An IAP Inhibitor,AR-406 Activates Apoptosis And Induces Radiosensitization In Normoxic And Hypoxic Cervical Cancer Cells

Objective: Radiation therapy resistance is directly correlated with the levels of the inhibitors of apoptosis protein(IAP)in cervical cancer.IAP antagonists increased the antitumor efficacy of X-irradiation in some cancers,but the effects of these antagonists on hypoxic tumor cells remain unclear.We aimed to investigate the radiosensitizing effect of AT-406,a small-molecule IAP inhibitor,on cervical cancer cell lines and the possible mechanism underlying radiosensitization under normoxic and hypoxic conditions.Methods: Hela and Siha human cervical cancer cell lines were treated with AT-406 at different concentration and radiation.The cell proliferation,apoptosis,and radiosensitivity under normoxic and hypoxic conditions were used to determine the effects of the drug with radiotherapy or not.Western Blotting analysis was subsequently performed to evaluated the efficacy of AT-406 on the expression of IAP family and related proteins.The apoptosis pathway was characterized by Caspase-3,-8,and-9 activity assays.Results: AT-406 increased sensitivity of both cervical cancer cell lines to radiation under normoxic condition,but a high AT-406 concentration was required to induce the same effect on Siha cells [survival enhancement ratio of Hela(SERHela)= 1.81,P < 0.05 at 10 ?M AT-406;SERSiha = 1.43,p < 0.05 at 10 ?M AT-406].Under hypoxic conditions,AT-406 enhanced radiation-induced reproductive cell death in both cell lines(SERHela = 2.07,P < 0.05 at 10 ?M AT-406;SERSiha = 1.72,p < 0.05 at 10 ?M AT-406).The radiosensitizing effect of AT-406 was more evident on hypoxic cells than on normoxic cells.Results of clonogenic survival assay and flow cytometry showed that AT-406 significantly enhanced radiation-induced apoptosis,especially in hypoxic cancer cells.Results obtained from Western Blotting analysis showed that AT-406 decreased the cellular IAP-1(c IAP1)level in Hela cells in a dose-dependent manner under normoxic condition and downregulated X-linked inhibitor of apoptosis protein(XIAP)expression in both two cell lines under hypoxic condition.The conflicting response of IAPs to AT-406 treatment under different oxygenation levels is associated with the preferential AT-406 radiosensitization on hypoxic cancer cells.It is also demonstrated that AT-406 works on both extrinsic death receptors and intrinsic mitochondrial apoptosis pathways to activate apoptosis by Caspase activity assays.Conclusions: AT-406 markedly radiosensitizes human cervical cancer cells by selectively inhibiting IAP family proteins and promoting Caspase activation to enhances apoptosis,particularly under hypoxic conditions.