Augmentation of sodium butyrate-induced apoptosis by phosphatidylinositol 3′-kinase inhibition in the KM20 human colon cancer cell line

Purpose: We have recently shown that inhibition of the phosphatidylinositol 3′-kinase (PI3k) pathway enhances sodium butyrate (NaBT)-mediated differentiation of human colon cancer cells. The purpose of this study was to determine whether PI3k inhibition can enhance the inhibitory effect of NaBT on an aggressive human colon cancer cell line, KM20. Experimental Design: The KM20 cell line, established from a metastatic colon cancer, was treated ill in vitro with NaBT, gemcitabine, or 5-fluorouracil either alone or in combination with the PI3k inhibitors wortmannin or LY294002; DNA fragmentation and cell viability were measured. As further indicators of apoptosis, protein was extracted to determine caspase-9 and caspase-3 activation and cleavage of poly(ADP-ribose) polymerase. In addition, the effect of NaBT and wortmannin on in vivo KM20 tumor growth was determined. Results: We demonstrate that inhibition of PI3k enhanced NaBT-mediated apoptosis and decreased KM20 cell viability; the nonspecific caspase inhibitor zVAD-fmk blocked the induction of apoptosis by the combination treatment. Either wortmannin or LY294002, combined with NaBT, enhanced activation of caspase-9 and caspase-3 and the subsequent cleavage of poly(ADP-ribose) polymerase. Furthermore, inhibition of PI3k increased the sensitivity of KM20 cells to gemcitabine and 5-fluorouracil. Wortmannin alone inhibited KM20 xenograft growth in vivo; the combination of wortmannin and NaBT demonstrated an enhanced effect compared with either agent alone. Conclusions: Our results are the first to show that inhibition of PI3k enhances NaBT-mediated colon cancer cell apoptosis through the activation of caspase-9 and caspase-3. Moreover, these findings suggest that agents that selectively target the PI3k pathway may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.