Both p53 dependent and independent pathways contribute to RAD001 mediated enhancement of chemo-sensitivity in hepatocellular carcinoma

Abstract

Objective: Chemo-resistance in hepatocellular carcinoma (HCC) hinders the application of systemic and local chemotherapy. Inhibition of mammalian target of rapamycin (mTOR) has been demonstrated to be able to sensitize tumor cells to cytotoxic drugs, but the mechanism remains unclear. Therefore, we design the present study to investigate the effects of mTOR inhibition on sensitization of HCC cells to chemo-cytotoxic drugs, with a focus on the potential interaction between mTOR and p53. Methods: In vitro, HCC cell lines with wild type (wt) p53 (HepG2), mutant/null p53 (PLC/Hep3B) and p53 stable transfectant (Hep3B-p53+) were treated with RAD001 alone, cisplatin alone or combination of RAD001 and cisplatin. Cell apoptosis was determined by Annexin V/ propidium iodine staining, and the molecular mechanism was explored by poly(ADP-ribose) polymerase (PARP) degradation. In vivo, the effects of RAD001 combined with cisplatin was evaluated in a HCC xenograft model. Results: The effect of mTOR inhibition was confirmed by dephosphorylation of 4E-BP1 and ribosomal protein S6. RAD001 enhanced cisplatin-induced PARP degradation and apoptosis in all the three cell lines tested. Transfection of p53 into Hep3B-p53- cell line could further enhance RAD001 mediated chemo-sensitization. RAD001, or cisplatin alone down-regulated the expression of pro-survival molecules, survivin, cyclin D1, and Bcl-2, and a synergistic effect was observed when RAD001 was combined with cisplatin. Prominent retardation of tumor growth was observed in the HCC xenografts treated with RAD001 and cisplatin. Conclusions: This study demonstrated that inhibition of mTOR enhanced cisplatin-induced chemo-cytotoxicity in HCC in both p53 dependent and independent manner.