Foods alternatives/complimentary to Gleevec (GIST)

Sil The precise book reference is "Cooking with fords that fight cancer" by Richard Beliveau and Denis Gingras, published by A&U. I got it from the ABC bookshop a few years ago but assume its widely available. There is also one which doesn't have the recipes by the same authors. They say in the book that it has recently been observed that lueolin and apigenin (found in mint, thyme and parsley) powerfully inhibit the activity of a key enzyme activated by the growth factor PDGF and involved in the establishment of new blood vessels in tumours. They go on to say that luteolin and apigenin posses an activity comparable to Gleevec. I am currently on Gleevec which thankfully is working, so I have not really looked into how much of parsley mint etc you need to eat to get an equivalent effect, nor whether it actually works on GIST. However, the book is very interesting in that it explains how cancer is currently thought to work, and explains how different foods may inhibit it. Also it suggests red wine and chocolate have ingredients that inhibit cancer so it has my vote!

natural alternative to HBOT to saturate cancer cells?

What would be a natural alternative to HBOT to saturate cancer cells with oxygen? Would exercise in a forest help or are there ways to increase our lung capacity? I understand the air we breathe is 21% oxygen.


There is nothing I know of that would be more effective than HBOT to saturate cancerous tissues with oxygen.
Professor Seyfried

Exercise in general or perhaps breathing from an oxygen tank and normobaric pressure.  100% O2
Dominic D'Agostino, PhD

Genistein vs Gleevec: natural TKI?

Genistein differs from Gleevec in that genistein is a natural phytoestrogen and targets tyrosine kinase inhibitors (among other things) while Gleevec is a man made small molecule generated to bind specifically to the fusion protein called bcr-abl created by the rearrangement occurring following the break and fusion between chromosomes 9 and 22 specific to CML (GIST also seem to be sensitive to Gleevec). Gleevec works because this fusion protein is present only in tumor cells and it is essential for tumor cell growth. Thus, Gleevec tends to have few side effects and unless the tumor cells develop resistance (which happens in a very small percentage of cases) the drug is effective in killing cells carrying the fusion protein. The presence of a unique protein like bcr-abl is not common in cancers (though others have been identified). So you have the difference between using an nonspecific tki like genistein which is likely to have significant side effects at higher doses as it targets many tkis and has estrogenic activity, and a specific molecule designed to inhibit one specific tyrosine kinase that is known to be involved in tumor cell survival and growth.

   So while the may be some benefits from eating foods containing genistein (besides isoflavones it has anti-oxidant activity) there are doses and side effects to consider - I am also not sure that the amounts present in foods are therapeutically significant. However, before you decide to do any of this and especially if your are thinking of taking supplements, you need to have a discussion with your doctor.


Traganos, Ph.D.
Emeritus Professor of Pathology

Ketogenic diet diary

Day 1. (6th July)
Transition phase, only replaced snack with low carb.
Headache fatigue at night

Day 2
110.0lbs
Ate 3 keto meals but added carbs squash

Headache fatigue
Constipation
Rectal pain

Felt less bloated, lighter

Day 3
110.4

Morning Headache
Legs tired
Stool hard and slow
Brain dead/mental unfocused
Throat clogged

Drank more bone broth, salt

Day 4 (9th July)
110.0
No headache
No fatigue
Ph 7.4
Stool long and easy pass
Stomach lump feels hard/bigger
Lung pain/shortness of breath (mild acidosis or reactive hypogleciemia, eat more carbs/protein)
More acne (not unusual symptom.)

shortness of breath while in ketosis?

Does anybody ele experience shortness of breath while in ketosis?

I have the last two times i tried the diet. As soon as i get back on a mod carb approach it goes away. its really stressing me out feeling like i cant breathe all day. I am not anemic and have 98-99% blood oxygen level.

is this common?

#2   08-05-2009, 11:23 AM

lylemcd Administrator   Join Date: Feb 2008 Posts: 19,795

First time I've heard of it. There is some loss of ketones and there was some weird data point (I'm going to forget the details) where resting RQ (a measure of fuel utilization) ould drop below 0.7 due to something related to extra CO2 being lost or some such. But I refer you to the parable of "Doc it hurts when I do this."

#3   08-05-2009, 11:34 AM

bretter Junior Member   Join Date: Aug 2009 Posts: 24

I finished carbing up sunday and I'm within a pound of baseline this morning.it is a symptom of ketoacidosis should I be concerned? Really enjoyed ur book btw.

#4   08-05-2009, 11:40 AM

lylemcd Administrator   Join Date: Feb 2008 Posts: 19,795

No, ketoacidosis doesn't develop in non-diabetics. Again, the details are hazy, it had something to do with how ketones are handled in the body and maybe a loss of base or something, it's been too many years and I never paid much attention to it. If it's causing problems, I'd say to continue dieting with a moderate carb non-keto diet (e.g. carbs around 1 g/lb bodyweight or 120-150 grams minimum) to avoid the issue entirely. Still low enough for various benefits, high enough to avoid ketosis, etc, etc.

#5   08-05-2009, 12:09 PM

ragiarn Member   Join Date: Jul 2009 Location: Connecticut Posts: 49

Ketoacidosis occurs in diabetics because there is not enough glucose within the cell mitochondria to allow the entry of the ketone bodies into the krebs cycle. Ketones are the result of partial metabolism of fat and requires the Krebs cycle to complete the total metobolism. The Krebs cycle needs glucose to keep it going. As the saying goes"Fat burns in the flame of glucose" (or something to that effect). As a result, in these diabetics, massive amount ketones enter the blood stream and overwhelm the buffering system of the blood. Despite the elevated blood glucose in diabetics ( most of these patients present with blood glucose levels in the range of 600-1400 mg/dl and blood pH as low as 7.1) the glucose is unable to enter the cell because of a lack of insulin. In non diabetics even on a low carb diet there is still enough glucose to run the krebs cycle. In low carb diets amino acids can, and often are, converted into glucose through gluconeogenesis to maintain a normal blood sugar level. Because insulin is still present the glucose can enter the cell and keep the krebs cycle going and thus prevent massive elevation of ketones and severe ketoacidosis. The buffering of the ketoacids is rather complicated since there are mulitple systems involved in this process. Many of these systems are compromised in diabetics and are not generally not a problem for an otherwise healthy person. This is a somewhat simplistic explanation of the process. For further clarification consutl with a bichemistry textbook. Ralph

#6   08-05-2009, 12:29 PM

lylemcd Administrator   Join Date: Feb 2008 Posts: 19,795

Or my first book. As well, in non-diabetics there are at least two failsafe systems to prevent runaway ketoacidosis First and foremost, when ketone concentrations get high enough, insulin is released, this shuts down FFA release from fat cells and ketone production in the liver. There'ss another one but the bottom line is that ketoacidosis in the sense of runaway ketone production as occurs in Type I diabetics, does NOT occur in non-diabetic humans. As long as there is some potential for insulin release, it won't ever get out of hand.

#7   08-05-2009, 02:33 PM

bretter Junior Member   Join Date: Aug 2009 Posts: 24

i think im going to throw in some carbs instead of fats for a few of my meals thanks

#8   10-02-2009, 11:17 PM

shwi Junior Member   Join Date: Oct 2009 Posts: 2

Quote: Originally Posted by bretter Does anybody ele experience shortness of breath while in ketosis? Yup, I've been doing a CKD for a few weeks now and I've definitely noticed a shortness of breath at times. I put it down to a loss in my cardiovascular fitness due to a decrease in the amount of cardio I've been doing. I realise I'm probably wrong.

#9   09-01-2010, 04:43 PM

Aminal Member   Join Date: Aug 2010 Posts: 80

Bump from the grave, but I searched and this was the only place on here I could find this, sorry if it's been discussed again. I am doing RFL (5 days now) and now seem to be experiencing the joys of ketosis (amongst other things). I am noticably short of breath but surprisingly energetic. A quick google seems to reveal that shortness of breath is a recognised symptom of ketosis in some (and ketoacidosis of course). I'm hoping it will pass as I enter the second week. People keep asking me why I'm sighing Last edited by Aminal : 09-01-2010 at 04:45 PM.

#10   09-07-2010, 07:06 AM

bretter Junior Member   Join Date: Aug 2009 Posts: 24

I bulked and got on HRT for low testosterone and it went away. I think I dieted too long and hard. dont do what i did.

Gist and ketogenic diet

Its not clear if GIST will respond to ketogenic diet. But, based on some general information, GIST are likely to because:
1. Neuro origin of cells.
2. "Tracing the roots of this disease to cellular respiration has yielded a promising lead on how GIST tumors might form,": cellular respiration typically means a defect in the mitochondrial function of oxidative phosphorylation. That would make this tumor dependent on fermentation of glucose for its energy.
3. The researchers examined tissue from 34 GIST patients for mutations in the genes for succinate dehydrogenase, an enzyme that processes oxygen to obtain energy for cells. [Again, cellular respiration of oxygen occurs in mitochondria-lacking this the cell metabolizes glucose with no alternatives]
4. Although the remaining patients did not have any of these mutations, succinate dehydrogenase in tissue from their tumors did not appear to be functioning and cellular respiration was disrupted. [disrupted CR means glucose is left for energy]
Therefore it is likely that these tumors are glucose sensitive. Deprivation of glucose with a Very Low Carbohydrate diet (ketogenic diet) may be effective in stopping progression or enhancing the effects of other cancer agents, like imatinib

Dr P.   M.D

Gleevec natural alternatives? Natural tyrosine kinase inhibitor

The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells.

Spinozzi F, Pagliacci MC, Migliorati G, Moraca R, Grignani F, Riccardi C, Nicoletti I.

Source

Istituti di Medicina Interna e Scienze Oncologiche, Università di Perugia, Italy.

Abstract

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitro cultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G2/M arrest at low genistein concentrations (5-10 micrograms/ml), while at higher doses (20-30 micrograms/ml) there was also a perturbation in S-phase progression. The derangements in cell-cycle control were followed by apoptotic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 micrograms/ml genistein effectively inhibit tyrosine kinase activity in cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Genistein-induced mitotic arrest of gastric cancer cells by downregulating KIF20A, a proteomics study.

Yan GR, Zou FY, Dang BL, Zhang Y, Yu G, Liu X, He QY.

Source

Institute of Life and Health Engineering, and National Engineering and Research Center for Genetic Medicine, Jinan University, Guangzhou, China.

Abstract

Genistein exerts its anticarcinogenic effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of action of genistein has not been completely elucidated. In this study, we used quantitative proteomics to identify the genistein-induced protein alterations in gastric cancer cells and investigate the molecular mechanism responsible for the anti-cancer actions of genistein. Total 86 proteins were identified to be regulated by genistein, most of which were clustered into the regulation of cell division and G2/M transition, consistent with the anti-cancer effect of genistein. Many proteins including kinesin family proteins, TPX2, CDCA8, and CIT were identified for the first time to be regulated by genistein. Interestingly, five kinesin family proteins including KIF11, KIF20A, KIF22, KIF23, and CENPF were found to be simultaneously downregulated by genistein. Significantly decreased KIF20A was selected for further functional studies. The silencing of KIF20A inhibited cell viability and induced G2/M arrest, similar to the effects of genistein treatment in gastric cancer. And the silencing of KIF20A also increased cancer cell sensitivity to genistein inhibition, whereas overexpression of KIF20A markedly attenuated genistein-induced cell viability inhibition and G2/M arrest. These observations suggested that KIF20A played an important role in anti-cancer actions of genistein, and thus may be a potential molecular target for drug intervention of gastric cancer.
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sources of Genistein

Genistein is found in plant foods such as soybeans, chickpeas, broccoli, cauliflower, alfalfa sprouts, clover sprouts, barley meal, sunflower seeds, and clover seeds. It is also found in many soy-based products such as soy milk, tempeh, miso, soy flour, infant formula, sports drinks, protein bars, and textured soy protein. Textured soy protein (TSP) is used as a meat substitute in vegetarian hamburgers, hot dogs, sausages, and meatballs. Though soy is by far the most common dietary source of genistein, it is not found in soy sauce or soybean oil. Genistein is also available as a dietary supplement in powder, pill, or capsule form.

Curcumin Inhibits Tyrosine Kinase Activity of p185^neu and Also Depletes p185^neu1

1. Ruey-Long Hong,
2. William H. Spohn and
3. Mien-Chie Hung2

+ Author Affiliations

1. Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan 10016 [R-L. H.], and Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [R-L. H., W. H. S., M-C. H.]

Next Section

Abstract

Curcumin, a natural compound present in turmeric, possessing both anti-inflammatory and antioxidant effects, has been studied vigorously as a chemopreventative in several cancer models. The erbB2/neu gene-encoded p185^neu tyrosine kinase is a potent oncoprotein. Overexpression of p185^neu in breast cancer is known to be a poor prognostic factor. We investigated the effect of curcumin on p185^neu tyrosine kinase and on the growth of breast cancer cell lines. Curcumin dose-dependently inhibited p185^neu autophosphorylation and transphosphorylation in vitro and depleted p185^neu protein in vivo. It dissociated the binding of p185^neu with GRP94 (glucose-regulated protein), a molecular chaperone, and enhanced the depletion of p185^neu. The amount of p185^neu protein on the cell membrane was drastically decreased after curcumin treatment. These data demonstrated a new mechanism of the anti-tyrosine kinase activity of curcumin. The growth of several breast cancer cell lines was inhibited; the IC₅₀ ranged from 7 to 18 μm, which, however, did not correlate with the expression level of p185^neu. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in p185^neu-overexpressing cell lines by 5 μm curcumin (% of control, basal level versus overexpression: 59.3 versus 16.7%; P < 0.001 by Student’s t test). Because curcumin effectively inhibited p185^neu tyrosine kinase activity by depleting p185^neu and potently suppressed the growth of multiple breast cancer cell lines, its therapeutic potential in advanced breast cancer is worthy of further investigation.

Emodin, a Protein Tyrosine Kinase Inhibitor from Polygonum cuspidatum

 

Emodin is being studied as a potential agent that could reduce the impact of type 2 diabetes. It is a potent selective inhibitor of the enzyme 11β-HSD1.^[2] In studies in obese mice, emodin limits the effect of glucocorticoids and may therefore ameliorate diabetes and insulin resistance.^[3]
Pharmacological studies have demonstrated that emodin when isolated from rhubarb exhibits anti-cancer effects on several human cancers, including human pancreatic cancer.^[4][5][6] Emodin in rhubarb extracts may also have neuroprotective properties against glutamate toxicity,^[7]
Aloe-emodin (1,3,8-trihydroxyanthraquinone) is a variety of emodin found in Socotrine, Barbados, and Zanzibar aloes, but not in Natal aloes.^{[citation needed]}
Emodin is also shown to block cytomegalovirus infections as well as herpes simplex. Research is currently being performed in this area.

List of plants that contain the chemical

• Senna obtusifolia^[8] (syn. Cassia obtusifolia^[9])
• Fallopia japonica^[10] (syn. Polygonum cuspidatum^[11])
• Ventilago madraspatana^[12]
• Kalimeris indica^[13]
• Rumex nepalensis^[14]
• Polygonum hypoleucum^[15]
• Cassia occidentalis^[16]
• Cassia siamea^[17]
• Acalypha australis^[18]
• Rheum palmatum^[19]

 

Oxygen, glucose, KD diet, hyperbaric oxygen therapies and cancer.

Oxygen, glucose, KD diet, hyperbaric oxygen therapies and cancer.

"Two years ago we were so excited about getting any type of clinical trial going. Unfortunately, the only clinical trial that could get any traction was one that the patients had no chance of living with standard care treatments. In other words, they had no chance of living. Today, there is at least one open clinical trial to evaluate the ketogenic diet in cancers that aren't advance, aka - not hopeless. Another trial is being designed to study metastatic cancer and will be looking at combining the ketogenic diet and hyperbaric oxygen therapy (HBOT) - this is based on a recent publication by Dr. Dominic D'agostino at USF. Simply put, there is no longer a question if the ketogenic diet works, there is only a question of how to make metabolic therapies more effective. I've written several times in our group about the need to do two things when you use a metabolic therapy. First, starve the cancer with the ketogenic diet and second, use therapeutics to "kill" the cancer cells and make them go into apoptosis once they are starving. We, as part of the Pet Cancer Trial, are using therapeutics like DCA, chloroquine and mebendazole to push starving cells toward death. So that's where we are today, looking at the first clinical trial and marveling how far we've come and how fast we're going. We simply have to find a way to convince people to stop using protocols that treat the symptoms of cancer and start using protocols that treat the cause of cancer. This Facebook group is the perfect place to grow disciples and groom ketogenic diet champions.

Nontoxic Cancer Therapy Proves Effective Against Metastatic Cancer

June 5, 2013 — A combination of nontoxic dietary and hyperbaric oxygen therapies effectively increased survival time in a mouse model of aggressive metastatic cancer, a research team from the Hyperbaric Biomedical Research Laboratory at the University of South Florida has found.

Dominic D'Agostino, Ph.D.
Assistant Professor
ddagosti@health.usf.edu

http://scienceblog.cancerresearchuk.org/2012/03/01/new-clue-to-how-cancer-cells-beat-oxygen-starvation/

Doesn't cancer need oxygen to grow?

http://www.drweil.com/drw/u/id/QAA322213

Can Oxygen Cure Cancer? I have heard that disease cannot live in the presence of oxygen. Is this true? If so, can oxygen therapy be used to treat cancer?
A	Answer (Published 1/5/2004)
Oxygen therapy is being promoted on the Internet as a treatment for cancer, but there is no evidence that it works. The notion that oxygen might destroy cancer cells goes back to the 1930s when Otto Warburg, MD, a Nobel Prize winner, discovered that compared to normal cells, cancer cells have a low respiration rate. He proposed that if cancer cells survive and grow in a low oxygen environment, they would die off if exposed to higher levels of oxygen. Since then, we've learned that Dr. Warburg was wrong. Oxygen doesn't slow cancer growth - tumors grow rapidly in tissues well supplied with oxygenated blood and the opposite is true, too: depriving tumors of oxygen doesn't stimulate their growth. Moreover, a study published in the Spring/Summer 1998 issue of Scientific Review of Alternative Medicine noted that since human tissues require 200 to 250 ml of oxygen per minute, an additional 20 ml that could be dissolved in all of the plasma of a normal weight adult would hardly be enough to make a difference in the amount of oxygen cancer cells would receive. Hyperbaric oxygen therapy and cancer--a review. Moen I, Stuhr LE. Source Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. ingrid.moen@biomed.uib.no Abstract Hypoxia is a critical hallmark of solid tumors and involves enhanced cell survival, angiogenesis, glycolytic metabolism, and metastasis. Hyperbaric oxygen (HBO) treatment has for centuries been used to improve or cure disorders involving hypoxia and ischemia, by enhancing the amount of dissolved oxygen in the plasma and thereby increasing O(2) delivery to the tissue. Studies on HBO and cancer have up to recently focused on whether enhanced oxygen acts as a cancer promoter or not. As oxygen is believed to be required for all the major processes of wound healing, one feared that the effects of HBO would be applicable to cancer tissue as well and promote cancer growth. Furthermore, one also feared that exposing patients who had been treated for cancer, to HBO, would lead to recurrence. Nevertheless, two systematic reviews on HBO and cancer have concluded that the use of HBO in patients with malignancies is considered safe. To supplement the previous reviews, we have summarized the work performed on HBO and cancer in the period 2004-2012. Based on the present as well as previous reviews, there is no evidence indicating that HBO neither acts as a stimulator of tumor growth nor as an enhancer of recurrence. On the other hand, there is evidence that implies that HBO might have tumor-inhibitory effects in certain cancer subtypes, and we thus strongly believe that we need to expand our knowledge on the effect and the mechanisms behind tumor oxygenation.

Does hyperbaric oxygen have a cancer-causing or -promoting effect? A review of the pertinent literature.

Feldmeier JJ, Heimbach RD, Davolt DA, Brakora MJ, Sheffield PJ, Porter AT.

Source

Department of Radiation Oncology, Wayne State University, Detroit, Michigan.

Abstract

We reviewed all known published reports or studies related to a possible cancer-causing or growth-enhancing effect by hyperbaric oxygen. Published articles were retrieved using Medline searches for the period 1960-1993. Additional references were obtained from bibliographies included in those articles discovered in the computer search. Also, hyperbaric medicine text books and the published proceedings of international hyperbaric conferences were visually searched. Studies and reports discovered in this fashion and related to the topic were included in the review. Twenty-four references were found: 12 were clinical reports, 11 were animal studies, and 1 reported both an animal study and a clinical report. Three clinical reports suggested a positive cancer growth enhancement, whereas 10 clinical reports showed no cancer growth enhancement. Two animal studies suggested a positive cancer-enhancing effect, and 10 animal studies showed no such effect. (The report that included both animals and humans is counted in both groups). The vast majority of published reports show no cancer growth enhancement by HBO exposure. Those studies that do show growth enhancement are refuted by larger subsequent studies, are mixed studies, or are highly anecdotal. A review of published information fails to support a cancer-causing or growth-enhancing effect by HBO.

Comment in

Comments on "Does hyperbaric oxygen have a cancer-causing or -promoting effect?". [Undersea Hyperb Med. 1996]

 

 

 

 

Lack of Oxygen in Cancer Cells Leads to Growth and Metastasis

Sep. 13, 2012 — It seems as if a tumor deprived of oxygen would shrink. However, numerous studies have shown that tumor hypoxia, in which portions of the tumor have significantly low oxygen concentrations, is in fact linked with more aggressive tumor behavior and poorer prognosis. It's as if rather than succumbing to gently hypoxic conditions, the lack of oxygen commonly created as a tumor outgrows its blood supply signals a tumor to grow and metastasize in search of new oxygen sources -- for example, hypoxic bladder cancers are likely to metastasize to the lungs, which is frequently deadly.

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A University of Colorado Cancer Center study recently published in the journal Cancer Research details a mechanism by which these hypoxic conditions create aggressive cancer, with possible treatment implications for cancers including breast, ovarian, colorectal, pancreatic, prostate, bladder and other cancers.
"We've known that the protein HIF-1a is overexpressed in hypoxic tumors. And we've known that the cancer stem cell marker CD24 is overexpressed in many tumors. This study shows a link between the two -- the HIF-1a of hypoxia creates the overexpression of CD24. And it's this CD24 that creates a tumor's aggressive characteristics of growth and metastasis," says Dan Theodorescu, MD, PhD, director of the University of Colorado Cancer Center and the paper's senior author.
Outgrowing the blood supply leads to tumor hypoxia, which leads to overexpression of HIF-1a, which signals the production of CD24, which makes tumors grow and metastasize. In addition to aggression, CD24 has also been shown to confer resistance to chemotherapy, allowing this small population of cells to regrow the tumor once chemotherapy ends, leading to relapse and disease progression.
"Now imagine we target CD24," Theodorescu says. "Either by removing a cell's ability to make CD24 or by killing cells marked by this protein, it's likely we could disarm this most dangerous population of cells."
Theodorescu and colleagues showed this by adjusting levels of HIF-1a and CD24 in cancer cell samples and animal models. With HIF-1a low and yet CD24 artificially high, cells retained the ability to grow and metastasize. With CD24 low and yet HIF-1a artificially high, cell survival and proliferation decreased.
"It seems CD24 overexpression in hypoxic cells drives growth and metastasis in these hypoxic tumors," Theodorescu says. "Now we have a rational target: CD24 for these hypoxic tumors."

New clue to how cancer cells beat oxygen starvation

Posted on March 1, 2012 by Simon Shears

CHCHD4 switches on a survival mechanism in low oxygen

We need oxygen to survive. Even the cells in the deepest, darkest parts of our body can’t live without it. But some cancer cells adapt to survive in very low oxygen levels, and these end up being some of the most difficult to treat.
Scientists in labs around the world are working to uncover the molecular machinery that allows cells to do this. And they hope to outsmart tumours by developing treatments that break these mechanisms.
This month our scientists discovered an important part of the oxygen-sensing machinery of tumour cells, which may be an early step towards a new way to treat cancer.

Oxygen sensor

As tumours rapidly grow and expand, the network of blood vessels bringing oxygen to their cells can’t keep up, leaving some cells starved of oxygen, or ‘hypoxic’. This would kill normal cells, but cancer cells have evolved to beat these conditions by switching on a protein called hypoxia-inducible factor (HIF), which in turn switches on other molecules inside the cell.
This cascade, called the HIF response, encourages new blood vessels to grow around and into the tumour. It also helps the tumour to adapt to hypoxic conditions by using alternative methods to produce energy.
A cell generates energy to grow and survive inside tiny ‘power stations’ called mitochondria, by using oxygen to fuel chemical reactions. And it’s been known for some time that mitochondria have an important role in flicking the ‘HIF’ switch.
But researchers were in the dark as to exactly how mitochondria are involved. So a team at University College London, led by Dr Margaret Ashcroft, set out to investigate.

Flicking the switch

The research, published in this month’s Journal of Clinical Investigation, delved into mitochondria’s inner workings and found that oxygen levels are monitored by a protein called CHCHD4. When oxygen levels fall below a critical level, this protein activates the HIF response.
In lab experiments, the researchers also varied the levels of CHCHD4 in cells starved of oxygen. When CHCHD4 was blocked entirely, it stopped the action of HIF in cells, stopping cancer cells from growing and – importantly – preventing the growth of new blood vessels.
But when too much CHCHD4 was switched on in oxygen-starved cancer cells, large amounts of HIF were activated. This kick started a sequence of events that allowed cells to survive in low oxygen.
And it seems CHCHD4 has this HIF-activating role in a number of different types of cancer. The team looked at samples from 236 breast cancer patients, and found that levels of CHCHD4 were greatest in samples that were most aggressive, and came from patients with poorer survival. They also found raised levels of CHCHD4 in samples from patients with pancreatic cancers and gliomas (a form of brain tumour).
Again, in these cancers, they found that increased levels of CHCHD4 were associated with more aggressive tumours and poorer survival.

New treatment opportunity?

As we said above, tumour cells that are able to thrive despite low oxygen levels are more likely to be resistant to treatment. Previous research has shown that targeting the HIF response can block tumour growth and spread, and improves the effect of drugs that halt the growth of new blood vessels (so-called ‘anti-angiogenics’), so these results could hold promise for more effective cancer treatments in the future.
Dr Ashcroft thinks the work opens a new area in our understanding of how tumour cells adapt and survive in an environment with little oxygen.
“We can now look at new ways of targeting this process by modulating the action of CHCHD4, hopefully leading to new drugs that could be used against those cancers that are difficult to treat,” she told us.
Her team now plans to continue this work in further studies, and we’ll be keeping an eager eye on her progress in this exciting field.
Reference
Yang, J. et al. (2012). Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression Journal of Clinical Investigation, 122 (2), 600-611 DOI: 10.1172/JCI58780

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.
- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.

Renal cell carcinoma patients with high levels of CDCP1 in their tumors survived, on average, shorter after their date of diagnosis than those with low levels of CDCP1 in their tumors.

The scientists went on to test levels of CDCP1 in cell lines from real human tumors. Whenever a cancer had high levels of HIF-2α (hypoxia-inducible factor-2α), a protein that cells produce in response to low oxygen, it also had high levels of CDCP1, helping solidify the connection between hypoxia and CDCP1. Bladder, breast, colorectal, kidney, ovarian, and pancreatic tumors showed particularly high levels of CDCP1, they found. Moreover, when the team analyzed the patient data corresponding to the tumor samples, they discovered that those with higher levels of CDCP1 had decreased survival times and an increased chance of the cancer spreading.
The results hint that CDCP1 could be an attractive drug target to block the spread of tumors stemming from hypoxic environments.  This could be particularly applicable to cases of clear cell renal cell carcinoma (ccRCC)—in many of these kidney cancers, a master oxygen-sensing protein is mutated, and hypoxia programs are constantly activated, whatever levels of oxygen the tumor is exposed to. Thirty percent of patients with ccRCC are diagnosed with metastatic tumors, and the cancer is notoriously hard to treat with chemotherapy. Shutting down CDCP1 might be one way to make these cancers less likely to spread, and more susceptible to treatments—it will take more work, though, to know for sure whether this approach has merit.
- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.
- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

 -------

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.
- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

Suffocating cancers that thrive without oxygen

Posted on February 7, 2013 by Sarah CP Williams

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.

Renal cell carcinoma patients with high levels of CDCP1 in their tumors survived, on average, shorter after their date of diagnosis than those with low levels of CDCP1 in their tumors.

The scientists went on to test levels of CDCP1 in cell lines from real human tumors. Whenever a cancer had high levels of HIF-2α (hypoxia-inducible factor-2α), a protein that cells produce in response to low oxygen, it also had high levels of CDCP1, helping solidify the connection between hypoxia and CDCP1. Bladder, breast, colorectal, kidney, ovarian, and pancreatic tumors showed particularly high levels of CDCP1, they found. Moreover, when the team analyzed the patient data corresponding to the tumor samples, they discovered that those with higher levels of CDCP1 had decreased survival times and an increased chance of the cancer spreading.
The results hint that CDCP1 could be an attractive drug target to block the spread of tumors stemming from hypoxic environments.  This could be particularly applicable to cases of clear cell renal cell carcinoma (ccRCC)—in many of these kidney cancers, a master oxygen-sensing protein is mutated, and hypoxia programs are constantly activated, whatever levels of oxygen the tumor is exposed to. Thirty percent of patients with ccRCC are diagnosed with metastatic tumors, and the cancer is notoriously hard to treat with chemotherapy. Shutting down CDCP1 might be one way to make these cancers less likely to spread, and more susceptible to treatments—it will take more work, though, to know for sure whether this approach has merit.

Categories: Medical Sciences

- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

Suffocating cancers that thrive without oxygen

Posted on February 7, 2013 by Sarah CP Williams

New findings on the protein CDCP1 provide a link between the low oxygen environments of some tumors, and their ability to spread between sites in the body. Despite the fact that many cancer cells thrive without oxygen, understanding the molecular pathways involved gives researchers a chance to suffocate these tumors for good.
Most cells in the human body can’t survive for long without oxygen—the element we gulp in with every breath is vital to building molecules, carrying out chemical reactions, and storing energy. But cancerous cells aren’t like other cells in the body. Many tumors, in fact, thrive under conditions of low oxygen, called hypoxia. And tumors depleted of oxygen have been found, time and time again, to be more dangerous, faster growing, faster spreading, and more resistant to treatments than other tumors. Why? Cells have built in programs that put them into crisis mode when oxygen levels in their neighborhood drop; altering their metabolism to make it more efficient, turning off cell death pathways in a desperate attempt to survive, and spurring the growth of new blood vessels toward the area. This hypoxia-induced ultra-survival program is just what tumor cells need to help them evade death and grow uncontrollably.
The new PNAS Early Edition paper by Emerling et. al. serves as a reminder that many unexpected cancer-linked genes may tie back to this hypoxia program in tumor cells. It also brings the results back to real patients, suggesting a potential new drug target for those with oxygen-depleted cancers.
Emerling et. al. were studying the response of tumor cells to hypoxia when they noticed that whenever the amount of oxygen dropped, levels of the protein CDCP1 increased. CDCP1 had never before been linked to oxygen, but in a 2011 PNAS paper by Liu et. al., CDCP1 had been connected to the ability of melanomas to spread throughout the body. More CDCP1 meant a higher chance of the melanoma spreading to the lung, for example. Until now, however, scientists didn’t have a good idea of why this was true, or why different cancers had different levels of CDCP1.
The team of researchers involved in the new paper—mostly based at Harvard Medical School and its associated hospitals—began testing the link between CDCP1 and oxygen to see if it could explain the protein’s role in helping cancers spread. Usually, cancer cells cut off from oxygen are more likely to migrate away from the tumor, the first step in spreading to distant organs. But when Emerling et. al. shut off CDCP1 in a group of cancer cells, low oxygen levels no longer increased the cells’ ability to migrate away—the protein acted as an off-switch against the cancer spreading. CDCP1, the results suggest, could be a missing link between hypoxia and tumor metastasis.

Renal cell carcinoma patients with high levels of CDCP1 in their tumors survived, on average, shorter after their date of diagnosis than those with low levels of CDCP1 in their tumors.

The scientists went on to test levels of CDCP1 in cell lines from real human tumors. Whenever a cancer had high levels of HIF-2α (hypoxia-inducible factor-2α), a protein that cells produce in response to low oxygen, it also had high levels of CDCP1, helping solidify the connection between hypoxia and CDCP1. Bladder, breast, colorectal, kidney, ovarian, and pancreatic tumors showed particularly high levels of CDCP1, they found. Moreover, when the team analyzed the patient data corresponding to the tumor samples, they discovered that those with higher levels of CDCP1 had decreased survival times and an increased chance of the cancer spreading.
The results hint that CDCP1 could be an attractive drug target to block the spread of tumors stemming from hypoxic environments.  This could be particularly applicable to cases of clear cell renal cell carcinoma (ccRCC)—in many of these kidney cancers, a master oxygen-sensing protein is mutated, and hypoxia programs are constantly activated, whatever levels of oxygen the tumor is exposed to. Thirty percent of patients with ccRCC are diagnosed with metastatic tumors, and the cancer is notoriously hard to treat with chemotherapy. Shutting down CDCP1 might be one way to make these cancers less likely to spread, and more susceptible to treatments—it will take more work, though, to know for sure whether this approach has merit.

Categories: Medical Sciences

- See more at: http://firstlook.pnas.org/suffocating-cancers-that-thrive-without-oxygen/#sthash.FPA0b1gi.dpuf

Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment, specifically, to decreases in oxygen, or hypoxia.^[1]

1. ^ Lee, K.; Zhang, H.; Qian, D. Z.; Rey, S.; Liu, J. O.; Semenza, G. L. (2009). "Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization". Proceedings of the National Academy of Sciences 106 (42): 17910. doi:10.1073/pnas.0909353106. edit

Hypoxia inducible factor-1α as a cancer drug target

1. Garth Powis1 and
2. Lynn Kirkpatrick2

+ Author Affiliations

1. ¹Arizona Cancer Center, University of Arizona, Tucson, Arizona and
2. ²ProlX Pharmaceuticals, Tucson, Arizona

1. Requests for Reprints: Garth Powis, Arizona Cancer Center, University of Arizona, Room 3977, 1515 North Campbell Avenue, Tucson, AZ 85724-5024. Phone: (520) 626-6408; Fax: (520) 626-4848. E-mail: gpowis@azcc.arizona.edu

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Abstract

The hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is an important regulator of the growing tumor's response to hypoxia. HIF-1 activity in tumors depends on the availability of the HIF-1α subunit, the levels of which increase under hypoxic conditions and through the activation of oncogenes and/or inactivation of tumor suppressor genes. HIF-1 activates genes that allow the cancer cell to survive and grow in the hostile hypoxic tumor environment. Increased tumor HIF-1α has been correlated with increased angiogenesis, aggressive tumor growth, and poor patient prognosis, leading to the current interest in HIF-1α as a cancer drug target. A number of anticancer agents have been reported to decrease HIF-1α or HIF-1 transactivating activity in cells in culture. However, more relevant to the agents' antitumor activity is whether HIF-1 is inhibited in tumors in vivo. This has been demonstrated for only a few of the reported HIF-1 inhibitors. Some of the agents are moving toward clinical trial where it will be important to demonstrate that the agents inhibit HIF-1α in patient tumors or, failing this, the downstream consequences of HIF-1 inhibition such as decreased vascular endothelial growth factor formation, and relate this inhibition to antitumor activity. Only in this way will it be possible to determine if HIF-1α is a valid cancer drug target in humans.

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression

Jun Yang¹, Oliver Staples¹, Luke W. Thomas¹, Thomas Briston¹, Mathew Robson², Evon Poon¹, Maria L. Simões¹, Ethaar El-Emir², Francesca M. Buffa³, Afshan Ahmed¹, Nicholas P. Annear¹, Deepa Shukla¹, Barbara R. Pedley², Patrick H. Maxwell^1,4, Adrian L. Harris³ and Margaret Ashcroft¹

¹Centre for Cell Signalling and Molecular Genetics, University College London, Division of Medicine, Rayne Institute, London, United Kingdom.
²Tumour Biology Section, UCL Cancer Institute, Paul O’Gorman Building, University College London, London, United Kingdom.
³Cancer Research UK Department of Medical Oncology, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
⁴Faculty of Medical Sciences, University College London, Division of Medicine, Rayne Institute, London, United Kingdom.

Address correspondence to: Margaret Ashcroft, Centre for Cell Signalling and Molecular Genetics, University College London, London, United Kingdom. Phone: 44.0.20.7679.6205; Fax: 44.0.20.7679.6211; E-mail: m.ashcroft@ucl.ac.uk.

Authorship note: Jun Yang and Oliver Staples contributed equally to this work.

First published January 3, 2012
Received for publication May 2, 2011, and accepted in revised form November 16, 2011.

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

NATURAL  HIF INHIBITORS

EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

Jian-Wei Gu^*, Kristina L Makey, Kevan B Tucker, Edmund Chinchar, Xiaowen Mao, Ivy Pei, Emily Y Thomas and Lucio Miele

• * Corresponding author: Jian-Wei Gu jgu@umc.edu

Author Affiliations

Cancer Institute, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA

For all author emails, please log on.

Vascular Cell 2013, 5:9 doi:10.1186/2045-824X-5-9

The electronic version of this article is the complete one and can be found online at: http://www.vascularcell.com/content/5/1/9

Received:	29 January 2013
Accepted:	16 April 2013
Published:	2 May 2013

© 2013 Gu et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The role of EGCG, a major green tea catechin in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Sixteen eight-wk-old female mice (C57BL/6 J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. Eight mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. EGCG treatment significantly reduced tumor weight over the control (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01), tumor CD (109 ± 20 vs. 156 ± 12 capillary #/mm^2; P < 0.01), tumor VEGF expression (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01), respectively. But, it has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. EGCG at 50 μg/ml significantly inhibited the activation of HIF-1α and NFκB as well as VEGF expression in cultured E0771 cells, compared to the control, respectively. These findings support the hypothesis that EGCG, a major green tea catechin, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression.

Introduction

The term ‘green tea’ refers to the product manufactured from fresh tea leaves by steaming or drying at elevated temperatures with the precaution to avoid oxidation of the polyphenolic components known as catechins [1]. The natural product (−)-epigallocatechin-3-gallate (EGCG) accounts for 50-80% of catechins in green tea, representing 200–300 mg in a brewed cup of green tea [2]. Several other catechins such as (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC) are found in lower abundance in green tea [3]. EGCG is defined as a major green tea catechin that contributes to beneficial therapeutic effects, including anti-oxidant, anti-inflammatory, anti-cancer, and immunomodulatory effects [4-6]. Studies conducted on cell-culture systems and animal models as well as human epidemiological studies show that EGCG in green tea could afford protection against a variety of cancer types [7]. Many studies have shown that EGCG produces anti-cancer effect by modulating the activity of mitogen-activated protein kinases (MAPKs), IGF/IGF-1 receptor, Akt, NFκB and HIF-1α [8-12]. A case–control study including 501 breast cancer cases and 594 controls shows that green tea consumption has a significant trend of decreasing risk in a dose-dependent manner, after adjusting for potential confounding factors [13]. However, the investigations of green tea or EGCG in breast cancer using animal model are very limited, and the role of EGCG in breast cancer therapy is poorly understood.

The growth and expansion of a tumor is mainly dependent on angiogenesis, the formation of new capillaries from pre-existing blood vessels. Avascular tumors are those that do not grow beyond a maximum size of 1 to 2 mm³ in the absence of neovascularization, and it may be eliminated by a normal immune system [14]. Angiogenesis requires stimulation of vascular endothelial cells through the release of angiogenic factors. Of these, the vascular endothelial growth factor (VEGF) is the most critical regulator in the development of the vascular system and is commonly overexpressed in a variety of human solid tumors including breast cancer [15]. Cancer cells are under greater hypoxia and oxidative stress than normal cells. Oxygen radicals and hypoxia co-operatively promote tumor angiogenesis [16]. Hypoxia causes the activation of HIF-1, in which it stimulates VEGF expression. HIF-1 levels are also increased by oxygen radicals. In addition, oxygen radicals activate NFκB that also increases VEGF expression. VEGF is a key angiogenic factor that stimulates the growth of tumors including breast cancer, in which VEGF exerts paracrine (especially angiogenesis) and autocrine (proliferation and migration) effects to promote progression of breast cancer [17]. As mentioned above, we believe that EGCG can block highly activated NFκB and HIF-1α pathways in breast tumor. Therefore, we hypothesizes that EGCG directly targets both of tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Also, EGCG treatment has no significant effects on the body weight, heart weight, angiogenesis and VEGF expression in normal tissues such as the heart and skeletal muscle.

To test this hypothesis, the present study aimed to determine the following: (a) whether a relative high oral dose of EGCG inhibits tumor growth, tumor angiogenesis, and VEGF expression in an immunocompetent mouse model (C57BL/6) of breast cancer; (b) whether oral EGCG treatment affects angiogenesis and VEGF expression in normal tissues such as the heart and skeletal muscle in the same mice; and (c) whether EGCG inhibits proliferation, migration, VEGF expression, the activation of HIF-1α and NFκB in cultured mouse and human breast cancer cells (E0771, MCF-7 and MDA-MB-231).

Materials and methods

Chemicals and cell lines

EGCG was purchased from Sigma Chemical Co. (St. Louis, MO). The mouse breast cancer cells (E0771) which were originally isolated from an immunocompetent C57BL/6 mouse, were provided by Dr. Sirotnak FM at Memorial Sloan Kettering Cancer Center, New York, NY [18]. Human estrogen-receptor positive breast cancer (MCF-7) cells and human triple negative breast cancer (MDA-MB-231) cells were purchased from the American Type Culture Collection (Rockville, MD). All breast cancer cells were maintained as monolayer cultures in RPMI Medium 1640 (GIBCO) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, and incubated at 37°C in a humidified 5%CO₂/air injected atmosphere.

Animal protocols

The protocols were carried out according to the guidelines for the care and use of laboratory animals implemented by the National Institutes of Health and the Guidelines of the Animal Welfare Act and were approved by the University of Mississippi Medical Center’s Institutional Animal Care and Use Committee. 16 female C57BL/6 mice at 7 weeks of age were purchased from Jackson Laboratory (Bar Harbor, Maine). The mice were allowed to acclimate for 1 week with standard chaw diet (Teklad, Harlan Sprague Dawley; Indianapolis, IN) and tap water before beginning the experiments. The eight week old female mice (n = 16) were inoculated with 10^6 E0771 cells suspended in 100 μl of phosphate-buffered saline into the left fourth mammary gland fat pad. Then, 8 mice received EGCG (25 mg/50 ml) in drinking water for 4 weeks and 8 control mice received drinking water only. Each mouse (20 g) usually drank 2 to 4 ml of water per day. Therefore, EGCG was given around 50 to 100 mg/kg/day to the mice. The body weight of the mice was monitored weekly. Tumor size was monitored every other day in two perpendicular dimensions parallel with the surface of the mice using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and average microvascular density (AMVD) or capillary density (CD) using CD31 immunohistochemistry.

Morphometric analysis of angiogenesis in tumor, the heart and limb muscles

The quantification of blood vessels in mouse breast tumor, the heart and limb muscle was determined with the modification of a previously reported method [17,19]. Briefly, the tissues were fixed in 4% neutrally buffered paraformaldehyde. For the heart left ventricular and limb muscle samples, consecutive thin transverse cryosections (5 μm) were cut along the base-apex axis. Consecutive thin cryosections (5 μm) of OCT compound (Sakura Finetek, Torrance, CA) embedded tissue samples were fixed in acetone at 4°C for 10 min. After washing in phosphate-buffered saline (PBS), the sections were treated with 3% H₂O₂ for 10 minutes to block endogenous peroxidase activity and were blocked with normal rabbit serum. Then, the sections were washed in PBS and incubated with rat anti-mouse CD31 (PECAM-1) monoclonal antibody (BD Pharmingen, San Diego, CA) at a 1:200 dilution overnight at 4°C. Negative controls were incubated with the rat serum IgG at the same dilution. All sections were washed in PBS containing 0.05% Tween-20, and were then incubated with a 2^nd antibody, mouse anti-rat IgG (Vector laboratories, Burlingame, CA) at a 1:200 dilution for 1 hour at room temperature again followed by washing with PBS containing 0.05% Tween-20. The sections were incubated in a 1:400 dilution of Extravadin Peroxidase (Sigma, St. Louis, MO) for 30 min. After washing in PBS containing 0.05% Tween-20, the sections were incubated in peroxidase substrate (Vector laboratories, Burlingame, CA) for 5 min. The sections were washed in PBS containing 0.05% Tween-20 and were counterstained with hematoxylin. A positive reaction was indicated by a brown staining. The microvascular vessels were quantified by manual counting under light microscopy. A microscopic field (0.7884 mm²) was defined by a grid laced in the eye-piece. At least 20 microscopic fields were randomly acquired from each tumor for analysis. Any endothelial cell or cell cluster showing antibody staining and clearly separated from an adjacent cluster was considered to be a single, countable microvessel. The value of average microvascular density (AMVD) or capillary density (CD) was determined by calculating the mean of the vascular counts per mm² obtained in the microscopic fields for each tissue sample.

Measurements of protein levels of VEGF by ELISA

Protein levels of VEGF in plasma, breast tumor, the heart, the limb muscle, and the medium cultured with E0771 cells were determined using mouse VEGF ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The total proteins of breast tumor, the heart, the limb muscle, and cultured E0771 cells were extracted using NE-PER Cytoplasmic Extraction Reagents (Pierce, Rockford, IL), according to the manufacturer’s protocol. The total protein concentration of these tissue extractions was determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein concentrations of VEGF were normalized and expressed as pictograms per milligram of total tissue or cell extraction protein.

Proliferation assay of cultured breast cancer cells

The E0771, MCF-7, and MDA-MB-231cells were seeded into 6-well tissue culture plates using RPMI Medium 1640 (GIBCO) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, and incubated at 37°C in a humidified 5%CO₂/air injected atmosphere. When the monolayer reached about 80% confluence, the cells were washed with PBS and incubated with fresh RPMI Medium 1640 with 10% FBS in the absence and presence of EGCG (0, 10, 50 μg/ml) for 18 hours. 3H-thymidine incorporation assay was used to determine the cell proliferation during the last 6 hours of incubation as previously described [20].

Migration assay

Migration was determined using BD BioCoat Matrigel Invasion Chamber (BD Bioscience Discovery Labware, Sedford, MA) according to a previous study, in which only invasive cells digested the matrix and moved through the insert membrane [21]. 1 × 10⁵ E0771 cells per well in 0.5 ml medium (RPMI Medium 1640) were seeded in the matrigel-coated upper compartment (insert) of a Transwell (24-well format, 8-μm pore) in the absence of and presence of EGCG (0, 10, 20, 50 μg/ml) and the medium with 10% FBS was added to the lower part of the well. After overnight incubation at 37°C and 5% CO₂, cells on the upper surface of the insert were removed using a cotton wool swab. Migrated cells on the lower surface of the insert were stained using DiffQuit (Dada Behring, Düdinen, Switzerland). The images of migrated cells were taken and the number of migrated cells was counted using a microscope (Leica, Germany) in a 20× objective.

HIF-1α and NFκB activation (motif binding) assays

We determined HIF-1α and NFκB activation in cultured E0771 cells in the absence and presence of EGCG (0 and 50 mg/ml) to investigate whether the down-regulation of VEGF by EGCG is associated with the inhibition of HIF-1α and NFκB activation (n = 6). The nuclear proteins were extracted by using Active Motif (Carlsbad, CA) nuclear extract kit. 20 μg nuclear proteins from each sample was used in the TransAM HIF-1α or NFκB p65 kit (Active Motif), which can measure the binding of activated HIF-1α or NFκB to its consensus sequence attached to a microwell plate, according the manufacturer’s instructions.

Statistical analysis

All determinations were performed in duplicated sets. Where indicated, data is presented as mean ± SE. Statistically significant differences in mean values between the two groups were tested by an unpaired Student’s t-test. Linear regression was performed by the correlation analysis between two continuous variables. A value of P < 0.05 was considered statistically significant. All statistical calculations were performed using SPSS software (SPSS Inc., Chicago, IL).

Results

A relative high oral dose of EGCG significantly inhibits the progression of breast cancer growth

We used a mouse breast cancer model that mimics the human disease, in which the mouse breast adenocinoma (E0771) cells were injected into the pad of the fourth mammary gland of female immunocompetent mice (C57BL/6). Immediately after the inoculation of E0771 cells, the eight week old female mice (n = 8) were given EGCG at 50 to 100 mg/kg/day in drinking water for four weeks and the control group (n = 8) was given regular drinking water only. Tumor size was then monitored every other day in two perpendicular dimensions parallel with the surface of the mice using dial calipers. As indicated in Figure 1A, the tumor cross section area was significantly reduced in the EGCG-treated group compared to the control group two weeks after the breast cancer inoculation. At the end of experiment, the tumor cross section area was reduced by 65% (P < 0.01) in EGCG-treated group compared to the control group (Figure 1A), which was consistent with the reduction in tumor weight (Figure 1B) in EGCG-treated group compared to the control group (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P <0.01). Clearly, EGCG treatment at 50 to 100 mg/kg/d in drinking water significantly inhibited the progression of breast cancer growth in the female mice by decreasing the tumor size and reducing the growth curve of breast cancer. However, there was no significant difference in the body weight, heart weight, kidney weight, or urinary protein between the EGCG-treated mice and the control mice.

Figure 1. The inhibition of the progression of breast cancer growth by oral EGCG in the immunocompetant female mice (C57BL/6) allografted with mouse breast cancer (E0771) cells. EGCG at 50 to 100 mg/kg/day in drinking water for four weeks significantly reduced a growth curve of breast cancer monitored by the tumor cross section area by 65% (Figure 1A, P < 0.01; n = 8) and tumor weight (Figure 1B, 0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01; n = 8), compared to the control group.

EGCG suppresses breast tumor angiogenesis and VEGF expression in mice

Growth and expansion of tumor mass are strictly dependent on angiogenesis because neovascularization permits rapid tumor growth by providing an exchange of nutrients, oxygen, and paracrine stimuli to the tumor [22]. Therefore, in this study, we used a morphometric analysis of immunohistochemical staining for CD31 to determine the effect of EGCG on breast tumor angiogenesis in mice. Representative images of CD31 staining of the breast cancer tumors showed that the EGCG-treated tumor had lesser microvessels than the control tumor (Figure 2A). Morphometric analysis (Figure 2A) indicated that PDTC treatment caused a significant decrease in average microvessel density (AMVD, the number of microvessels per mm2 area) of breast tumors compared to the control breast tumors (109 ± 20 vs. 156 ± 12 microvessels number per mm^2; n = 8; P < 0.01). These results also suggest that a pronounced decrease in tumor angiogenesis is associated with a decrease in tumor size of breast cancer tumor in the female mice treated with EGCG compared to those in the control mice. Figure 2B also demonstrated that EGCG treatment reduced plasma VEGF levels over the control mice (26.48 ± 3.76 vs. 40.79 ± 3.5 pg/ml; n = 8; P < 0.01) and tumor VEGF expression over the control mice (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; n = 8; P < 0.01). These findings suggest that the inhibition of tumor angiogenesis in mice by EGCG is due to the down-regulation of VEGF because VEGF is a key angiogenic factor.

Figure 2. Oral EGCG at 50–100 mg/kg/d in drinking water significantly reduced intratumoral microvessel density (Panel A: 109 ± 20 vs. 156 ± 12 microvessel #/mm^2; P < 0.01), plasma VEGF levels (Panel B; 26.48 ± 3.76 vs. 40.79 ± 3.5 pg/ml; P < 0.01), and tumor VEGF expression (Panel B; 45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01) over the control, respectively in mice (n = 8). The digital images show CD31 immunohistochemistry staining in OCT-embedded cryosections of mouse breast cancer tumors obtained from a control (Figure 2A) or EGCG-treated (Figure 2A) mouse.

EGCG directly inhibits proliferation and migration of breast cancer cells

We used a 3H-thymidine incorporation assay to determine the effects of EGCG on the proliferation of cultured mouse breast cancer cells (E0771), human estrogen receptor positive breast cancer cells (MCF-7), and triple negative breast cancer cells (MDA-MB-231). Figure 3A showed that E0771 cells treated with EGCG caused a dose-related decrease in 3H-thymidine incorporation, decreasing by 22% at 10 μg/ml and by 77% at 50 μg/ml, compared to the control group (n = 6; P < 0.01). We examined the inhibitory effect of EGCG on E0771 cell migration using BD BioCoat Matrigel Invasion Chamber. Figure 3B demonstrates that EGCG at 10, 20, and 50 μg/ml caused a dose-dependent reduction of migrated breast cancer (E0771) cells, decreasing by 25%, 48%, and 71%, respectively, compared to the control group (n = 6; P < 0.01). In the another experiment, as shown in Figure 3C, we demonstrated that EGCG at 50 μg/ml significantly inhibited the proliferation of human estrogen receptor positive breast cancer cells (MCF-7) and triple negative breast cancer cells (MDA-MB-231) by 91% and 52%, respectively, compared to the control group (n = 6; P < 0.01), but not at 10 μg/ml. These in vitro findings illustrate that EGCG can directly target breast cancer cells by inhibiting the proliferation and migration.

Figure 3. EGCG caused a dose-related inhibition in 3H-thymidine incorporation, decreasing by 22% at10 μg/ml and by 77% at 50 μg/ml (Panel A, n = 6, P < 0.01), and in migration (Panel B, n = 6, P < 0.01) in cultured E0771 cells, compared to the control group. In Panel C, EGCG at 50 μg/ml significantly inhibited the proliferation in cultured MCF-7 and MDA-MB-231 cells, compared to the control group (n = 6; P < 0.01), respectively.

The down-regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1α and NFκB activation

HIF-1 and NFκB pathways are highly activated in breast tumor, in which they can co-operatively promote tumor angiogenesis by increasing VEGF expression [16]. We used VEGF ELISA kit and HIF-1α and NFκB activation (Motif Binding) assays to determine whether EGCG could suppress HIF-1α and NFκB activation and VEGF expression in cultured mouse breast cancer (E0771) cells. Figure 4A showed that EGCG at 50 μg/ml significantly inhibited VEGF expression (1752 ± 49 vs. 2254 ± 91 pg/mg; n = 6; P < 0.01) in cultured E0771 cells, compared to the control. In the same experiment, EGCG at 50 μg/ml also significantly suppressed the activation of HIF-1α (0.11 ± 0.02 vs. 0.24 ± 0.02; P < 0.01; Figure 4B) and NFκB (1.15 ± 0.21 vs. 1.61 ± 0.32; n = 6; P < 0.01; Figure 4C), compared to the control, respectively. These results suggest that the inhibition of HIF-1α and NFκB activation contributes to the down-regulation of VEGF expression.

Figure 4. EGCG at 50 μg/ml significantly inhibited VEGF expression (Panel A, 1752 ± 49 vs. 2254 ± 91 pg/mg, n = 6, P < 0.01), the activation of HIF-1α (Panel B, 0.11 ± 0.02 vs. 0.24 ± 0.02, n = 6, P < 0.01) and NFκB (Panel C, 1.15 ± 0.21 vs. 1.61 ± 0.32; n = 6, P < 0.01) in cultured E0771 cells, compared to the control, respectively.

Oral EGCG treatment has no effects on angiogenesis and VEGF expression in normal tissues such as the heart and skeletal muscle in mice

The data showed that there was no significant difference in the body weight (22.38.25 ± 0.51 vs. 22.94 ± 0.57; n = 8; P = 0.9437), heart weight (84.7 ± 11.2 vs. 85.1 vs. 10.6 mg; n = 8; P = 0.3546), or kidney weight (237.5 ± 9.2 vs. 240.1 ± 8.9 mg; n = 8; P = 0.3735) between the EGCG-treated mice and the control mice. Figure 5A showed that EGCG treatment did not affect the capillary density (number of capillary/mm^2 area) (3270 ± 162 vs. 3103 ± 226 #/mm^2; n = 8; P = 0.5215) analyzed by CD31 immunochemistry and morphometric analysis, and VEGF expression (261 ± 22 vs. 245 ± 19 pg/mg; n = 8; P = 0.4517) determined by ELISA in the mouse heart, compared to the control group, respectively. Figure 5B showed that there was no significant difference in the capillary density (370 ± 55 vs. 381 ± 44 #/mm^2; n = 8; P = 0.5401), and VEGF expression (225 ± 16 vs. 214 ± 20 pg/mg; n = 8; P = 0.7825) in the limb skeletal muscles between the EGCG-treated mice and the control mice, respectively. These findings illustrate that EGCG does not significantly affect angiogenesis and VEGF expression in the normal tissues such as the heart and skeletal muscles.

Figure 5. EGCG treatment did not affect the capillary density (3270 ± 162 vs. 3103 ± 226 #/mm^2; n = 8; P = 0.5215), and VEGF expression (261 ± 22 vs. 245 ± 19 pg/mg; n = 8; P = 0.4517) in the mouse heart, compared to the control group (Panel A), respectively. There was no significant difference in the capillary density (370 ± 55 vs. 381 ± 44 #/mm^2; n = 8; P = 0.5401), and VEGF expression (225 ± 16 vs. 214 ± 20 pg/mg; n = 8; P = 0.7825) in the limb skeletal muscles between the EGCG-treated mice and the control mice (Panel B), respectively. The digital images show CD31 immunohistochemistry staining in OCT-embedded cryosections of the heart (Panel A) and the limb muscle (Panel B) of control mouse and EGCG-treated mouse, respectively.

Discussion

The major new findings from this study include: 1) a relative high oral dose of EGCG significantly inhibits the progression of mouse breast cancer growth in female immunocompetent mice; 2) EGCG significantly suppresses breast tumor angiogenesis and VEGF expression in these mice; 3) EGCG treatment does not significantly affect angiogenesis and VEGF expression in the normal tissues such as the heart and skeletal muscles in the same experiment; 4) EGCG directly inhibits proliferation and migration of cultured mouse and human breast cancer cells; and 5) the down-regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1α and NFκB activation. These findings support the hypothesis that EGCG, a major green tea catechin directly targets both of tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Also, EGCG treatment has no significant effects on angiogenesis and VEGF expression in normal tissues such as the heart and skeletal muscle.

An important finding of this study is that a relative high oral dose of EGCG treatment at 50 to 100 mg/kg/day in drinking water significantly slows a growth curve of breast cancer in C57BL/6 female mice compared to the control group, which is characterized by 65% and 68% reduction in the tumor cross section area and tumor weight, respectively. Clearly, oral EGCG treatment is very effective in suppressing progression of breast cancer in a wild type immunocompetent mouse model. Ullmann et al. reported that peak plasma concentrations were greater than 3 μg/ml after oral dose of 1600 mg in healthy human subjects [23]. We believe that oral dose of 50 to 100 mg/kg/day in human can reach the effective plasma concentrations of EGCG against breast cancer. Recent methods developed for the stereoselective total synthesis of EGCG, and structurally related catechins, could provide new sources of these compounds for biomedical use [24]. Our next step is clinical trial for EGCG in breast cancer therapy.

That's 6 pills (400mg) for a 110lbs person.




Alternative to oxygen therapies on HBOT? How to increase oxygen uptake?

Effects of qigong on cardiorespiratory changes: a preliminary study.

Lim YA, Boone T, Flarity JR, Thompson WR.

Source

Life College, Sports Health Science, Marietta, GA 30060.

Abstract

Qigong, a special form of breathing exercise, was investigated to examine its effect on cardiorespiratory changes. Ten volunteers (five males and five females) participated in a 20-minute group instructional session for 10 consecutive days before testing of its treatment effects. The testing protocol followed a C1-T-C2 design, where C1, T, and C2 represented the first, treatment, and second control period, respectively. Each period consisted of a 5-minute interval, and thus each testing session consisted of 15 minutes. The results indicated there were no statistically significant differences (p > 0.05) in heart rate or tidal volume for the three 5-minute periods. There was a significant decrease (p < 0.05) in respiratory exchange ratio between T and C2. A significant increase in ventilatory efficiency for carbon dioxide production was found between C1 and T. Statistically significant differences (p < 0.05) were found in the volume of oxygen consumed and carbon dioxide produced, frequency of breath, expired ventilation, and ventilatory efficiency for oxygen produced between the T and the two control periods. This preliminary study of Qigong demonstrates that the subjects were able to learn the technique in a short period of time. The data also suggest that, with an improvement of nearly 20% in ventilatory efficiency for oxygen uptake and carbon dioxide production, this technique may have useful therapeutic value.