Modulating autophagy to influence apoptosis induced by dihydroartemisinin in bladder cancer cell lines in vitro

Objective To investigate whether combined treatment of dihydroartemisinin (DHA) and autophagy inhibitors results in enhancing the cyotoxic effects of DHA upon human bladder cells, T24, in vitro, and to explore the mechanism of autophagy on DHA-induced T24 bladder cancer cell death. Methods DHA with or without autophagy inhibitor (3-MA, 5 mmol/L) was added to treat human bladder cell, T24, at varying concentrations (0, 12.5, 25.0, 50.0, 100.0 μmol/L)for 24, 48 and 72 h. To measure cell viability, we performed methyl thiazol tetrazolium (MTT) assay.We examined apoptosis by flow cytometry.After administration of the lower concentration of DHA for 24 h, the morphologic analysis of T24 cells autophagy after monodansylcadaverine staining was performed using fluorescence microscopy.Transmission electron microscope was employed to observed the subcellular structure of T24 cells.Western blotting analysis was used to detect the expression levels of autophagy-related proteins [microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and p62] and apoptosis marker proteins [procaspase-3, total poly adenosine diphosphate-ribose polymerase (PARP) and cleaved-PARP]. Results The results showed that DHA significantly inhibited the proliferation and induced apoptosis of T24 bladder cancer cells in a dose-and time-dependent manner as compared with control group (P=0.003) (The inhibition rate of 12.5 μmol/L concentration of DHA were 5.79%, 6.61%, 11.09% at the time of 24, 48 and 72 h; the inhibition rate of 25.0 μmol/L concentration of DHA were 11.25%, 15.32%, 21.35% at the time of 24, 48 and 72 h; the inhibition rate of 50.0 μmol/L concentration of DHA were 27.16%, 30.66%, 47.58%, at the time of 24, 48 and 72 h; the inhibition rate of 100.0 μmol/L concentration of DHA were 41.52%, 57.52%, 74.29%, at the time of 24, 48 and 72 h. The apoptosis rate of 12.5 μmol/L concentration of DHA were 3.66%, 6.44%, 9.78%, at the time of 24, 48 and 72 h; the apoptosis rate of 25.0 μmol/L concentration of DHA were 7.02%, 13.4%, 17.55%, at the time of 24, 48 and 72 h; the apoptosis rate of 50.0 μmol/L concentration of DHA were 20.53%, 26.97%, 38.69%, at the time of 24, 48 h and 72 h; the apoptosis rate of 100.0 μmol/L concentration of DHA were 29.82%, 39.26%, 44.92%, at the time of 24, 48 and 72 h). The combination of the autophagy inhibitor 3-MA and DHA resulted in enhancing DHA-induced apoptosis cell death in T24 bladder cancer cells.Under the fluorescence microscopy, we found that condensed punctate autophagic vacuoles stained by monodansylcadaverin (MDC) were located in the cytoplasm.Electron microscopy demonstrated formation of multiple autophagic vacuoles in T24 cells after DHA treatment.Western blotting analysis showed that the expression levels of both autophagy-related proteins (LC3, beclin-1) and apoptosis marker proteins (cleaved-PARP) were increased compared with the control group.On the contrary, the levels of p62, procaspase-3 and total PARP were decreased.Moreover, the combined treatment with 3-MA increased the detection of cleaved-PARP, while decreasing procaspase-3 and total PARP, compared to either group treated with a single agent. Conclusion DHA has great anti-cancer efficacy on human bladder cancer T24 cells.Autophagy was induced and a crucial survival mechanism for cancer cells during DHA-induced apoptotic cell death in human bladder cancer cells. Key words: Urinary bladder neoplasms; Apoptosis; Autophagy; Dihydroartemisinin