AB44. Akt inhibition attenuates rasfonin (A304)-induced autophagy and casapase-dependent apoptosis via mTOR complex signaling in renal cancer cells

Abstract: Rasfonin (A304) was originally extracted from the Taleromyces sp. and Trichurus terrophilus, and was found to have the inhibitory activity on cancer cells. However, the underlying mechanism of its regulatory role in autophagy and programmed cell death remains largely unknown. In present study, the A304-induced cell death was first detected using human renal cancer cell line ACHN. A304 caused cell viability loss of ACHN in a time and dose-dependent manner. This finding was confirmed by colony growth in a survival assay, in which A304 was found to kill the cell depending on the stimulus concentration. Flow cytometry data revealed that the A304-induced cell death of ACHN could be either apoptotic or necrotic (may be either necrosis or secondary necrosis). Using electron and confocal microscopy, we demonstrate that A304 enhances the formation of autophagosome. In immunoblotting assay, we observed that treatment with A304 increased the ratio of LC3-II to actin relative to the control in a concentration dependent manner. Meanwhile, we found that p62/SQSTM1, which is considered to be a selective substrate of autophagy and will be accumulated when autophagy is inhibited, was decreased in A304-exposed cells. Although Akt inhibition attenuates A304-induced autophagy, apoptosis and second necrosis, yet, it failed to rescue cell viability loss upon prolonged stimulation. Interestingly, different from former report, we found that Akt could play a positively regulatory role in autophagy through the mTOR complex. Moreover, Akt isforms appeared to function differentially in the mediation of autophagy and apoptosis induced by A304 depending on the stimulation duration.

Keywords: Rasfonin; A304; mTOR complex; autophagy