Calcitriol Sensitizes Cervical Cancer Cells To Irradiation By Regulating Autophagy And Apoptosis

Abstract

Currently, there is limited investigation on the role of active vitamin D3 (calcitriol) for radiosensitization of cervical cancer. This study aims to examine the influence of calcitriol on the reaction to ionizing radiation (IR) of cervical cancer cells and its mechanism. The radiosensitive effect of calcitriol was confirmed by colony formation assay and CCK8 assay. AnexinV/PI flow cytometry and western blot were detected for the identification of apoptosis. Confocal microscopy and transmission electron microscope were performed to evaluate the autophagic flux. Possible downstream signaling pathways affected by the calcitriol and IR were assayed by label free proteomic analyses. Clonogenic survival assays demonstrated a clear radio-enhancement when the cells were treated with calcitriol pre-treatment to IR, indicated by significant lower survival fractions compared to radiation alone in SiHa and CaSki cells. Furthermore, the reduced clonogenic survival was confirmed by the most significant proliferation inhibition after combination treatments in CCK8 assay. Flow cytometric analysis exhibited that calcitriol induced cell apoptosis and irradiation alone showed a non-specific effect on apoptosis. However, apoptotic rate is significantly upregulated when irradiation was involved in pre-treatment with calcitriol. By testing levels of Bcl-2, Bax, caspase-3 and cleaved-caspase-3, an even greater extent apoptosis execution was further proved when a combination of both treatments was used. For irradiation group, significant promotion in autophagy was detected according to the increased level of LC3B-II, with a modest increase in the treatment with calcitriol. Intriguingly, we observed that calcitriol almost abolished LC3B-II expression induced by radiation. On the basis of that, treatment with the autophagy/lysosome inhibitor chloroquine induced a further decrease in LC3B-II accumulation in calcitriol combined irradiation group. To elucidate the decreased autophagy, we observed LC3B puncta formation and autophagosome vesicle. As expected, these changes were consistent with the data from western blot. Consistent with the results observed in cells treated with calcitriol or irradiation, proteomic analyses indicated that calcitriol had a clear effect on elevating caspase-8 and Ambra1(autophagic factor Activating Molecule in Beclin1-Regulated Autophagy) reduction in the combination treatment group. Calcitriol sensitizes cervical cancer cells to irradiation by increasing apoptosis and decreasing autophagy. For the underlying mechanism, as Ambra1 was reported to be cleaved by caspases, we imply that decrease in autophagy induced by calcitriol combined with IR is a result of caspase-mediated Ambra1 degradation, which needs further study.