Dr. Weeks’ Comment: Foods are your best medicines – that is a truism since Hippocrates preached it centuries ago. Here we see that bitter melon can kill the most lethal aspect of a cancer- the cells which can replicate and proliferate and kill – the cancer STEM cells.
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“… These data suggest that BMW extracts are potent inhibitors of colon cancer stem cells. …..”
Methanolic Extracts of Bitter Melon Inhibit Colon Cancer Stem Cells by Affecting Energy Homeostasis and Autophagy
Deep Kwatra, Dharmalingam Subramaniam, […], and Shrikant Anant
Abstract
Bitter melon fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes. However its effects on cancer progression are not well understood. Here, we have determined the efficacy of methanolic extracts of bitter melon on colon cancer stem and progenitor cells. Both, whole fruit (BMW) and skin (BMSk) extracts showed significant inhibition of cell proliferation and colony formation, with BMW showing greater efficacy. In addition, the cells were arrested at the S phase of cell cycle. Moreover, BMW induced the cleavage of LC3B but not caspase 3/7, suggesting that the cells were undergoing autophagy and not apoptosis. Further confirmation of autophagy was obtained when western blots showed reduced Bcl-2 and increased Beclin-1, Atg 7 and 12 upon BMW treatment. BMW reduced cellular ATP levels coupled with activation of AMP activated protein kinase; on the other hand, exogenous additions of ATP lead to revival of cell proliferation. Finally, BMW treatment results in a dose-dependent reduction in the number and size of colonospheres. The extracts also decreased the expression of DCLK1 and Lgr5, markers of quiescent, and activated stem cells. Taken together, these results suggest that the extracts of bitter melon can be an effective preventive/therapeutic agent for colon cancer.
1. Introduction
Colorectal cancer is the second leading cause of cancer related deaths and the third most commonly occurring noncutaneous carcinoma in the United States of America [1]. Although early diagnosis often leads to a complete cure, in most cases the polyps go undetected. In such cases, therapies such as surgical intervention, chemotherapy, and radiation are often not sufficient to tackle the disease, thus needing other prevention-related or nonconventional therapeutic strategies. Hence there is a need of better options for therapy and prevention of the disease.
A number of studies have shown that diet can play a significant role in the development of colon cancer as a higher risk is associated with consumption of high-fat, low-fiber diet and red meat [2, 3]. Use of certain foods and condiments (such as curcumin) in food may be responsible for lower prevalence of the disease in certain parts of India and central Asia [4, 5]. Bitter melon (Momordica charantia) is a tropical and subtropical vine, widely grown in Asia, Africa, and the Caribbean for its edible fruit. The fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes [6, 7], though all parts of the plant (fruit, seed, and leaves) have been shown to possess hypoglycemic properties [8]. Studies have shown that bitter melon extracts are well tolerated in both acute and chronic doses in animals [9–11]. Recent studies have demonstrated that aqueous extracts of bitter melon can inhibit the growth of breast and prostate cancers [12–14]. Though the mechanisms behind the antitumor activity are poorly known, the previous studies hint towards induction of apoptosis as one potential mechanism [15, 16].
Not much is known about the active principles in bitter melon that have anticancer activity. A 30
kDa ribosome-inactivating protein (RIP) termed Momordica or MAP30 was identified in bitter melon. Addition of MAP30 toMDA-MB-231 breast cancer cells in culture reduced proliferation of the cells. In addition, there was a reduction in expression of Human Epidermal Growth Factor Receptor 2 (HER2) [15–17]. Moreover, modification of MAP30 resulted in reduced immunogenicity while retaining the anti-proliferative activity against prostate cancer cell xenografts in nude mice [15, 16]. Thirteen cucurbitane-type triterpene glycosides have also been identified in bitter melon extracts with potential anti-proliferative activity [18]. α-eleostearic acid is found in high amounts in the seeds of bitter melon and may be effective in some (MDA-MB-231, ER +ve MDA-ERα7) tumor cells but not in others [19, 20]. However, none of these studies have been comprehensive, and no major studies have been performed using colon cancer cells.
In this paper, we present the results of our in vitro experiments showing that methanolic extracts of bitter melon (BMW) inhibit cell proliferation, prevent colony formation, and promote S phase cell cycle arrest of colon cancer cells. We also show that these extracts suppress cancer cell spheroid formation suggesting that the extracts target stem cells within the cancer. Mechanistically, we have determined that while the extracts do not induce apoptosis, there is autophagy via the AMPK signaling pathway. In addition, the extracts modulate energy homeostasis to affect the viability of the colon cancer cells.
3. Results
3.1. Bitter Melon Extracts Inhibit the Proliferation and Colony Formation of Colon Cancer Cells
We first determined the sensitivity of HT-29 and SW480 colon cancer cells to bitter melon extracts. To determine whether bitter melon extracts affect cell proliferation, the cells were grown in the presence of varying concentrations of BMW and BMSk (0-500μg/mL) for 48h and the cell proliferation was measured by hexoseminidase assay. Both BMW and BMSk inhibited the proliferation of HT-29 and SW480 cells in a dose-dependent manner (Figure 1(a)). The IC50 dose of BMW for proliferation of HT-29 cells was observed to be 57μg/mL, which was much less than that observed for BMSk (105μg/mL). Similar trend was observed in SW480 cells where IC50 dose of BMW for proliferation was 85μg/mL, which was again found to be much lower than IC50 for BMSk (108μg/ML) (Figure 1(a)). Under similar conditions, the IC50 for the noncancerous human foreskin fibroblast (HFF) cells could not be calculated as neither BMW nor BMSk significantly inhibited proliferation of the cells when compared to controls (Figure 1(b)).
μg/mL) of bitter melon whole …To further study the effect of BMW on cell growth and proliferation in colon cancer cells, clonogenicity assay was performed. SW480 and HT-29 cells were treated with 100μg/mL BMW and BMSK extracts. Both extracts reduced colony formation of the two cell lines in a concentration dependent manner (Figure 1(b)). However, there was a significantly less number of surviving colonies following BMW treatment as compared to BMSk treated samples, suggesting that BMW was more potent in inhibiting the growth of colon cancer cells (Figure 1(b)).
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3.6. Bitter Melon Extracts Possess Anticancer Stem Cell Activity
Recent studies have demonstrated that a small population of cells contains tumor initiating potential while the majority of cells within a tumor have undergone differentiation and lost this potential [30]. Colonospheres are spheroids that are grown in ultralow binding plates and are believed to represent the growth of cells from stem cells [31]. Hence, the colonosphere cultures are used extensively to determine the effect of agents on stem cells. Accordingly, to determine the effect of bitter melon extracts on 3D cultures, cells treated with BMW were used for spheroid formation. The BMW-treated cells showed marked decrease in spheroid formation, when compared to control-treated cells (Figure 6(a)). Moreover, BMW treatment resulted in a concentration dependent decline in both the size and number of spheroids formed (Figure 6(b)).
We have recently demonstrated that Doublecortin Calmodulin-like Kinase 1 (DCKL1) is a marker of quiescent stem cells in a variety of cancers including colon cancers [32]. Since, BMW extracts inhibited colonosphere formation, we determined whether DCLK1 expression was affected. Western blot analyses demonstrated that DCLK1 expression was markedly reduced in cells treated with BMW extracts (Figure 6(c)). Another stem cell marker in the colon Lgr5 was also studied to further confirm the activity of BMW on colon cancer stem cells. Similar to DCLK1, there was a concentration dependent decline in Lgr5 expression in BMW-treated cells (Figure 6(c)). Further confirmation was obtained by flow cytometry, where a significant reduction on DCLK1+ cells was observed in cells treated with BMW extracts (Figure 6(d)). These data suggest that BMW extracts are potent inhibitors of colon cancer stem cells.
4. Discussion
The data presented in this paper demonstrates that the methanolic extract of bitter melon is potent in inhibiting the growth of colon cancer cells. Since, cancer is a disease involving uncontrolled proliferation of cells, if a drug product is able to deter this cell division, it can potentially possess anticancer activity. Given the potent inhibition of proliferation of HT-29 and SW480 colon cancer cells by the bitter melon extracts, we proceeded with determining the mechanism of action. As a first step, we tested whether the activity resides in the skin or in the flesh. We did this because previous studies have demonstrated potent anticancer activity in the skin of grapes and peanuts [33, 34]. In bitter melon, our data suggests that the active ingredient most probably resides in the flesh as it inhibited cell proliferation with a much higher efficiency than the extracts from the skin.
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Though recent studies have reported a potential role of bitter melon in both preventing as well as attenuating the cancer progression, its potential mechanism of action has not been properly elucidated [15, 16]. Bitter melon has been used conventionally as an antidiabetic phyto-product in Ayurvedic and Traditional Chinese systems of medicine. The mechanism by which most antidiabetic medications work is either by acting as an insulin mimetic or by affecting the energy homeostasis of the cells by modulating the metabolic pathways. Bitter melon has been suspected to utilize the second strategy and our results suggest the same. Some energy modulators as well as calorie control in cancer patients have been shown to have positive effect on tumor progression [37]. Upon treatment of colon cancer cells with increasing concentration of bitter melon flesh extracts, there was a marked decrease in ATP production. This reduction in ATP production was similar to the ATP reduction seen with AICAR (a known AMPK activator). Further confirmation that the energy modulating activity of bitter melon extracts is in part responsible for cell death was obtained when ectopically added ATP was able to partially rescue the antiproliferative activity.
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It is being increasingly understood that there are a rare population of cells within a tumor that have the capacity to initiate and sustain tumorigenesis. These cancer stem cells, also termed cancer-initiating cells exhibit properties such as drug resistance and label retention, and are phenotypically undifferentiated. Stem cells have been increasingly recognized as the cause for not only primary tumorigenesis, but also for relapse of a tumor. A method of growing cells that represents growth from the stem cells is the colonosphere assay, where the cells are allowed to grow and form spheroids in ultralow attachment plates. So for a therapeutic entity to be a successful anticancer agent it needs to be effective against cancer stem cells, and reduction in spheroid formation capacity is used as one measure. Similar to that seen with proliferation assays, the effect of bitter melon flesh extracts was again found to be much stronger than the skin extracts in colonosphere assays. The size and number of the spheres were found to significantly decrease in cells treated with the extracts from the flesh as compared to control cells, indicating that the extracts can potentially act against cancer stem cells limiting their propagation and proliferation.