BI-69A11 promotes autophagy as an early event followed by the induction of apoptosis.

Abstract

Background: Autophagy, a highly conservative cellular degradation process recently acquires much attention due to its role in regulation of cancer development and progression and in determination of the response of tumor cells to anticancer therapy. Recent studies suggest that induction of autophagy is an early event that can lead into apoptosis after treatment with chemotherapeutic drugs via inhibition of Akt and its downstream target mTOR. Aberrant activation of PI3K/Akt/mToR pathway is responsible in cancer development, including colorectal cancer. BI-69A11, an imidazole derivative, was found to be effective in inhibiting AKT and eliciting cell death in the colon cancer by induction of apoptosis. The aim of our study is to investigate the effect of BI-69A11 in inducing autophagy as an early event of cell death. Material and Methods: Western blotting was performed after treatment HCT116 cells with BI-69A11 in dose dependent and time dependent manner to check the expression profile of LC3 and p62 and caspase-3 and -9. Morphological analysis was performed by staining the cells with acridine orange and lysotracker dye. Western blotting analysis was performed to investigate the profile of p-Akt and its downstream targets. HCT116 cells were transfected with myr-akt-pcDNA with Turbofect and treated with BI-69A11 and western blotting analysis was performed. Results: Here we found that BI-69A11 induced autophagy followed by induction of apoptosis via inhibition of PI3K/Akt/mTOR pathway and activation of caspase cascade HCT116 cells. The conversion of microtubule associated protein LC3-I to LC3-II, decreased level of p62 and the accumulation of Autophagic Vacuoles (AVs) and lysosomal vesicle in response of BI-69A11, suggested either an increase in the synthesis of autophagosomes or a blockage of lysosomal fusion and degradation as an early event. The activation of Caspase-3 and -9 proved the induction of autophagy was followed by apoptosis due to BI-69A11 treatment.BI-69A11 inhibited the phosphorylation of Akt at the Ser 473 residue without affecting total akt level. It also reduces the expression profile of the downstream targets of Akt. Moreover, transfection with myr-Akt, prevented the decrease in p-Akt levels, and partially blocked BI-69A11-induced autophagy in HCT116 cells. Conclusion: BI-69A11 inhibits tumor cell growth by a mechanism involving early autophagy and late apoptosis through the suppression of Akt/mTOR signaling. Here we reported that early autophagy is mainly dependent on induction of autophagosomes and conversion of LC3 that can be caused due to inhibition of Akt/mTOR signaling.