Molecular mechanisms of resveratrol induced cell death in MDA-MB-231 breast cancer cells
Prabhu, Varun Vijay
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Metastatic breast cancer is one of the leading causes of cancer related death in women. Surgery in combination with radiation and chemotherapy has shown promising results for breast cancer therapy. However, toxicity and development of resistance in response to chemotherapeutic drugs has resulted in a need for alternative anti-cancer agents for breast cancer. Resveratrol (trans-3, 5, 4'-trihydroxystilbene) is a plant polyphenol found at high levels in the skin of grapes and red wine. It possesses the ability to target a wide range of signaling pathways that impair the growth and survival of cancer cells with relatively low toxicity. Resveratrol has been shown to induce apoptotic cell death in breast cancer cells, but the mechanism of resveratrol induced apoptosis in highly metastatic and treatment unresponsive breast cancer cells such as MDA-MB-231 is unclear. Current evidence suggests that resveratrol targets the mitochondria causing loss of mitochondrial membrane potential to induce cytochrome c release. However, the mechanism of cytochrome c release, apoptosome formation and subsequent caspase activation is not well characterized. In addition, resveratrol is known to induce autophagic cell death in breast cancer cells. However, autophagy is also reported to contribute to cell survival. Thus, the molecular mechanism of resveratrol induced cell death in breast cancer cells remains unclear. The primary goal of this thesis is to determine the mechanism of apoptotic cell death as well as the role of autophagy in regulating cell death in response to resveratrol in breast cancer cells. To accomplish this, we used MDA-MB-231 cells which were subjected to resveratrol treatment for various time points followed by isolation of the cytosolic and mitochondrial fractions using differential centrifugation. The cytosolic and mitochondrial fractions were used to determine the molecular mechanism of apoptosis upon resveratrol treatment. Mechanistic analysis of the fractions using chemical crosslinking, gel filtration chromatography and Western blotting revealed that resveratrol induces Bax translocation to the mitochondria where Bax and Bak form oligomers to form the channels that induce cytochrome c release from the mitochondria. This process coincides with Bim upregulation and Bid cleavage on the mitochondria. Surprisingly, cytochrome c release into the cytosol fails to induce oligomerization of APAF-1, thus preventing apoptosome formation. In addition, Western blotting and caspase activity measurement revealed that active caspase 9 primarily associates with the mitochondria. Finally, resveratrol also induces autophagy in MDA-MB-231 cells as the levels of LC3-II increase upon resveratrol treatment. Furthermore, inhibition of autophagy by silencing essential autophagy genes such as ATG-5 and Beclin-1 with lentiviral shRNA enhanced caspase activation. These findings suggest that resveratrol causes apoptotic cell death by inducing caspase activation within the mitochondria in an apoptosome independent manner and the induction of autophagy forms a part of a survival response. Thus, inhibition of autophagy along with induction of apoptosis could represent a new strategy for breast cancer therapy.