Patients with type II diabetes mellitus are more susceptible to colorectal cancer (CRC) incidence than non-diabetics. The anti-diabetic drug metformin is most commonly prescribed for the treatment of this disease and has recently shown antitumor effect in preclinical studies. The aberrant mutational activation in the components of RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathway is very frequently observed in CRC. We previously reported that metformin inhibits the phosphorylation of ERK and BEZ235, a dual inhibitor of PI3K and mTOR, has anti-tumor activity against HCT15 CRC cells harboring mutations of KRAS and PIK3CA. Therefore, we hypothesized that simultaneous inhibition of two pathways by combining metformin with BEZ235 could be more effective in the suppression of proliferation than single agent treatment in HCT15 CRC cells. Here, we investigated the combinatory effect of metformin and BEZ235 on the cell survival in HCT15 CRC cells. Our study shows that both of the two signaling pathways can be blocked by this combinational strategy: metformin suppressed both pathways by inhibiting the phosphorylation of ERK, 4E-BP1 and S6, and BEZ235 suppressed PI3K/AKT/ mTOR pathway by reducing the phosphorylation of 4E-BP1 and S6. This combination treatment synergistically reduced cell viability. The combination index (CI) values ranged from 0.44 to 0.88, indicating synergism for the combination. These results offer a preclinical rationale for the potential therapeutic option for the treatment of CRC.

Molecular targeting for the altered signaling pathways has been proven to be effective for the treatment of many types of human cancer, including colorectal cancer (CRC). The dual phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 has shown to exhibit potent antitumor activity against solid tumors. Autophagy is a cellular lysosomal catabolic process to maintain metabolic homeostasis, which has been known to be induced in response to many therapeutic agents in cancer cells. This process is negatively regulated by mTOR and often acts as prosurvival or prodeath mechanism following cancer therapeutics. The current study was designed to investigate the antiproliferation activity of BEZ235 and to evaluate the role of autophagy induced by BEZ235 using HCT15 CRC cells bearing ras oncogene mutation. We found that BEZ235 decreases cell viability, which was mostly dependent on G1 arrest of cell cycle via suppression of cyclin A expression. BEZ235 affects PI3K/Akt/mTOR signaling pathway by increasing the phosphorylation of AKT at Ser473 and RAS/RAF/MEK/ERK pathway by decreasing the phosphorylation of ERK at Tyr204. BEZ235 also stimulated autophagy induction as evidenced by the increased expression of LC3-II and abundant acidic vesicular organelles (AVOs) in the cytoplasm. In addition, the combination of BEZ235 with autophagy inhibitor chloroquine, a known antagonist of autophagy, counteracted the antiproliferation effect of BEZ235. Thus, our study indicates that autophagy induced in response to BEZ235 treatment appears to act as cell death mechanism in HCT15 CRC cells.