects on Cancer and Normal Stem Cells.” ANTICANCER RESEARCH 35 (2015): 599-614.
IL-6 (also known as interferon (IFN)-β2) is a multi-functional
cytokine involved in the immune and inflammatory response
and progression from inflammation to cancer. Increased IL-6
activity has been found in multiple cancers, including multiple
myeloma, as well as breast, colon and prostate carcinoma, and
IL-6 has been associated with decreased survival and more
aggressive disease in these patients

Furthermore, IL-6 has been shown to convert regular cancer cells to CSCs in established breast and prostate cancer cell lines.  One mechanism by which curcumin targets CSCs is inhibition of IL-6 release from cells, thus preventing CSC stimulation. Curcumin has been shown to decrease IL-6 levels or inhibit IL-6 function in multiple experimental systems.

IL-8 (CXCL8) is an important cytokine, which increases after tumor cell death, stimulates CSCs and results in tumor regrowth and resistance to chemotherapy
Curcumin is a potent inhibitor of IL-8 production, as well as of numerous IL-8 cancer-promoting bio-activities. Curcumin was found to reduce chronic non-bacterial prostatitis in rats by blocking IL-8 release

The Wnt signaling pathways regulate multiple processes during embryonic development, as well as gene transcription, cell migration, cell proliferation and tissue homeostasis in the adult organism (103-107). These pathways occur in multiple species, including drosophila, where much of the original work was done, as well as mice and humans

Wnt signaling regulates levels of the protein β-catenin. Wnt signaling is associated with a decrease in β-catenin phosphorylation, so β-catenin accumulates and, in turn, stimulates the genes for VEGF, cyclin D1 and c-Myc.
Aberrant Wnt signaling and excessive levels of β-catenin can result in carcinogenesis and uncontrolled cell proliferation.

Curcumin modulates Wnt signaling. Karkarala et al. have shown that curcumin can inhibit Wnt signaling and the formation of mammospheres in breast cancer cell lines, as well as in normal breast cell lines Evidence indicates that curcumin can act at multiple points along the Wnt pathway Like the Wnt pathways, the Notch pathway has been conserved among species through evolution. The Notch signaling pathway plays a critical role in regulating cell differentiation, cell proliferation and apoptosis.

Curcumin acts to suppress tumor cells at multiple sites along the Notch pathway.
The question arises as to why curcumin does not seem to have the same
deleterious effects on normal stem cells (NSCs) as it does on CSCs.

Curcumin has been shown to have a much greater uptake by malignant cells
compared to normal cells.

Curcumin appears to shift the microenvironment around these cells to one that is adverse to proliferation of CSCs, but conducive to NSCs. As noted,
curcumin has been shown to suppress the release of proinflammatory
cytokines induction of CSC differentiation may be one of the ways curcumin depletes CSCs.

part of curcumin’s toxicity to CSCs involves suppression of molecular abnormalities in the Wnt pathway, such as its inhibition of β-catenin (122, 125-126). Curcumin has opposite effects on neural stem cells as it
stimulates neurogenesis.
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11)  Anticancer Drugs. 2014 Sep 16. [Epub ahead of print]
Curcumin inhibits aerobic glycolysis and induces mitochondrial-mediated apoptosis through hexokinase II in human colorectal cancer cells in vitro.
Wang K1, Fan H, Chen Q, Ma G, Zhu M, Zhang X, Zhang Y, Yu J.
1aJiangsu Institute of Cancer Research bJiangsu Research Institute of Geriatrics, Nanjing, China.

Curcumin, the major pigment of the dietary spice turmeric, has the potential for chemoprevention by promotion of apoptosis. Here, we investigated the molecular mechanisms of curcumin in glycolytic inhibition and apoptotic induction in human colorectal cancer HCT116 and HT29 cells. On the one hand, curcumin downregulated the expression and activity of hexokinase II (HKII) in HCT116 and HT29 cells in a concentration-dependent manner, but had little effect on the other key glycolytic enzymes (PFK, PGM, and LDH).

On the other, curcumin induced dissociation of HKII from the mitochondria, resulting in mitochondrial-mediated apoptosis. Furthermore, the phosphorylation of mitochondrial HKII through AKT was responsible for the curcumin-induced dissociation of HKII, which was different from the mechanism of HKII inhibitor 3-BrPA. These results have important implications for the metabolism reprogramming effect and the susceptibility to curcumin-induced mitochondrial cytotoxicity through the regulation of HKII, and provide a molecular basis for the development of naturally compounds as novel anticancer agents for colorectal carcinoma.
berberine synergy with curcumin

full free pdf

11A )  Balakrishna, Acharya, and M. Hemanth Kumar. “Evaluation of synergetic anticancer activity of berberine and curcumin on different models of A549, Hep-G2, MCF-7, Jurkat, and K562 cell lines.” BioMed research international 2015 (2015).

In conclusion, we confirmed that the combination of Curcumin and Berberine synergistically generates anticancer effects in A549, Hep-G2, MCF-7, Jurkat, and K562 cells in vitro, possibly mediated by inducing apoptosis. With regard to A549, Hep-G2, MCF-7, Jurkat, and K562 Curcurmin and Berberine are of extreme antitumor agents. The combination of Curcumin and Berberine is a novel strategy that has potential in the treatment of cancer patients.

The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure Curcumin with <54% and pure Berberine with <45% on average on all cell line models.

11B)   Anticancer Res. 2015 Dec;35(12):6373-8.
Curcumin for the Treatment of Glioblastoma. Sordillo LA1, Sordillo PP1, Helson L2.

Glioblastoma multiforme is a highly aggressive primary cancer of the brain associated with a poor prognosis. Modest increases in survival can sometimes be achieved with the use of temozolomide and radiation therapy after surgery, but second-line therapy after recurrence has a limited efficacy. Curcumin has demonstrated promising results against this form of cancer in experimental models. The reported activity of curcumin against cancer stem cells, a major cause of glioblastoma resistance to therapy, and its ability to augment the apoptotic effects of ceramides, suggest it would have a synergistic effect with cytotoxic chemotherapy agents currently used in second-line therapy, such as lomustine.

Curcumin AML

11C) Rao, Jia, et al. “Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells.” J Transl Med 9.1 (2011): 71. Curcumin reduces Bcl2 induces apoptosis in CD34pos acute myeloid leukemia cells Rao Jia 2011

 

Review – Many Natural Products That Target Cancer Stem Cells

12) Moselhy, J., et al. “Natural Products That Target Cancer Stem Cells.” Anticancer research 35.11 (2015): 5773.
Epigallocatechin-3-gallate (EGCG) – Green Tea
6-Gingerol – Ginger
β-Carotene – Carrot, Leafy Greens
Baicalein – Chinese Skullcap
Curcumin – Turmeric
Cyclopamine – Corn Lilly
Delphinidin – Blueberry, raspberrry
Flavonoids (Genistein) – Soy, red clover, coffee
Gossypol – Cottonseed
Guggulsterone – Commiphora (myrrh tree)
Isothiocyanates – Cruciferous vegetables
Linalool – Mint
Lycopene – Grapefruit, tomato
Parthenolide – Feverfew
Perylill alcohol – Mint, cherry, lavender
Piperine – Black pepper
Placycodon saponin – Playycodon grandifloruim
Psoralidin – Psoralea corylilyfolia
Quercetin – Capers, onion
Resveratrol – Grapes, plums, berries
Salinomycin – Streptomyces albus
Silibinin – Milk Thistle

Ursolic acid – Thyme, basil, oregano
Vitamin D3
– Fish, egg yolk, beef, cod liver oil
Withaferin A – Withania somnifera (ashwaganda)

Sulforaphane – Cancer Stem Cells – Breast CA

13) 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.  A NOD/SCID xenograft model was employed to determine whether sulforaphane could target breast CSCs in vivo,

Sulforaphane eliminated breast CSCs in vivo, thereby abrogating tumor growth after re-implantation of primary tumor cells into the secondary mice
These findings support the use of sulforaphane for chemoprevention of breast cancer stem cells and warrant further clinical evaluation.

Breast Cencer CAncer stem cells in vitro and in vivo xenograft mouse model

14) Kallifatidis G, Rausch V, Baumann B, et al. Sulforaphane targets pancreatic tumour-initiating cells by NF-kappaB-induced antiapoptotic signalling. Gut. 2009;58:949–63. [PubMed]
An interesting observation is that sulforaphane was able to inhibit stem/progenitor cells at the concentrations (0.5~ 5 μM) that hardly affected the bulk population of cancer cells, implying that sulforaphane is likely to preferentially target stem/progenitor cells compared to the differentiated cancer cells.

These results suggest that sulforaphane was able to eliminate breast CSCs in primary xenografts, thereby abrogating the re-growth of tumors in secondary mice. Taken together with the in vivo Aldefluor assay results, these findings suggest that sulforaphane targets breast CSCs with high potency.
sulforaphane decreased the protein level of β-catenin by up to 85% in MCF7 and SUM159 cells; and the expression of cyclin D1, one of the Wnt/β-catenin target genes, declined by up to 77% as well.

As a chemoprevention agent, sulforaphane possesses many advantages, such as high bioavailability and low toxicity

A recent pilot study detected an accumulation of sulforaphane in human breast tissue, with 1.45 ± 1.12 pmol/mg for the right breast and 2.00 ± 1.95 pmol/mg for the left, in eight women who consumed broccoli sprout preparation containing 200 μmol sulforaphane about 1 hr before the surgery (36). These concentrations of sulforaphane are expected to be effective against breast CSCs, based on our in vitro results

15) 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.

16) Wu, S., et al. “Sulforaphane produces antidepressant-and anxiolytic-like effects in adult mice.” Behavioural brain research 301 (2015): 55.

17) Sestili, Piero, and Carmela Fimognari. “Cytotoxic and Antitumor Activity of Sulforaphane: The Role of Reactive Oxygen Species.” BioMed Research International 2015 (2015).

18) Shang, Hung-Sheng, et al. “Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.” Environmental toxicology (2016).

19) Drug Deliv Transl Res. 2013 Apr 1; 3(2): 165–182.
Novel strategies targeting cancer stem cells through phytochemicals and their analogs.  Prasad Dandawate, Subhash Padhye, Aamir Ahmad, and Fazlul H. Sarkarcorresponding author

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Sulfasalazine

Cancer Stem Cells in gastric cancer- tumor cells evaluated before and after sulfasalazine for CD44.  Optimal dose of SSZ was considered as 8g/day

20) Shitara, Kohei, et al. “Effect of sulfasalazine (SSZ) on cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205).” ASCO Annual Meeting Proceedings. Vol. 32. No. 15_suppl. 2014.

Effect of sulfasalazine (SSZ) on cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205).
2014 ASCO Annual Meeting Abstract Number: 2545 Citation: J Clin Oncol 32:5s, 2014 (suppl; abstr 2545) Author(s): Kohei Shitara et.al.

Background: CD44 is an adhesion molecule expressed in cancer stem-like cells (CSCs). Our group recently reported that CD44 splice variant (CD44v) is expressed in CSCs and interacts with xCT, a glutamate-cystine transporter, keeping high levels of the intracellular reduced glutathione (GSH). Thus, CSCs with a high expression of CD44v have an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS), resulting in resistance to various therapeutic stresses. Sulfasalazine (SSZ) as an xCT inhibitor suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in vivo study.

Methods: A phase 1 dose escalation study in patients with advanced gastric cancer was conducted to determine the optimal dose. SSZ was given fourth-daily oral administration with 2 weeks as one cycle. A 3+3 escalation was used to evaluate a MTD. Tumor tissues were obtained pre- and post SSZ administration to evaluate expression of CD44v and intra-tumor level of GSH by immunohistochemistry and boron doped diamond microelectrode, respectively.

Results: Eleven patients were dosed from 8 g to 12 g/day; median age: 71 years (61-78); median number of prior chemotherapies: 3 (1-4). There was two DLT of grade 3 anorexia and nausea among patients who were treated with 12 g/day. One additional patients required frequent dose interruption with grade 2 anorexia and nausea. Therefore 12g/day was judged as MTD. No DLT was observed among patients with 8g/day.

Patients with high CD44v expression patients achieved reduced expression of CD44v after the administration of SSZ for 2 weeks as well as decreased level of GSH.

The individual variability of SSZ exposure was explainable in terms of the genotypes of ABCG2 and NAT2 which influence SSZ pharmacokinetics. Conclusions: Optimal dose of SSZ was considered as 8g/day. Down regulation of CD44v expression and decreased level of GSH migh be a pharmacodynamic marker of drug-on-target effect and mode of action of SSZ for CSCs, which warrants further investigation for combination with chemotherapy or other targeting agents. Clinical trial information: UMIN000010254.

stem cells – gastric cancer ccell line   colon cancer cell line

full free pdf
21) Ishimoto, Takatsugu, et al. “CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc− and thereby promotes tumor growth.” Cancer cell 19.3 (2011): 387-400.  CD44 regulates redox status in cancer cells by stabilizing xCT Cancer cell 2011 Ishimoto Takatsugu

The xCT Inhibitor Sulfasalazine Suppresses CD44-Dependent Tumor Growth and Promotes Activation of p38MAPK in Tumor Cells In Vivo –  Stem Cells !!!

CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor

CD44v interacts with and stabilizes xCT, a subunit of a glutamate-cystine transporter

Cells deficient in xCT or depleted of GSH have recently been found to exhibit p38MAPK activation even at low levels of oxidative stress (Chen et al., 2009; Sato et al., 2005), indicating that xCT-mediated cystine transport for GSH
synthesis plays a key role in prevention of such stress signaling.

Sulfasalazine is a well-characterized specific inhibitor of xCT-mediated cystine transport and has been shown to inhibit the growth, invasion, and metastasis
of several types of cancer.  sulfasalazine treatment stimulated the phosphorylation of p38MAPK in HCT116 tumor cells in vivo. Finally, we examined whether suppression of xCT function by sulfasalazine might enhance the effect of the anticancer drug CDDP (Cis-Platin) on tumor growth. The antitumor effect of CDDP at a low dose (2 mg/kg) was significantly enhanced by treatment with sulfasalazine (Figure 8E), suggesting that sulfasalazine reduces the ROS defense capacity of cancer cells and sensitizes them to available chemotherapeutic drugs.

xCT is a component of a plasma membrane transporter (the xc system) that mediates the cellular uptake of extracellular cystine in exchange for intracellular glutamate a key role in GSH synthesis.

The activity of xCT-mediated cystine uptake in cancer cells is highly associated with cell proliferation, chemoresistance, and tumor growth
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2013

sulfasalazine with 9-cis-retinoic acid (Panretin®, Alitretinoin, or 9-cis-retinoic acid, is a form of vitamin A. It is also used in medicine as an antineoplastic (anti-cancer) agent developed by Ligand Pharmaceuticals. It is a first generation retinoid. Ligand gained Food and Drug Administration (FDA) approval for alitretinoin in February 1999.)

22) http://cancerres.aacrjournals.org/content/73/6/1855.long
Yoshikawa, M., et al. “xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma.” Cancer research 73.6 (2013): 1855.

Sulfasalazine has been reported to inhibit not only the xCT-mediated cystine transport but also NF-κB signaling.

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Sulfasalazine – Inhibits Mantle Cell Lymphoma

23) Bebb, G., et al. “Sulfasalazine, inhibits growth of mantle cell lymphoma (MCL) cell cultures via cyst (e) ine starvation and delays tumour growth in a newly developed murine MCL model.” BLOOD. Vol. 102. No. 11. 1900 M STREET. NW SUITE 200, WASHINGTON, DC 20036 USA: AMER SOC HEMATOLOGY, 2003.

Introduction: Cyst(e)ine deficiency within lymphoid cells leads to a rapid decline in their levels of glutathione (a major free radical scavenger), loss of defense against oxidative stress, and subsequently apoptosis. Lymphoid cells cannot synthesize the amino acid and depend for growth and viability on its uptake from their micro-environment. Since lymphomas have been shown to retain the inability to synthesize cyst(e)ine they are potentially susceptible to cyst(e)ine starvation-based therapy. We have previously demonstrated that sulfasalazine (SASP), a drug used for treatment of severe inflammatory bowel disease and rheumatoid arthritis, is a potent inhibitor of the cystine/glutamate antiporter, xc-, a major plasma membrane cystine transporter. SASP abrogated growth of T and B lymphoma cell cultures via cystine starvation; SASP, administered intraperitoneally, markedly inhibited growth of rat Nb2-U17 lymphoma transplants in Nb rats without major toxicity to the hosts (Anti-Cancer Drugs 14:21, 2003).

In the present study we investigated the usefulness of SASP in our newly developed model of MCL, a B-cell non-Hodgkin lymphoma (NHL), characterized by cyclin D1 and BCL2 over-expression. Results: Growth of human MCL cultures in Fischer’s medium, supplemented with 10% fetal bovine serum and antibiotics, was markedly inhibited by SASP at therapeutic concentrations, showing IC50s of 0.13 and 0.30 for Z138C and Granta MCL cultures, respectively. Culture growth arrest could be largely prevented by enhancing cellular cystine uptake using 66 uM 2-mercaptoethanol (reported to promote cystine uptake via the leucine transporter), indicating that the SASP-induced inhibition resulted from cyst(e)ine starvation.

MCL In vivo study in mice

A study into the efficacy of SASP in vivo was initiated using SCID/Rag2-M mice injected subcutaneously with Z138C cells (5 million cells/mouse); such a procedure leads to consistent development of tumours within 28 days. When tumours had reached a weight of about 0.1 gr, groups of six mice were treated for 10 consecutive days with saline (controls) or SASP (250 mg/kg body weight i.p., b.i.d.), a dosage well below the maximally tolerated dosage (300 mg/kg every 8 hr). It was found that treatment with SASP inhibited tumour growth, showing a delay in growth of at least 7 days, without major toxicity.

Conclusions: SASP has a marked inhibitory effect on growth of MCL cell lines in vitro, an effect also seen in vivo in our murine SC MCL model. SASP may represent a novel approach for MCL treatment. The precise molecular consequences of SASP treatment on MCL cells warrant further investigation. Additional studies on the effect of SASP at higher dosages and in combination with cyclophosphamide and targeted therapies, eg. ASO and monoclonal antibodies against bcl-2, are in progress.

Sulfasalazine in Lymphoma

24) Gout, Peter W., Chris R. Simms, and May C. Robertson. “In vitro studies on the lymphoma growth-inhibitory activity of sulfasalazine.” Anti-cancer drugs 14.1 (2003): 21-29.

Sulfasalazine (SASP) is a novel, potent inhibitor of cellular cystine uptake mediated by the x(c)- cystine/glutamate antiporter. Lymphoid cells cannot synthesize cyst(e)ine and depend for growth on its uptake from their micro-environment. We previously showed that SASP (0.2 mM) can abrogate lymphoma cell proliferation in vitro by specifically inhibiting x(c)- -mediated cystine uptake. Intraperitoneal administration of SASP to Noble rats markedly suppressed Nb2-U17 rat lymphoma transplant growth, notably without major toxicity to the hosts. Since Nb2-U17 cells are x(c)- -deficient, the growth arrest was apparently not due to SASP-tumor cell interaction, but possibly to interference with x(c)- -mediated cysteine secretion by somatic cells. In this study we found that replication of x(c)- -deficient Nb2-11 lymphoma cells can be sustained in vitro, in the absence of cystine uptake enhancers, by co-culturing with IMR-90 fibroblasts known to secrete cysteine. SASP, at 0.15 and 0.2 mM, arrested replication of fibroblast-driven Nb2-11 cells by 93 and 100%, respectively, without impeding fibroblast proliferation. Addition of 2-mercapto-ethanol (60 microM), a cystine uptake enhancer, almost completely prevented this growth arrest, indicating that SASP specifically inhibited cysteine secretion by the fibroblasts, a process based on x(c)- -mediated cystine uptake. It is proposed that the lymphoma growth-inhibitory activity of SASP in vivo involves inhibition of cysteine secretion by tumor-associated somatic cells (macrophages, dendritic cells), leading to cysteine starvation of the tumor cells and apoptosis. The difference between the lymphoma cells and fibroblasts in sensitivity to SASP treatment is consistent with the marked antitumor effect of SASP lacking significant side effects.

Small cell Lung cancer  full pdf

25) Guan, Jun, et al. “The x c- cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine.” Cancer chemotherapy and pharmacology 64.3 (2009): 463-472. The x c cystine glutamate antiporter as a potential therapeutic target for small-cell lung cancer_sulfasalazine Guan Cancer 2009
Conclusions The xc- cystine/glutamate antiporter is potentially useful as a target for therapy of SCLC based on glutathione depletion. Sulfasalazine may be readily used for this approach, especially in combination chemotherapy.

pancreatic full pdf

26) Lo, M., et al. “Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer.” Current Oncology 17.3 (2010): 9-16.Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer Lo Gout Current Oncology 2010

Breast

27) 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.

Prostate

28) Doxsee, Daniel W., et al. “Sulfasalazine-induced cystine starvation: Potential use for prostate cancer therapy.” The Prostate 67.2 (2007): 162-171. Sulfasalazine Induced Cystine Starvation Prostate CancerTherapy Doxsee 2007

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Toxicity sulphasalazine

29) Liedorp, M., A. E. Voskuyl, and BW Van Oosten. “Axonal neuropathy with prolonged sulphasalazine use.” Clinical & Experimental Rheumatology 26.4 (2008): 671. Ann Med Interne (Paris). 2001 Jun;152(4):283-4. free pdf.
[Sulfasalazine neurotoxicity]. [Article in French] Chadenat ML1, Morelon S, Dupont C, Dechy H, Raffin-Sanson ML, Dorra M, Rouveix E.
We report a case of seizures with acute encephalopathy in a female patient under sulfasalazine treatment for polyarthritis. Neurotoxicity secondary to sulfasalazine was suspected. This side effect has seldom been reported in the literature.

30) Clin Ter. 1997 Jan-Feb;148(1-2):7-13. [Sulfasalazine: side effects and duration of therapy in patients with rheumatoid arthritis]. [Article in Italian] Mundo A1, Pedone V, Lamanna G, Cervini C.

Sulphasalazine (SSZ) is now recognised to be a useful agent in the management of rheumatoid arthritis (RA). We studied SSZ toxicity (2 g/die) and duration of therapy in 102 patients with RA. Adverse events occurred in 25.4% of all patients. In all patients the reactions subsided on either discontinuation of the drug or decrease of the dose. Gastrointestinal was the most common. At 5 years of follow-up the percentage of patients treated with SSZ still on drug was 29%, the inefficacy was 40% of the total drop-out.

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31) Lewerenz, Jan, et al. “The cystine/glutamate antiporter system xc− in health and disease: from molecular mechanisms to novel therapeutic opportunities.” Antioxidants & redox signaling 18.5 (2013): 522-555.  The cystine glutamate antiporter system xc in health and disease Lewerenz Jan 2013

Early evidence suggested that nonsteroidal anti-inflammatory drugs also inhibit system xc – (15). On this basis, the Gout lab identified the FDA-approved drug sulfasalazine, commonly used to treat chronic inflammatory diseases such as rheumatoid arthritis, as a potent system xc – inhibitor (79). However, this compound is also a potent inhibitor of nuclear factor kappa B (NF-jB) activation (283).

32) Gout, P. W., et al. “Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x c-cystine transporter: a new action for an old drug.” Leukemia (08876924) 15.10 (2001).  1Department of Cancer Endocrinology, BC Cancer Agency, Vancouver, BC, Canada

33) Doxsee, Daniel W., et al. “Sulfasalazine‐induced cystine starvation: Potential use for prostate cancer therapy.” The Prostate 67.2 (2007): 162-171.

34) Chung, W. Joon, and Harald Sontheimer. “Sulfasalazine inhibits the growth of primary brain tumors independent of nuclear factor‐κB.” Journal of neurochemistry 110.1 (2009): 182-193.

35) Guan, Jun, et al. “The x c− cystine/glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine.” Cancer chemotherapy and pharmacology 64.3 (2009): 463-472.

36) Dixon, Scott J., et al. “Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis.” Elife 3 (2014): e02523.Pharmacological inhibition of cystine glutamate exchange induces endoplasmic reticulum stress and ferroptosis.Elife 2014 Dixon SCott

37) 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. Sulfasalazine induced reduction of glutathione levels in breast cancer cells Narang 2007 Chemotherapy

Background: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the xc– cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. Methods: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. Results: Incubation of the mammary cancer cells with SASP (0.3–0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). Conclusions: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.

We also showed, for the first time, that sulfasalazine (salicylazosulfapyridine, SASP), an anti-inflammatory drug used against inflammatory bowel disease
and rheumatoid arthritis, is a potent inhibitor of x c – – mediated cystine uptake [16] , and that it can markedly inhibit proliferation of human breast carcinoma cells at relatively low concentrations (0.2–0.5 m M ) via cystine starvation [17] .  The human breast carcinoma cell lines, MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative, highly invasive),

38) From Rheumatoid artritis.net:  How is sulfasalazine taken?

Sulfasalazine comes in a 500-mg tablet for oral administration and it typically started at a dose of 500 mg per day and increased by 500 mg every week, while monitoring for side effects, until a daily target dose (this is determined by your weight, approximately 40 mg/kg) is reached. For most adult patients with RA, the final daily dose ranges from 2000-3000 mg (2-3 grams). If gastrointestinal (GI) side effects are problematic, divided doses can be used or a special enteric-coated form (Azulfidine EN-tabs) to protect against GI effects. To help prevent stomach upset, you should take sulfasalazine with food, followed by a full glass of water.1,2

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Mefloquin – AML cells and Progenitors (Stem Cells)

39) Sukhai, Mahadeo A., et al. “Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors.” Journal of Clinical Investigation 123.1 (2013): 315.

mefloquin and artemisinin combination

However, strikingly, 10 of these, including the artemisinin class of antimalarials (27, 28), proved to be compounds that are known to increase the production of ROS.

Synergistic combinations of mefloquine and artenimol or artesunate also synergistically increased ROS production

mefloquine specifically targets lysosomal function. This finding is consistent with mefloquine’s known ability to preferentially accumulate in lysosomes of the malarial parasite

Mefloquine directly disrupted lysosomes isolated from AML cell lines and primary AML patients’ samples, in a dose-dependent manner, as measured by release of cathepsins B and L.

The effects of mefloquine were specific to lysosomes, as mefloquine treatment did not disrupt isolated mitochondria

In a dose-dependent manner, mefloquine disrupted lysosomes in TEX leukemia cells and mefloquine-sensitive cells from AML patients, but not normal hematopoietic cells.

Taken together, these data point to mefloquine-mediated lysosomal disruption as the cellular mechanism underlying antileukemic action.

AML cells have increased lysosomal mass compared with normal hematopoietic cells.

TEM revealed that lysosomes are larger in primary human AML cells, including the CD34+ AML cells, as well as in AML cell lines, in comparison to the lysosomes found in normal human CD34+ hematopoietic cells

Here we report that mefloquine, a quinoline approved for the treatment and prevention of malaria (23, 24), has toxicity for human AML cells including AML progenitors, while sparing normal human hematopoietic cells treated with the same doses. 

Serum concentrations of mefloquine up to 5 μM have been reported in individuals receiving 250 mg weekly for malaria prophylaxis (59, 60). Thus, antileukemia concentrations of mefloquine may be pharmacologically achievable.

The antimalarial chloroquine is structurally similar to mefloquine and inhibits the degradation of autophagy targets in the autophagolysosome. Through this mechanism, chloroquine can induce cell death and sensitize cells to chemotherapy (including imatinib mesylate in chronic myeloid leukemia; refs. 63–66) and radiation. However, as it involves induction of lysosome disruption, the mechanism of action of mefloquine appears distinct from that of chloroquine and other inhibitors of autophagy.

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Targeting progenitor cells   Parthenolide (Feverfew)

41) Guzman, Monica L., et al. “The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells.” Blood 105.11 (2005): 4163-4169.  Recent studies have described malignant stem cells as central to the initiation, growth, and potential relapse of acute and chronic myelogenous leukemia (AML and CML). Because of their important role in pathogenesis, rare and biologically distinct leukemia stem cells (LSCs) represent a critical target for therapeutic intervention. However, to date, very few agents have been shown to directly target the LSC population. The present studies demonstrate that parthenolide (PTL), a naturally occurring small molecule, induces robust apoptosis in primary human AML cells and blast crisis CML (bcCML) cells while sparing normal hematopoietic cells. Furthermore, analysis of progenitor cells using in vitro colony assays, as well as stem cells using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft model, show that PTL also preferentially targets AML progenitor and stem cell populations. Notably, in comparison to the standard chemotherapy drug cytosine arabinoside (Ara-C), PTL is much more specific to leukemia cells. The molecular mechanism of PTL-mediated apoptosis is strongly associated with inhibition of nuclear factor κ B (NF-κB), proapoptotic activation of p53, and increased reactive oxygen species (ROS). On the basis of these findings, we propose that the activity of PTL triggers LSC-specific apoptosis and as such represents a potentially important new class of drugs for LSC-targeted therapy.

Parthenolide (PTL) is a sesquiterpene lactone found as the major active component in Feverfew (Tanacetum parthenium), an herbal medicine that has been used to treat migraine and rheumatoid arthritis for centuries.12 More recently, PTL has been found to have several other properties, including antitumor activity, inhibition of DNA synthesis, and inhibition of cell proliferation in different cancer cell lines.13-16 In addition, PTL sensitizes cancer cells to other antitumor agents17-20 and acts as a chemopreventive agent in a UVB-induced skin cancer animal model.21 PTL is a potent inhibitor of NF-κB activation and has been shown to directly bind IκB-kinase (IKK)22,23 and to modify the p50 and p65 NF-κB subunits.24,25 PTL can also block signal transducers and activators of transcription 3 (STAT3) phosphorylation on Tyr705,26 sustain c-Jun N-terminal kinase (JNK) activation,17,18 and increase intracellular reactive oxygen species (ROS).13,27

parthenolide AML depletion of glutathione

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42) J Biol Chem. 2013 November 22; 288(47): 33542–33558.
Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells* Shanshan Pei,‡

Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells.

Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. Our findings indicate agents such as parthenolide (PTL) and piperlongumine (PLM) have a dramatic inhibitory effect on the leukemic glutathione system, whereas only a limited and transient perturbation in normal cells. This preferential effect is strongly linked to their selective toxicity toward leukemia and other cancer cell types. Importantly, we have previously shown that PTL effectively eradicates AML stem and progenitor populations (11), cells that are typically resistant/refractory to conventional chemotherapy (12, 13).

Thus, we propose that therapeutic targeting of glutathione metabolism represents a potentially powerful strategy to induce selective toxicity toward a broad range of primary leukemia cells, including malignant stem and progenitor populations.

We first studied PTL, which contains an active α,β-unsaturated-γ-lactone group (Fig. 3B, red circular area) that should readily react with the free thiol group of glutathione. Indeed, PTL induced a dose-dependent decrease of cellular glutathione within 2 h of treatment in primary AML cells

Targeting cancer stem cells – Salinomycin Pathenolide

43) Naujokat, Cord, and Roman Steinhart. “Salinomycin as a Drug for Targeting Human Cancer Stem Cells.” Journal of Biomedicine and Biotechnology 2012 (2012).

In particular, the biomolecules salinomycin and parthenolide as well as the biguanide metformin have been demonstrated to induce apoptosis in various types of human cancer cells [108, 123, 124], suggesting that these compounds may contribute to the eradication of cancer more effectively than compounds targeting either CSCs or regular cancer cells. Moreover, the ionophore antibiotic salinomycin seems to have even extended capabilities of eliminating cancer (Table 1), because this compound has been demonstrated to effectively target regular cancer cells [16, 125–127], highly multidrug and apoptosis-resistant cancer cells [16, 85, 125], and CSCs [16, 84, 87, 88, 128–131].

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44)  Chapter 3  Wnt/β-Catenin Signaling Pathway in Canine Skin Melanoma and a Possibility as a Cancer Model for Human Skin Melanoma  By Jae-Ik Han and Ki-Jeong Na

45)  full free pdf
Mathur, Rohit, et al. “Targeting Wnt pathway in mantle cell lymphoma-initiating cells.” Journal of hematology & oncology 8.1 (2015): 63. Targeting Wnt pathway in mantle cell lymphoma initiating cells Mathur 2015

46)  Li, Yanyan, et al. “Implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds.” The Journal of nutritional biochemistry 22.9 (2011): 799-806.

Sulforaphane

47) Rodova, Mariana, et al. “Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.” PloS one 7.9 (2012): e46083.

Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer.

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Berberine  – Berberine inhibits WNT Pathways

48) Biofactors. 2013 Nov-Dec;39(6):652-62. Berberine acts as a natural inhibitor of Wnt/β-catenin signaling–identification of more active 13-arylalkyl derivatives. Albring KF1, Weidemüller J, Mittag S, Weiske J, Friedrich K, Geroni MC, Lombardi P, Huber O.

Aberrant activation of the canonical Wnt/β-catenin signaling pathway has been reported for numerous tumors of different origins. In most cases, mutations in components of the Wnt signaling pathway or in β-catenin itself were detected which ultimately induce a genetic program that promotes cell proliferation and attenuates apoptosis. Thus, targeting of Wnt/β-catenin signaling is of specific therapeutic interest.  As a result of berberine treatment, cellular levels of active β-catenin were reduced concomitant with an increase in the expression of E-cadherin.

48A) Cell Physiol Biochem. 2013;32(5):1213-24.  Berberine induces apoptosis in p53-null leukemia cells by down-regulating XIAP at the post-transcriptional level. Liu J1, Zhang X, Liu A, Liu S, Zhang L, Wu B, Hu Q.

Berberine exerts anticancer activities both in vitro and in vivo through different mechanisms. However, the underlying molecular mechanisms of berberine induced p53-independent apoptosis remain unclear.The p53-null leukemia cell line EU-4 cells were exposed to berberine. Then the cell viability and apoptosis were determined. Western blot and PCR were employed to detect the expression of apoptosis related protein, XIAP and MDM2. Small interfering RNA (siRNA) was applied to knock down endogenous expression of MDM2 and XIAP.
RESULTS:Berberine induced p53-independent, XIAP-mediated apoptotic cell death in p53-null leukemia cells. Treatment with berberine resulted in suppression of XIAP protein in a dose- and time- dependent manner. Berberine induced down-regulation of XIAP protein involving inhibition of MDM2 expression and a proteasome-dependent pathway. Moreover, inhibition of XIAP by berberine or siRNA increased the sensitivity of leukemia cells to doxorubicin-induced apoptosis.
CONCLUSION:  Our findings characterize the molecular mechanisms of berberine-induced caspase activation and subsequent apoptosis, and berberine may be a novel candidate inducer of apoptosis in leukemia cells, which normally lack p53 expression.

Berberine

49)  Eur J Pharmacol. 2014 Oct 5;740:584-95. Targets and mechanisms of berberine, a natural drug with potential to treat cancer with special focus on breast cancer.Jabbarzadeh Kaboli P1, Rahmat A2, Ismail P3, Ling KH4.

Berberine was shown to be effective in inhibiting cell proliferation and promoting apoptosis invarious cancerous cells. Some signaling pathways affected by berberine, including the MAP (mitogen-activated protein) kinase and Wnt/β-catenin pathways, are critical for reducing cellular migration and sensitivity to various growth factors.

Epiphany Against cancer

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50) Guamán Ortiz, Luis Miguel, et al. “Berberine, an epiphany against cancer.” Molecules 19.8 (2014):  12349-12367. Epiphany Against Cancer

BBR treatment promotes cell cycle arrest and death in human cancer cell lines, coupled to an increased expression of apoptotic factors

BBR functions as an inhibitor of the telomere elongation by blocking the telomerase activity through formation of a G-quadruplex with telomeric DNA
BBR has the potential to modulate and regulate Wnt/β-catenin pathway [56], which in normal cells is inactivated by ubiquitination and subsequent degradation of the β-catenin protein,

BBR was proved to alter the mitochondrial membrane potential (MMP), inhibit mitochondrial respiration leading to mitochondrial dysfunction and regulate the expression of Bcl-2 family members, as Mcl-1 [45,47]. Alterations in mitochondrial membrane stimulate the release of cytochrome c promoting the formation of reactive oxygen species (ROS) that trigger apoptosis that requires the activation of caspases and poly(ADP-ribose) polymerase-1 (PARP-1)
cleavage.

Integrity of P53 relevant

In general, the integrity of p53 is relevant because cells with p53wt were found to be very sensitive to BBR, whereas cell lines lacking functional p53 do not respond to BBR treatment.

BBR functions as an inhibitor of the telomere elongation by blocking the telomerase activity through formation of a G-quadruplex with telomeric DNA

BBR has the potential to modulate and regulate Wnt/ß-catenin pathway [56], which in normal cells is inactivated by ubiquitination and subsequent degradation of the ß-catenin protein,

Berberine Inhibits Cancer Stem Cells – Patent

51) Hsieh, Hsiu-Mei, et al. “Berberine-containing pharmaceutical composition for inhibiting cancer stem cell growth or carcinoma metastasis and application thereof.” U.S. Patent Application No. 14/790,154.

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Silibinin – Inhibits WNT pathway

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52) Tiwari, Prabha, and K. P. Mishra. “Silibinin in cancer therapy: A promising prospect.” Cancer Research Frontiers 1.3 (2015): 303-318.

Silibinin also inhibited Wnt/β-catenin signaling by suppressing Wnt co-receptor LRP6 expression in human breast cancer cells MDA-MB-231 and T-47D

Exogenous SOD markedly enhanced silibinin-induced apoptosis

clinical trials for the treatment of hepatotoxicity in childhood acute lymphoblastic leukemia (ALL). Silymarin (The target dose of silibinin was 5.1 mg/kg/day) was administered orally for 28 days and it significantally reduced liver toxicity in children with ALL (74). Recently a new silibinin drug formulation Legasil® administration improved hepatic failure due to extensive liver infiltration in a breast cancer patient (75).
A Phase II Study to Assess Efficacy of Combined Treatment with Erlotinib (Tarceva) and Silybin-phytosome (Siliphos) in Patients with EGFR mutant lung adenocarcinoma is going on (ClinicalTrials.gov Identifier:

Silibinin has also shown promising results against cancer stem cells, supporting further development of anti-cancer therapeutics that target tumor stem cells.

53) Siegel, Abby B., et al. “A phase I dose-finding study of silybin phosphatidylcholine (milk thistle) in patients with advanced hepatocellular carcinoma.” Integrative cancer therapies (2013): 1534735413490798.

Targeting Cancer Stem Cells – Pterostilbene

54) 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.

Breast cancer stem cells (BCSCs) constitute a small fraction of the primary tumor that can self-renew and become a drug-resistant cell population, thus limiting the treatment effects of chemotherapeutic drugs. The present study evaluated the cytotoxic effects of five phytochemicals including 6-gingerol (6-G), 6-shogaol (6-S), 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-HF), nobiletin (NOL), and pterostilbene (PTE) on MCF-7 breast cancer cells and BCSCs. The results showed that 6-G, 6-S, and PTE selectively killed BCSCs and had high sensitivity for BCSCs isolated from MCF-7 cells that expressed the surface antigen CD44(+)/CD24(-). 6-S and PTE induced cell necrosis phenomena such as membrane injury and bleb formation in BCSCs and inhibited mammosphere formation. In addition, 6-S and PTE increased the sensitivity of isolated BCSCs to chemotherapeutic drugs and significantly increased the anticancer activity of paclitaxel. Analysis of the underlying mechanism showed that 6-S and PTE decreased the expression of the surface antigen CD44 on BCSCs and promoted β-catenin phosphorylation through the inhibition of hedgehog/Akt/GSK3β signaling, thus decreasing the protein expression of downstream c-Myc and cyclin D1 and reducing BCSC stemness.

Pterostilbene – human lung adenocarcinoma

55) Yang, Yang, et al. “Pterostilbene exerts antitumor activity via the Notch1 signaling pathway in human lung adenocarcinoma cells.” PloS one 8.5 (2013): e62652.

antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway.  PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS).

Chloroquin and Pterostilbene – autophagy inhibitor potentiated pterostilbene effect

56) Papandreou, Ioanna, et al. “Plant stilbenes induce endoplasmic reticulum stress and their anti-cancer activity can be enhanced by inhibitors of autophagy.” Experimental cell research 339.1 (2015): 147-153.

We performed a screen of 1726 small, drug like molecules to identify those that could activate an ER-stress responsive luciferase gene. After secondary screening, we determined that the plant stilbenes pterostilbene and piceatannol were the most potent inducers of ER stress from this group. ER stress can be particularly toxic to cells with high ER load, so we examined their effect on cells expressing the Wnt family of secreted glycoprotein growth factors. Molecular analysis determined that these ER stress-inducing stilbenes could block Wnt processing and also induce autophagy in acute lymphoblastic leukemia cells expressing Wnt16. Combining pterostilbene (to induce ER stress) with chloroquine (to inhibit autophagy) lead to significant cellular toxicity in cells from aggressive acute lymphoblastic leukemia.
CONCLUSIONS:  Plant stilbenes are potent inducers of ER stress. However, their toxicity is more pronounced in cancer cells expressing Wnt growth factors. The toxicity of stilbenes in these ALL cells can be potentiated by the addition of autophagy inhibitors, suggesting a possible therapeutic application.

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Chloroquin- Mefloquin

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

Interestingly, CQ has been identified as a cancer stem cell targeting agent for other aggressive cancers including breast cancer11, 12, glioblastoma multiforme13, and chronic myeloid leukemia14. However, the mechanism by which CQ affects the CD44+/CD24-/low CSCs remains unclear.

2016
58) Eur J Pharmacol. 2016 Jan 15;771:139-44. Epub 2015 Dec 11.
Time to use a dose of Chloroquine as an adjuvant to anti-cancer chemotherapies.
Pascolo S1.

Chloroquine, a drug used for over 80 years to treat and prevent malaria and, more recently, to treat autoimmune diseases, is very safe but has a plethora of dose-dependent effects. By increasing pH in acidic compartments it inhibits for example lysosomal enzymes. In the context of cancer, Chloroquine was found to have direct effects on different types of malignancies that could potentiate chemotherapies. For example, the anti-malaria drug may inhibit both the multidrug-resistance pump and autophagy (mechanisms that tumor cells may use to resist chemotherapies), intercalate in DNA and enhance the penetration of chemotherapeutic drugs in cells or solid cancer tissues. However, these activities were mostly demonstrated at high doses of Chloroquine (higher than 10mg/kg or 10mg/l i.e. ca. 31µM). Nevertheless, it was reported that daily uptake of clinically acceptable doses (less than 10mg/kg) of Chloroquine in addition to chemo-radio-therapy increases the survival of glioblastoma patients (Sotelo et al., 2006; Briceno et al., 2007). However, the optimal dose and schedule of this multi-active drug with respect to chemotherapy has never been experimentally determined. The present article reviews the several known direct and indirect effects of different doses of Chloroquine on cancer and how those effects may indicate that a fine tuning of the dose/schedule of Chloroquine administration versus chemotherapy may be critical to obtain an adjuvant effect of Chloroquine in anti-cancer treatments. We anticipate that the appropriate (time and dose) addition of Chloroquine to the standard of care may greatly and safely potentiate current anti-cancer treatments.

Mefloquin – Gastric Cancer 2016

59) Liu, Yanwei, et al. “Mefloquine effectively targets gastric cancer cells through phosphatase-dependent inhibition of PI3K/Akt/mTOR signaling pathway.” Biochemical and biophysical research communications (2016).

Deregulation of PI3K/Akt/mTOR pathway has been recently identified to play a crucial role in the progress of human gastric cancer. In this study, we show that mefloquine, a FDA-approved anti-malarial drug, effectively targets human gastric cancer cells. Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC50 ~0.5-0.7 µM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors. The combination of mefloquine with paclitaxel enhances the activity of either drug alone in in vitro and in vivo. In addition, mefloquine potently decreased phosphorylation of PI3K, Akt, mTOR and rS6. Overexpression of constitutively active Akt significantly restored mefloquine-mediated inhibition of mTOR phosphorylation and growth, and induction of apoptosis, suggesting that mefloquine acts on gastric cancer cells via suppressing PI3K/Akt/mTOR pathway. We further show that mefloquine-mediated inhibition of Akt/mTOR singaling is phosphatase-dependent as pretreatment with calyculin A does-dependently reversed mefloquine-mediated inhibition of Akt/mTOR phosphorylation. Since mefloquine is already available for clinic use, these results suggest that it is a useful addition to the treatment armamentarium for gastric cancer.

pancreatic CA chloroquin

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60) Frieboes, Hermann B., et al. “Chloroquine-Mediated Cell Death in Metastatic Pancreatic Adenocarcinoma Through Inhibition of Autophagy.” JOP. Journal of the Pancreas 15.2 (2014): 189-197. Chloroquine Mediated Cell Death in Metastatic Pancreatic Adenocarcinoma Through Inhibition of Autophagy 2014 Hermann Frieboes

Melanoma-chloroquin

61) Egger, Michael E., et al. “Inhibition of autophagy with chloroquine is effective in melanoma.” journal of surgical research 184.1 (2013): 274-281.
BACKGROUND:  Cancer cells adapt to the stress resulting from accelerated cell growth and a lack of nutrients by activation of the autophagy pathway. Two proteins that allow cell growth in the face of metabolic stress and hypoxia are hypoxia-inducible factor-1a (HIF-1a) and heat shock protein 90 (Hsp 90). We hypothesize that chloroquine (CQ), an antimalarial drug that inhibits autophagosome function, in combination with either echinomycin, a HIF-1a inhibitor, or 17-dimethylaminoethylamino-17-dimethoxygeldanamycin (17-DMAG), an Hsp 90 inhibitor, will result in cytotoxicity in melanoma.
MATERIALS AND METHODS:  Multiple human melanoma cell lines (BRAF wild-type and mutant) were tested in vitro with CQ in combination with echinomycin or 17-DMAG. These treatments were performed in hypoxic (5% O2) and normoxic (18% O2) conditions. Mechanism of action was determined through Western blot of autophagy-associated proteins HIF-1a and Hsp 90.
RESULTS:  Chloroquine, echinomycin, and 17-DMAG each induced cytotoxicity in multiple human melanoma cell lines, in both normoxia and hypoxia. Chloroquine combined with echinomycin achieved synergistic cytotoxicity under hypoxic conditions in multiple melanoma cell lines (BRAF wild-type and mutant). Western blot analysis indicated that echinomycin reduced HIF-1a levels, both alone and in combination with CQ. Changes in LC3 flux indicated inhibition of autophagy at the level of the autophagosome by CQ therapy.
CONCLUSIONS:  Targeting autophagy with the antimalarial drug CQ may be an effective cancer therapy in melanoma. Sensitivity to chloroquine is independent of BRAF mutational status. Combining CQ with the HIF-1a inhibitor echinomycin improves cytotoxicity in hypoxic conditions.

Breast Cancer cell line

62) 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.

Prostate Cancer

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63) Yan, Kun-Huang, et al. “Mefloquine induces cell death in prostate cancer cells and provides a potential novel treatment strategy in vivo.” Oncology letters 5.5 (2013): 1567-1571.

Krudsood et al(7) reported that MQ caused a blood plasma concentration of 5,796 ng/ml (15.35 µM) in a clinical study on Plasmodium falciparum-infected adults. Dow et al(8) noted that higher blood levels of MQ were reached under therapeutic regimens (2.1–23 µM) rather than in prophylaxis (3.8 mM) (9,10).

Mefloquin in Lysosomes

64) Glaumann, Hans, Anne-Marie Motakefi, and Helena Jansson. “Intracellular distribution and effect of the antimalarial drug mefloquine on lysosomes of rat liver.” Liver 12.4 (1992): 183-190.

Abstract: Mefloquine was administered in a single dose (1–30 mg/100 g) to rats in order to study its subcellular distribution and effects on rat liver lysosomal structure and function. Subcellular fractionation showed a significant enrichment of mefloquine in lysosomes. Even repeated administration of mefloquine did not affect the levels of cytochrome-P-450 or its reductase, indicating, although not proving, that it is not metabolized by this mono-oxygenase system. Mefloquine caused an expansion of the lysosomal apparatus, earliest seen by 24 h and lasting for some 7 days. Initially, cytoplasmic constituents were seen inside the lysosomes. Later, the lysosomes harboured myelin-like figures (multilamellar bodies) disappearing after 7–10 days. The proteolytic and lipolytic capacity was assessed in isolated lysosomes. Mefloquine caused increased protein degradation but decreased breakdown of lipids. Concomitantly, all five major phospholipids (phosphatidyl-choline, -ethanolamine, -inositol, -serine and sphingomyelin) increased in the lysosomes. It is concluded that: (1) mefloquine is a lysosomotropic drug that accumulates in lysosomes; (2) mefloquine impairs lipid degradation with ensuing accumulation of lipids in lysosomes; and (3) lysosomal trapping explains the high volume distribution of mefloquine.

chloroquin

65) Eur J Pharmacol. 2009 Dec 25;625(1-3):220-33.Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies.  Solomon VR1, Lee H.

Chloroquine (CQ), N’-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine, is widely used as an effective and safe anti-malarial and anti-rheumatoid agent. CQ was discovered 1934 as “Resochin” by Andersag and co-workers at the Bayer laboratories. Ironically, CQ was initially ignored for a decade because it was considered too toxic to use in humans. CQ was “re-discovered” during World War II in the United States in the course of anti-malarial drug development. The US government-sponsored clinical trials during this period showed unequivocally that CQ has a significant therapeutic value as an anti-malarial drug. Consequently, CQ was introduced into clinical practice in 1947 for the prophylaxis treatment of malaria (Plasmodium vivax, ovale and malariae). CQ still remains the drug of choice for malaria chemotherapy because it is highly effective and well tolerated by humans. In addition, CQ is widely used as an anti-inflammatory agent for the treatment of rheumatoid arthritis, lupus erythematosus and amoebic hepatitis. More recently, CQ has been studied for its potential as an enhancing agent in cancer therapies. Accumulating lines of evidence now suggest that CQ can effectively sensitize cell-killing effects by ionizing radiation and chemotherapeutic agents in a cancer-specific manner. The lysosomotrophic property of CQ appears to be important for the increase in efficacy and specificity. Although more studies are needed, CQ may be one of the most effective and safe sensitizers for cancer therapies. Taken together, it appears that the efficacy of conventional cancer therapies can be dramatically enhanced if used in combination with CQ and its analogs.

Randomized human trial chloroquin prolonged survival in Glioblastoma.

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66) Ann Intern Med. 2006 Mar 7;144(5):337-43. Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double-blind, placebo-controlled trial. Sotelo J1, Briceño E, López-González MA.  Adding Chloroquine to Conventional Treatment for Glioblastoma Multiforme Sotelo Julio 2006

Malignant cell clones resistant to chemotherapy and radiotherapy frequently lead to treatment failure in patients with glioblastoma multiforme. Preliminary studies suggest that adding chloroquine to conventional therapy may improve treatment outcomes.
OBJECTIVE:   To examine the effect of adding chloroquine to conventional therapy for glioblastoma multiforme.
DESIGN: Randomized, double-blind, placebo-controlled trial.
SETTING: National Institute of Neurology and Neurosurgery of Mexico.
PATIENTS: 30 patients with surgically confirmed glioblastoma confined to 1 cerebral hemisphere, with a Karnofsky performance score greater than 70, no comorbid disease, and age younger than 60 years.
INTERVENTIONS: Oral chloroquine at 150 mg/d for 12 months beginning on postoperative day 5 or placebo. All patients received conventional chemotherapy and radiotherapy.
MEASUREMENTS: Primary outcome was survival after surgery; surviving patients were followed up to October 2005. Periodic evaluation using the Karnofsky scale and imaging studies, as well as hematologic tests and ophthalmologic examinations, was done in all patients.
RESULTS: Median survival after surgery was 24 months for chloroquine-treated patients and 11 months for controls. At the end of the observation period, 6 patients treated with chloroquine had survived 59, 45, 30, 27, 27, and 20 months, respectively; 3 patients from the control group had survived 32, 25, and 22 months, respectively. Although not statistically significantly different, the rate of death with time was approximately half as large in patients receiving chloroquine as in patients receiving placebo (hazard ratio, 0.52 [95% CI, 0.21 to 1.26]; P = 0.139).
LIMITATIONS: The observed advantage of chloroquine may be due to chance; differences in pretreatment characteristics and conventional treatment regimens could not be adjusted for because of the small sample size.
CONCLUSIONS:  Chloroquine may improve mid-term survival when given in addition to conventional therapy for glioblastoma multiforme. These results suggest that larger, more definitive studies of chloroquine as adjuvant therapy for glioblastoma are warranted.

67) Kimura, Tomonori, et al. “Chloroquine in cancer therapy: a double-edged sword of autophagy.” Cancer research 73.1 (2013): 3-7.
The dosage of chloroquine usually ranges between 100 and 500 mg/day. Side effects are minimal at low doses, while many more toxic effects occur at higher doses, such as visual disturbances, gastrointestinal upset, electrocardiographic changes, headache, and pruritus.

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Mefloquine  –  AML-stem cells killing Acute Myeloid Leukemia

2014
68) Zhang, Hui, Hai Fang, and Kankan Wang. “Reactive oxygen species in eradicating acute myeloid leukemic stem cells.” Stem Cell Investigation 1.6 (2014).

Mefloquine  –  AML-stem cells killing

Most recently, Sukhai et al. an antimalarial agent called ‘mefloquine’ for its capacity in targeting newly AML-stem cells (32). This study reveals a previously unappreciated mechanism for AML-stem cells killing. Mefloquine is able to disrupt lysosome integrity releasing hydrolases, lipases, proteases and cathepsins. This disruption subsequently increases the levels of ROS and triggers death of AML cells and stem cells in a caspase-independent manner. Mefloquine is widely used for malaria therapy and chemoprevention, its clinical safety has been already characterized in large cohorts of clinical trials. Its AML targeting efficacy will be next for evaluation.

Fenretinide Vit A –Also from Zhang, Hui,  Stem Cell Investigation 1.6 (2014).

Fenretinide is a well-tolerated vitamin A derivative that lacks a carboxyl functional group likely necessary for retinoid receptor activity. Our most recent study has shown that it is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells, at physiologically achievable concentrations (5 µM). Fenretinide-induced AML-stem cells death is associated with the rapid generation of ROS, induction of genes responsible for stress responses and apoptosis, and repression of genes involved in NF-?B and Wnt signaling (34). Though there is no clinical trial ongoing for AML treatment, fenretinide has been verified its safety and low toxicity in phase II-III clinical trials for solid tumors such as small cell lung cancer, breast cancer and prostate cancer (82-85). Moreover, via bioinformatics analysis we have observed that fenretinide down-regulated genes are significantly correlated with genes related to a poor prognosis/relpase of AML. We anticipate that fenretinide is a potent AML-stem cells targeting candidate in the treatment of AML.

Parthenolide (PTL) and dimethylaminoparthenolide (DMAPT)

PTL is a naturally occurring small molecule that has been evaluated for in vitro and in vivo efficacy on AML progenitor and stem cell populations. However, low soluble feature makes its pharmacologic potential less attractive. Instead, DMAPT, a dimethylamino analog of PTL, demonstrates 1,000-fold greater solubility in water than PTL. Both of DMAPT and PTL show similar efficacy on AML-stem cells through the similar molecular mechanisms, including the elevated ROS, p53 activation and NF-?B inactivation. Canine xenograft experiments, an equivalent to phase I clinical trials, show that the pharmacologic properties of DMAPT are superior to PTL (26,27). These data call for further studies on clinical safety and translational efficacy of DMAPT/PTL in AML treatment.

Mefloquin thioridazine (Mellaril) – AML cell line

69) Sachlos, Eleftherios, et al. “Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.” Cell 149.6 (2012): 1284-1297.

compounds that induce differentiation can be identified based on the reduction of GFP intensity in neoplastic hPSC reporter lines and could be exploited for chemical screening.

This hPSC-screening platform was validated by using an assembled library of 51 defined compounds with established stem cell and anticancer activity

Compounds that can induce differentiation of neoplastic hPSCs are potential anticancer candidates.  the ideal compound should only differentiate neoplastic hPSCs while not affecting normal hPSCs.  we extended our tests to chemical libraries composed of 590 well-established annotated compounds from the NIH Clinical Collection and Canadian Compound Collection

11 compounds were identified to induce differentiation as indicated by a reduction in both GFP percentage residual activity (%RA) and Hoechst %RA (Figures 2B and 2C). Four of these compounds (indatraline, thioridazine, azathioprine, and mefloquine) were identified as candidate compounds based on clustering and levels of Hoechst %RA in excess of 30%.

Only thioridazine and mefloquine were found to possess EC50 values lower than the 10 µM target threshold

At both 1 µM and 10 µM salinomycin reduced AML-blast CFU potential (Figure 3J) but also reduced HSPC CFU potential over all doses tested (Figure 3I), indicative of nonspecific toxicity in the hematopoietic system. In contrast, mefloquine and thioridazine reduced AML-blast CFU formation (Figure 3J) while having little effect on HSPC CFU potential (Figure 3I) and multilineage composition (Figure S3D), indicating that mefloquine and thioridazine do not alter normal hematopoiesis.

Alternatively, the combination of thioridazine at 10 µM with AraC at 100 nM demonstrates almost complete elimination of AML-blast-CFUs while preserving HSPC function (Figure 7F), suggesting that these specified concentrations can induce remission and prevent relapse of AML in patients. Collectively, these data show the synergistic benefit of combining an anti-LSC agent (thioridazine) with an antiproliferative agent (AraC) currently used as a single first-line treatment for human AML and to targeting CSCs in addition to other cells in the leukemogenic hierarchy. This combined effect with thioridazine is likely to have significant benefit to AML patients as it can reduce the severe cytotoxic effects associated with high-dose AraC therapy, as illustrated in

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Mefloquin Patent 2002

70) Treatment of cancer with mefloquine, its purified enantiomers, and mefloquine analogs  US 20030216426 A1
Regents Of The University Of California
Cancers, particularly solid tumors (e.g., breast, colon and ovarian cancers) and cancers of the hematologic system, e.g., hemopoietic cancers such as leukemias, lymphomas or myelomas, are treated by administration of a therapeutically effective amount of a compound having the formula (I):

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Antibiotics Eradicate Cancer Stem Cells

2015 OncoTarget

73) Lamb, Rebecca, et al. “Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease.”  Lamb Rebecca Antibiotics that target mitochondria effectively eradicate cancer stem cells 2015 OncoTarget

Finally, recent clinical trials with doxycycline and azithromycin (intended to target cancer-associated infections, but not cancer cells) have already shown positive therapeutic effects in cancer patients, although their ability to eradicate cancer stem cells was not yet appreciated.

Doxycycline for Lymphoma

2015
74) Ann Hematol. 2015 Apr;94(4):575-81.  Long-term outcomes of first-line treatment with doxycycline in patients with previously untreated ocular adnexal marginal zone B cell lymphoma.

Han JJ1, Kim TM, Jeon YK, Kim MK, Khwarg SI, Kim CW, Kim IH, Heo DS.
Author information 1Department of Internal Medicine, Seoul National University Hospital, 101  Daehak-ro, Jongno-gu, Seoul, 110-744, South Korea.

Ocular adnexal lymphoma (OAL) has been associated with Chlamydophila psittaci infection, for which  doxycycline has been suggested as a treatment option. We conducted this study to evaluate the long-term results of first-line doxycycline treatment in patients with OAL. Ninety patients withhistologically confirmed OAL with marginal zone B cell lymphoma were enrolled. Each patient received one or two cycles of doxycycline (100 mg bid) for 3 weeks. After a median follow-up period of 40.5 months (8-85), the 5-year progression-free survival (PFS) rate was 60.9 %. All patients were alive at the last follow-up date. Thirty-one patients (34 %) showed local treatment failure without systemic spread. However, PFS rate in these patients was 100 % after salvage chemotherapy and/or radiotherapy.

PFS was independently predicted in multivariate analysis by the tumor-node-metastasis (TNM) staging (hazard ratio [HR], 4.35; 95 % confidence interval [CI], 2.03-9.32; P < 0.001) and number of cycles of doxycycline (HR, 0.31; 95 % CI, 0.14-0.69; P = 0.004). No serious adverse event was reported during doxycycline therapy. In conclusion, first-line doxycycline therapy was effective and safe.

Patients who failed to respond to doxycycline therapy were successfully salvaged with chemotherapy and/or radiotherapy without compromising long-term outcomes. Patients with T1N0M0 disease could be considered good candidates for first-line doxycycline.

2015

13 patients antibiotics alone for gastric lymphoma – HP eradication regimen

75) Ann Hematol. 2015 Jun;94(6):969-73. doi: 10.1007/s00277-014-2298-3. Epub 2015 Jan 13. Antibiotic treatment as sole management of Helicobacter pylori-negative gastric MALT lymphoma: a single center experience with prolonged follow-up. Raderer M1, Wöhrer S, Kiesewetter B, Dolak W, Lagler H, Wotherspoon A, Muellauer L, Chott A.

Relatively little is known about the long-term outcome of patients with Helicobacter pylori (HP)-negative gastric lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) with antibiotic therapy as sole management. We have analyzed all patients with HP-negative gastric MALT lymphoma undergoing antibiotic therapy as sole management of their disease. HP negativity was defined as negative histology, breath test and serology, and response to treatment, survival and long-term outcome was assessed together with clinico-pathological characteristics including t(11; 18) (q21; q21) translocation. Out of 97 patients with gastric MALT lymphoma, 24 were HP-negative, and 13 (5 females and 8 males) underwent only antibiotic management for initial therapy. Eight had stage I and five were found to have stage II disease, with three patients suffering from an underlying autoimmune disease. Antibiotic therapy consisted of standard HP eradication regimens consisting of clarithromycin in all patients, along with metronidazole in seven and amoxicillin in six plus a proton-pump inhibitor. After a median follow-up of 95 months (42-, 181+), 12/13 patients are alive. Six patients with stage I disease achieved an objective response (five complete (CR) and one partial remission, 46 %), four had stable disease (lasting 11-27 months), and three progressed. All patients with stable disease received chemotherapy, but only one patient due to clear cut progression. One patient relapsed 23 months after initial CR, and achieved a second CR with antibiotics now lasting 87 months. These results indicate that a relevant percentage of patients with HP-negative gastric MALT lymphoma may benefit from antibiotic therapy and do not require additional oncological therapies. Our data suggest that the remissions seen in these patients might be durable as evidenced by prolonged follow-up in our series.

2013

76) Kiesewetter, Barbara, and Markus Raderer. “Antibiotic therapy in nongastrointestinal MALT lymphoma: a review of the literature.” Blood 122.8 (2013): 1350-1357.

A single course of oral doxycycline at a dose of 100 mg given twice a day for 3 weeks was the most popular regimen and was used by most investigators.14⇓⇓-17,19⇓⇓-22 By contrast, Kim and coworkers19 added a second course after an interval of 3 weeks for patients with residual eye-related symptoms after the initial cycle. The activity of a 6-month oral application of 500 mg clarithromycin twice a day was assessed in an Italian pilot study,18 assuming potential additional direct anticancer effects of macrolide antibiotics through changes in apoptotic mechanisms of tumor cells. In addition, 1 patient received HP eradication as first-line treatment of OAML.  CR was achieved in 23 patients (18%) out of the collective of all 131 patients reported. Thirty-six (27%) had a PR

2006

77) Ferreri, Andrés JM, et al. “Bacteria-eradicating therapy with doxycycline in ocular adnexal MALT lymphoma: a multicenter prospective trial.” Journal of the National Cancer Institute 98.19 (2006):1375-1382.

Background: An association between ocular adnexal MALT lymphoma (OAL) and Chlamydia psittaci (Cp) infection has been proposed, and recent reports suggest that doxycycline treatment causes tumor regression in patients with Cp-related OAL. The effectiveness of doxycycline treatment in Cp-negative OAL has not been tested. Methods: In a prospective trial, 27 OAL patients (15 newly diagnosed and 12 having experienced relapse) were given a 3-week course of doxycycline therapy. Objective lymphoma response was assessed by computerized tomography scans or magnetic resonance imaging at 1, 3, and 6 months after the conclusion of therapy and every 6 months during follow-up. Cp infection in patients was determined by touchdown enzyme time-release polymerase chain reaction (TETR-PCR). Statistical tests were two-sided. Results: Eleven patients were Cp DNA–positive and 16 were Cp DNA negative. Doxycycline was well tolerated. At a median follow-up of 14 months, lymphoma regression was complete in six patients, and a partial response (≥50% reduction of all measurable lesions) was observed in seven patients (overall response rate [complete and partial responses] = 48%). Lymphoma regression  was observed in both Cp DNA–positive patients (seven of 11 experienced regression) and Cp DNA–negative patients (six of 16 experienced regression) (64% versus 38%; P = .25, Fisher’s exact test). The three patients with regional lymphadenopathies and three of the five patients with bilateral disease achieved objective response. In relapsed patients, response was observed both in previously irradiated and nonirradiated patients. The 2-year failure-free survival rate among the doxycycline- treated patients was 66% (95% confidence interval = 54 to 78), and 20 of the 27 patients were progression free. Conclusions: Doxycycline is a fast, safe, and active therapy for Cp DNA–positive OAL that was effective even in patients with multiple failures involving previously irradiated areas or regional lymphadenopathies. The responses observed in PCR-negative OAL may suggest a need for development of more sensitive methods for Cp detection and investigation of the potential role of other doxycycline-sensitive bacteria.

Ferreri et al conducted a prospective phase 2 clinical trial of 27 patients (15 newly diagnosed and 12 relapsed) with OAML, using doxycycline 100 mg orally twice daily for 3 weeks.  Partial or complete lymphoma regression after antibiotic therapy was observed in 7 of 11 Cp-positive and 6 of 16 Cp-negative patients, with an overall response rate of 48%. The 2-year failure-free survival rate among patients treated with doxycycline was 66%

Abramson et al84 treated 3 patients with biopsy-proven conjunctival MALT lymphoma with antibiotic therapy, resulting in 2 complete remissions and 1 partial response.

Husain et al43 conducted a meta-analysis, identifying 4 studies with a total of 42 patients who had

been treated with oral doxycycline.

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full free pdf
78) Husain, Amina, et al. “Meta–analyses of the association between Chlamydia psittaci and ocular adnexal lymphoma and the response of ocular adnexal lymphoma to antibiotics.” Cancer 110.4 (2007): 809-815.
Abramson DH, Rollins I, Coleman M. Periocular mucosa-associated lymphoid/low grade lymphomas: treatment with antibiotics. Am J Ophthalmol. 2005;140:729–730. Am J Ophthalmol. 2005 Oct;140(4):729-30.
Periocular mucosa-associated lymphoid/low grade lymphomas: treatment with antibiotics. Abramson DH1, Rollins I, Coleman M.

To report on the treatment of primary mucosa-associated lymphoid tumors (MALT)/low grade lymphomas of the conjunctiva/orbit treated solely with systemic antibiotics. DESIGN: Retrospective interventional case series.
METHODS: Three adult patients with biopsy/marker proven MALT lymphomas of the conjunctiva/orbit were treated with systemic antibiotics and followed for signs of local or systemic relapse.
RESULTS: All three patients showed a response to antibiotics based on clinical, ultrasonographic, and MRI/CT imaging studies. Two patients have had complete remissions (42 months follow-up) and one a partial remission (18 months). No systemic relapses have occurred.
CONCLUSION: MALT/low grade lymphomas of the conjunctiva/orbit respond to systemic antibiotic therapy and may have complete remissions.

Ivermectin

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Ivermectin inhibits WNT-TCF pathway

Ivermectin – WNT pathway

79) Melotti, Alice, et al. “The river blindness drug Ivermectin and related macrocyclic lactones inhibit WNT‐TCF pathway responses in human cancer.” EMBO molecular medicine (2014): e201404084.

Ivermectin used as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.

We find that macrocyclic lactones of the Avermectin family have specific anti-WNT-TCF response activity in human cancer cells and that the clinically approved compound Ivermectin (EMEA- and FDA-approved) is a specific WNT-TCF response blocker at low micromolar concentrations.

cancer stem cells
Pre-treatment with Ivermectin and Selamectin inhibits colon cancer stem cell self-renewal in clonogenic spheroid assays.  These results suggest an action on both the bulk of the tumor and its cancer stem cells.

Moreover, they might also be useful as routine prophylactic agents, for instance against nascent TCF-dependent intestinal tumors in patients with familial polyposis and against nascent sporadic colon tumors in the general aging population.

Constitutive activation of canonical WNT-TCF signaling is implicated in multiple diseases, including intestine and lung cancers, but there are no WNT-TCF antagonists in clinical use. We have performed a repositioning screen for WNT-TCF response blockers aiming to recapitulate the genetic blockade afforded by dominant-negative TCF. We report that Ivermectin inhibits the expression of WNT-TCF targets, mimicking dnTCF, and that its low concentration effects are rescued by direct activation by TCFVP16. Ivermectin inhibits the proliferation and increases apoptosis of various human cancer types. It represses the levels of C-terminal ß-CATENIN phosphoforms and of CYCLIN D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases.In vivo, Ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without obvious side effects. Analysis of single semi-synthetic derivatives highlights Selamectin, urging its clinical testing and the exploration of the macrocyclic lactone chemical space. Given that Ivermectin is a safe anti-parasitic agent used by > 200 million people against river blindness, our results suggest its additional use as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.

Wingless/integrase-1 (WNT) signaling.  The name Wnt was a portmanteau of int and Wg and stands for “Wingless-related integration site.  Other cancers also show an active canonical WNT pathway; these include carcinomas of the lung, stomach, cervix, endometrium, and lung as well as melanomas and gliomas

We have used a transcriptional reporter assay for TCF activity driven by APC-insensitive N’?ß-CATENIN, to test a collection of clinical-trial tested small molecules (Microsource 1040 library).  Of the 4 putative antagonists, only one, 4B5 (Avermectin B1), perfectly tracked the gene expression profile induced by dnTCF4.  anti-helmintic agent Avermectin B1, which belongs to the 16-membered Avermectin macrocyclic lactone family derived fromStreptomyces avermitilis.

The drug is used in humans against insect and worm infections, including river blindness caused by Onchocerca volvulus.  The dominant negative forms of TCF (dn-TCF) that can be used to block Wnt signaling in the nucleus. as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.

We find that macrocyclic lactones of the Avermectin family have specific anti-WNT-TCF response activity in human cancer cells and that the clinically approved compound Ivermectin (EMEA- and FDA-approved) is a specific WNT-TCF response blocker at low micromolar concentrations.

Ivermectin Inhibits cancer stem cells

Pre-treatment with Ivermectin and Selamectin inhibits colon cancer stem cell self-renewal in clonogenic spheroid assays

These results suggest an action on both the bulk of the tumor and its cancer stem cells.

Moreover, they might also be useful as routine prophylactic agents, for instance against nascent TCF-dependent intestinal tumors in patients with familial polyposis and against nascent sporadic colon tumors in the general aging population.

commercial form from the pharmacy, Stromectol™,

Selamectin, which scored as toxic in the primary screen at 10 µM, was ˜ tenfold more potent than ivermectin.

Here we report that Ivermectin (Campbellet al, 1983), an off-patent drug approved for human use, and related macrocyclic lactones, have WNT-TCF pathway response blocking and anti-cancer activities. Whereas the exact molecular target for Ivermectin and Selamectin that affects WNT-TCF responses remains to be identified, the present findings show that these drugs block WNT-TCF pathway responses, likely acting at the level of ß-CATENIN/TCF function, affecting ß-CATENIN phosphorylation status.

Cell toxicity appears at doses greater (> 10 µM for 12 h or longer or > 5 µM for 48 h or longer for Ivermectin) than those required to block TCF responses and induce apoptosis.

This drug does not cross the blood–brain barrier.

Indications may include treatment for incurable ß-CATENIN/TCF-dependent advanced and metastatic human tumors of the lung, colon, endometrium, and other organs.

Moreover, they might also be useful as routine prophylactic agents, for instance against nascent TCF-dependent intestinal tumors in patients with familial polyposis and against nascent sporadic colon tumors in the general aging population.

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80) NEW ZEALAND DATA SHEET STROMECTOL ivermectin 3 mg tablet 2011

81) Chhaiya, Sunita B., et al. “IJBCP International Journal of Basic & Clinical Pharmacology.”  International Journal 2.6 (2013): 799. Chhaiya, Sunita. Ivermectin pharmacology and therapeutic applications Sunita Chhaiya 2012

82) The Pharmacokinetics and Interactions of Ivermectin in Humans.  Canga, Aránzazu González, et al.”The pharmacokinetics and interactions of ivermectin in humans—a mini-review.” The AAPS journal 10.1 (2008): 42-46.

Ivermectin is exceptionally potent, with effective dosages
levels that are unusually low. In the treatment of onchocerciasis,
the optimal dose of ivermectin is 150 µg/kg, but the
frequency of administration is still controversial, ranging from
150 µg/kg once to three times yearly. The optimal duration of
treatment has not been established (6).

It is effective in most patients with scabies after a single oral dose of 200 µg/kg, but often the regimen involves two or three repeated doses, separated by interval of 1 or 2 weeks (7).

prolonged prothrombin ratios were observed in 148  subjects given ivermectin orally. Although no patients suffered bleeding complications, factor II and VII levels were reduced in most of them, suggesting interference with vitamin K
metabolism.

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Take Ivermectin with FOod every 4 days.

83) J Clin Pharmacol. 2002 Oct;42(10):1122-33.
Safety, tolerability, and pharmacokinetics of escalating high doses of ivermectin in healthy adult subjects.  Guzzo CA1, Furtek CI, Porras AG, Chen C, Tipping R, Clineschmidt CM, Sciberras DG, Hsieh JY, Lasseter KC.

Safety and pharmacokinetics (PK) of the antiparasitic drug ivermectin, administered in higher and/or more frequent doses than currently approved for human use, were evaluated in a double-blind, placebo-controlled, dose escalation study. Subjects (n = 68) were assigned to one of four panels (3:1, ivermectin/placebo): 30 or 60 mg (three times a week) or 90 or 120 mg (single dose). The 30 mg panel (range: 34 7-594 microg/kg) also received a single dose with food after a 1-week washout. Safety assessments addressed both known ivermectin CNS effects and general toxicity. The primary safety endpoint was mydriasis, accurately quantitated by pupillometry. Ivermectin was generally well tolerated, with no indication of associated CNS toxicity for doses up to 10 times the highest FDA-approved dose of 200 microg/kg. All dose regimens had a mydriatic effect similar to placebo. Adverse experiences were similar between ivermectin and placebo and did not increase with dose. Following single doses of 30 to 120 mg, AUC and Cmax were generally dose proportional, with t(max) approximately 4 hours and t1/2 approximately 18 hours. The geometric mean AUC of 30 mg ivermectin was 2.6 times higher when administered with food. Geometric mean AUC ratios (day 7/day 1) were 1.24 and 1.40 for the 30 and 60 mg doses, respectively, indicating that the accumulation of ivermectin given every fourth day is minimal. This study demonstrated that ivermectin is generally well tolerated at these higher doses and more frequent regimens.

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84) Editorial Commentary: Ivermectin as a Complementary Strategy to Kill Mosquitoes and Stop Malaria Transmission? Richard W. Steketee1 and Feiko O. ter Kuile2

Repeated doses of up to 800 µg/kg have been used in the treatment of onchocerciasis [8–10]. Furthermore, earlier dose-escalation studies with ivermectin have shown that doses up to 2000 µg/kg (ie, 5 times the highest US Food and Drug Administration–approved dose) are well tolerated with no indication of central nervous system or general toxicity [11]. Additional dosing during the third day of the ACT treatment (as done in this trial) or at day 7 (and perhaps at day 14)

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Ivermectin safely given incombination with Artemisinin Derivative Artemether

85)  Efficacy and Safety of the Mosquitocidal Drug Ivermectin to Prevent Malaria Transmission After Treatment: A Double-Blind, Randomized, Clinical Trial
André Lin Ouédraogo1,a, Guido J. H. Bastiaens2,a, Alfred B. Tiono1, Wamdaogo M. Guelbéogo1, Kevin C. Kobylinski3,4, Alphonse Ouédraogo1, Aïssata Barry1, Edith C. Bougouma1, Issa Nebie1, Maurice San Ouattara1, Kjerstin H. W. Lanke2, Lawrence Fleckenstein5, Robert W. Sauerwein2, Hannah C. Slater6, Thomas S. Churcher6, Sodiomon B. Sirima1, Chris Drakeley7, and Teun Bousema2,7
Background. Artemisinin combination therapy effectively clears asexual malaria parasites and immature gametocytes but does not prevent posttreatment malaria transmission. Ivermectin (IVM) may reduce malaria transmission by killing mosquitoes that take blood meals from IVM-treated humans.
Methods. In this double-blind, placebo-controlled trial, 120 asymptomatic Plasmodium falciparum parasite carriers were randomized to receive artemether-lumefantrine (AL) plus placebo or AL plus a single or repeated dose (200 µg/kg) of ivermectin (AL-IVM1 and AL-IVM2, respectively). Mosquito membrane feeding was performed 1, 3, and 7 days after initiation of treatment to determine Anopheles gambiae and Anopheles funestus survival and infection rates.
Results. The AL-IVM combination was well tolerated. IVM resulted in a 4- to 7-fold increased mortality in mosquitoes feeding 1 day after IVM (P < .001). Day 7 IVM plasma levels were positively associated with body mass index (r = 0.57, P < .001) and were higher in female participants (P = .003), for whom An. gambiae mosquito mortality was increased until 7 days after a single dose of IVM (hazard rate ratio, 1.34 [95% confidence interval, 1.07–1.69]; P = .012). Although we found no evidence that IVM reduced Plasmodium infection rates among surviving mosquitoes, the mosquitocidal effect of AL-IVM1 and AL-IVM2 resulted in 27% and 35% reductions, respectively, in estimated malaria transmission potential during the first week after initiation of treatment.
Conclusions. We conclude that IVM can be safely given in combination with AL and can reduce the likelihood of malaria transmission by reducing the life span of feeding mosquitoes.

86) Chaccour, Carlos J., et al. “Ivermectin to reduce malaria transmission: a research agenda for a promising new tool for elimination.” Malar J 12.153 (2013): 10-1186.

Recent publications have highlighted the likely benefit of combining ivermectin with drugs such as artemisinin combination therapy (ACT). ACT is
highly effective in most malaria-endemic settings but does not prevent malaria-transmission in the first weeks after treatment [53,54].
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87)  Ivermectin Use in Scabies ROBERT S. FAWCETT, M.D., M.S., York Hospital Family Practice Residency, York, Pennsylvania.  Am Fam Physician. 2003 Sep 15;68(6):1089-1092.

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ivermectin cancer cell death

2015

88)  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).

We found 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.
FIg 7 Model of P2X4/P2X7/Pannexin-1-induced cancer cell death.

Ivermectin induces P2X4/P2X7-dependent activation of Pannexin-1 channels and release of ATP. The release of ATP might be transiently protective, but only in cell types that are highly sensitive to Ivermectin-induces cell swelling when ATP and Ca2+ signaling are essential for control of cell volume. In cancer cells where no cell size changes can be observed (for example human TNBC MDA-MB-231 cells), high concentrations of ATP (1–3?mM) immediately enhance Ivermectin cytotoxicity. Potentiated P2X7 receptor signaling drives a fast progressing necrotic/pyroptotic mechanism driven by NADPH oxidases-generated ROS, cytosolic Ca2+/CaMKII activation, and MPTP, and characterized by caspase-1 cleavage, due to possible NLRP3 inflammasome activation. Necrotic killing is followed by a slower progressing apoptotic cell death program mediated by caspase-3 activation. The failure of the default apoptotic pathway might be attributed to faster activation of caspase-1, inadequate autophagic control of mitochondrial MPTP, collapse of cellular energy metabolism, resulting in rapid progression of necrotic cell death. Damage to mitochondria and ER stress as well as potential depletion of cellular ATP reserves simultaneously promote autophagy that might render even the slower apoptotic pathway immunogenic.
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2004

89) Drinyaev, Victor A., et al. “Antitumor effect of avermectins.” European journal of pharmacology 501.1 (2004): 19-23.

90) Searching for Ivermectin Deficiency Syndrome by Dr Simon Yu author of Accidental Cure.

91) 2012 Patent for Ivermectin as treatment for hematologic malignancy (including mantle cell lymphoma.)  Use of synergistic combinations of an avermectin and an antineoplastic compounds for the treatment of hematological malignancies EP 2498785 A1 (text from WO2011054103A1)

92) Sharmeen, Sumaiya, et al. “The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.” Blood 116.18 (2010): 3593-3603.

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

curcumin Beta Cateninin

93)  FEBS Lett. 2005 May 23;579(13):2965-71. Epub 2005 Apr 21.
The inhibitory mechanism of curcumin and its derivative against beta-catenin/Tcf signaling.  Park CH1, Hahm ER, Park S, Kim HK, Yang CH.
We investigated the inhibitory mechanism of curcumin and its derivative (CHC007) against beta-catenin/T-cell factor (Tcf) signaling in various cancer cell lines. Curcumin is known to inhibit beta-catenin/Tcf transcriptional activity in HCT116 cells but not in SW620 cells. To clarify the inhibitory effect of curcumin against beta-catenin/Tcf signaling, we tested several cancer cell lines. In addition, in order to verify the inhibitory mechanism, we performed reporter gene assay, Western blot, immunoprecipitation, and electrophoretic mobility shift assay. Since inhibitors downregulated the transcriptional activity of beta-catenin/Tcf in HEK293 cells transiently transfected with S33Y mutant beta-catenin gene, whose product is not induced to be degraded by adenomatous polyposis coli-Axin-glycogen synthase kinase 3beta complex, we concluded that the inhibitory mechanism was related to beta-catenin itself or downstream components. Western blot analysis suggested that no change in the amount of cytosolic and membranous beta-catenin in a cell occurred; however, nuclear beta-catenin and Tcf-4 proteins were markedly reduced by inhibitors and this lead to the diminished association of beta-catenin with Tcf-4 and to the reduced binding to the consensus DNA. In the present study, we demonstrate that curcumin and its derivative are excellent inhibitors of beta-catenin/Tcf signaling in all tested cancer cell lines and the reduced beta-catenin/Tcf transcriptional activity is due to the decreased nuclear beta-catenin and Tcf-4.

In all cell lines tested, β-catenin’s transcriptional activity was suppressed by inhibitors dependent on the concentration  In this regard, we assume that the antitumor activity of curcumin occurs via the inhibition of the β-catenin/Tcf signaling pathway by reducing the nuclear β-catenin and Tcf-4 proteins.

94) PHI Test Dr Emil Schandl AMerican Metabolic Lab Hollywood Florida
Published on Jun 25, 2015
The Role of Human Autocrine Motility Factor in Tumor Malignancy.
Recorded at the A4M 23rd Annual World Congress on Anti-Aging Medicine, in Hollywood, FL on May 2015.  PHI, the Human Autocrine Motility Factor (AMF) is an extremely important tumor marker .

95) Clarke, John D., Roderick H. Dashwood, and Emily Ho. “Multi-targeted prevention of cancer by sulforaphane.” Cancer letters 269.2 (2008): 291-304.

ROS: The production of reactive oxygen species (ROS) has been postulated to be a key mechanism by which SFN induces apoptosis. SFN administration to PC3 prostate cancer cells resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome C, and apoptosis. All of these effects were reversed with administration of the antioxidant N-acetylcysteine and overexpression of catalase [48]. The authors indicated that conjugation of SFN with GSH, a necessary step in SFN metabolism, depletes the intracellular concentration of GSH and potentially lowers the oxidative stress threshold of the cell. In their experiments SFN treatment increases mitochondrial ROS production and induces apoptosis as indicated by release of cytochrome C via both death-receptor and mitochondrial caspase cascades [48].

96) Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, Xiao D, Zeng Y, Watkins SC, Johnson CS, Trump DL, Lee YJ, Xiao H, Herman-Antosiewicz A. Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species. J. Biol. Chem. 2005;280:19911–19924.

SFN treatment caused a rapid decline in the level of GSH. For instance, the GSH levels in PC-3 cells treated for 3 and 6 h with 40 μm SFN were reduced by about 90 and 94%, respectively, compared with control (Fig. 2C). We also found that SFN treatment causes a rapid and marked depletion of intracellular GSH levels.

97) Chuang, Linus, et al. “Sulforaphane Induces Cell Cycle Arrest, Migration, Invasion, and Apoptosis in Epithelial Ovarian Cancer Cells.” American Journal of Cancer Review 1.1 (2013): 9-24.

98)  Cancer Prev Res (Phila). 2015 Aug;8(8):712-9.
Effect of Sulforaphane in Men with Biochemical Recurrence after Radical Prostatectomy.  Cipolla BG1, Mandron E2, Lefort JM3, Coadou Y4, Della Negra E5, Corbel L5, Le Scodan R6, Azzouzi AR7, Mottet N8.

Increases in serum levels of prostate-specific antigen (PSA) occur commonly in prostate cancer after radical prostatectomy and are designated “biochemical recurrence.” Because the phytochemical sulforaphane has been studied extensively as an anticancer agent, we performed a double-blinded, randomized, placebo-controlled multicenter trial with sulforaphane in 78 patients (mean age, 69 ± 6 years) with increasing PSA levels after radical prostatectomy. Treatment comprised daily oral administration of 60 mg of a stabilized free sulforaphane for 6 months (M0-M6) followed by 2 months without treatment (M6-M8). The study was designed to detect a 0.012 log (ng/mL)/month decrease in the log PSA slope in the sulforaphane group from M0 to M6. The primary endpoint was not reached. For secondary endpoints, median log PSA slopes were consistently lower in sulforaphane-treated men. Mean changes in PSA levels between M6 and M0 were significantly lower in the sulforaphane group (+0.099 ± 0.341 ng/mL) than in placebo (+0.620 ± 1.417 ng/mL; P = 0.0433). PSA doubling time was 86% longer in the sulforaphane than in the placebo group (28.9 and 15.5 months, respectively). PSA increases >20% at M6 were significantly greater in the placebo group (71.8%) than in the sulforaphane group (44.4%); P = 0.0163. Compliance and tolerance were very good. Sulforaphane effects were prominent after 3 months of intervention (M3-M6). After treatment, PSA slopes from M6 to M8 remained the same in the 2 arms. Daily administration of free sulforaphane shows promise in managing biochemical recurrences in prostate cancer after radical prostatectomy.

The PSA doubling time was 28.9 months for the sulforaphane group and 15.5 months in the placebo group (+86%).

QuadriGuard

99) QuadriGuard™ (ivermectin/pyrantel pamoate/praziquantel)
CHEWABLE TABLETS FOR DOGS For oral use in dogs only.

100) Parasites, Allergies and Autism:Paradise Lost in a Parallel Universe . Simon Yu, MD Internal Medicine Autism One Chicago, May 25-26. 2013  See pdf file.  Simon Yu Parasites Autism One Chicago 2013

Most Common Parasite Medications & Doses Dr SImon YU
! Ivermectin 12mg 3-4 x/day with pyrantel pamoate
250 mg 3-4 x/day for 10days to up to 30 days
! Tinadazole 500 mg at bed time up to 30 days or
! Alinia 500 mg 2-3 x/day for 3-10 days
! Albendazole 200 mg 4 x/day for skin problems
! Praziquantel 600 mg 4 x/day 2-4 wks or Levamisole
50 mg 4 x/day 2-4 wks for cancer

Ivermectin + pyrantel pamoate +praziquantel for 7
days, then follow by tinidazole+ levamisole for 10
days and repeat the cycle every month for 3 times.

Ivermectin and albendazole for 3 wks and
repeat 3 wks later for total 3 rounds

one month period on Ivermectin + pyrantel
pamoate for 2 wks and follow by
parziquantel + tinidazole for 2 weeks.

101) Drugs for Parasitic Infections The Medical Letter 2013 University of Alabama  Useful Compendium of Drug Treatment of Parasitic Infections

102) Ivermectin Dosage Guidelines Parasites Protocol Gant 2012

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Summary
Article Name
Targeting Cancer Stem Cells with NonToxic Therapies
Description
Targeting Cancer Stem Cells with NonToxic Therapies
Author
Jeffrey Dach MD