Abstract LB-171: Dietary agent induced AMPK activation: Targeting stem cells for the prevention of colon cancer

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. Although early diagnosis often leads to cure, most cases go undetected, thus needing other prevention-related strategies. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis. It has also been shown to control tumor progression by regulation of cell cycle, protein synthesis and cell growth and/or survival. Dietary agents can affect AMPK signaling thereby reducing risk of cancer progression. Bitter melon is recommended in ancient Indian/Chinese medicine for treatment of metabolic disorders. Bitter melon extracts (BME) have been shown to affect breast and prostate cancers, though the mechanism is not well known. Here, we report the inhibitory effect of BME on colon cancer stem cells via AMPK activation. We tested the effect of aqueous BME on two CRC lines HT-29 and SW480. It significantly inhibited the proliferation and clonogenic capacity of both the lines. In addition, cell cycle analysis showed a significant increase in cells arrested at the G2/M phase. Moreover, the whole extract was found to be more effective when compared to the skin extracts, suggesting that the active ingredient is present at higher levels in the flesh and seeds of the fruit than in the skin. We next determined the mechanism by which BME affects the growth of the colon cancer cells. Western blot analyses suggested that there was no caspase-3 activation. However, western blots demonstrated a significant induction in LC3B cleavage, which was further confirmed by immunocytochemistry. This suggests that the cells were undergoing autophagy. To confirm this, we performed Real Time RT-PCR and western blot analyses for autophagy markers along with flow based autophagy analyses. While BCL 2 (pro-apoptotic marker) was reduced, there was an increase in Beclin-1 (marker for autophagy) in the two cell lines. Real Time RT-PCR analyses demonstrated increased levels of Atg5, 7 and 12 mRNA in BME treated cells when compared to control. Western blot analyses also showed enhanced phosphorylation of AMPK, and its downstream target proteins. BME were also found to reduce cellular ATP levels, which might be the cause of AMPK signaling. Cell proliferation was found to be reactivated in the presence of exogenous supply of ATP. To study the inhibitory effect of BME on colon cancer stem cells, spheroid formation assay was performed. There was a dose-dependent reduction in the number and size of the spheres. Furthermore, western blot analyses demonstrated a reduction in the expression of DCLK1, a quiescent stem cell marker which was further verified by immunocytochemistry and flow cytometry. Taken together, these results suggest that dietary agents such as BME can target colon cancer cells and mark them for autophagy. Further studies are warranted for developing bitter melon extract as an effective chemopreventive/therapeutic agent in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-171. doi:1538-7445.AM2012-LB-171