Abstract 5721: Grape seed extract induces both intrinsic and extrinsic apoptotic pathways in its efficacy against human colorectal cancer cells harboring varying degrees of metastatic potential

There are 150,000 new cases of colorectal cancer (CRC) each year in the United States alone; one-third of these patients die. Traditional methods of treatment are known to result in severe toxicity suggesting that additional strategies are needed for CRC control. Grape seed extract (GSE); a health supplement rich in polyphenolic compounds has been previously shown by us to exhibit anti-cancer effects in in-vitro and in-vivo models of CRC. In general, as cancer progresses it becomes more resistant to conventional treatments; partly due to resistance to apoptosis, suggesting that the use of agents which are able to induce cancer cell death can be an effective approach to control various malignancies, including CRC. Accordingly, the present study focuses on eluting the efficacy and associated mechanisms of apoptotic death associated with GSE treatment in human CRC cell lines, differing in their metastatic potential. Three human CRC cell lines were chosen based on phenotypic and genetic variations; SW480, SW620 and HCT116, representing cancer stages II-IV, respectively. In SW480 cells, GSE treatment (25-100μg/mL) resulted in a time and a concentration-dependent increase in cell death; from 17-37% death after 12h, to 19-38% after 24h, and maximum of 60% death observed at 48h. Annexin-PI staining revealed that the cell death induced by GSE was apoptotic in nature. At the molecular level, GSE induced caspase-9, −3, and −8 cleavage indicating the involvement of both intrinsic and extrinsic pathways of apoptosis. However, we did not observe any changes in the Bcl-2 family members. Unlike SW480, much lower concentrations of GSE (10-30μg/mL) were effective in causing 50% cell death as early as 12h in SW620 CRC cells, and involved specifically the activation of the extrinsic apoptosis pathway via DR4 up regulation, caspase-8, −3, and PARP cleavage, as a robust mechanism of apoptotic cell death. In HCT116 cells, GSE treatment (10-30μg/mL) resulted in 42-60% death after 12h, which did not increase, significantly, at later time points. Cell death ELISA revealed that almost 60% apoptotic death occurred as early as 12h at 30μg/mL concentration of GSE. Mechanistic studies revealed that GSE activates the intrinsic pathway of apoptosis, via cleavage of caspase-9 and −3. We also observed caspase-8 cleavage with no alteration of Bid levels, indicating secondary activation of this caspase through the intrinsic pathway. Together, these results clearly show that the apoptosis inducing effect of GSE involves both intrinsic and extrinsic pathways, and the apoptotic activity of GSE increases with an increase in the metastatic potential of the CRC cell lines; HCT116 being the most sensitive. In conclusion, our findings further support the notion that GSE could be an effective chemopreventive and chemotherapeutic agent for different stages of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5721.