P21-Related Autophagy Precedes Apoptosis In Oridonin-treated Human Prostate Cancer

Objective:Previous studies have shown that Oridonin (ORI) was able to inhibit proliferation and induce apoptosis in several cell types. It also is one of crucial compounds in PC-SPES, which is a potent eight-herb formulation widely used in prostate cancer treatment. However, the mechanism is not fully clear. The aim of present study is to further understand the potential of ORI as a treatment for prostate cancer.Methods:Human prostate cancer (HPC) cells was cultured in vitro, and the inhibitory ratio of oridonin on HPC cells was assayed by CCK-3 kit, the ultrastructures of the cells was observed under light microscope and transmission electron microscope (TEM). AO and MDC were used to detecte autophagosomes and The change of DNA was detected by DAPI staining. Expression of LC3 and P21 proteins were detected using western blot.Results:In this study, ORI inhibited proliferation of PC-3, DU145 and LNCaP prostate cancer lines in a time-and dose-dependent manner. Treated with ORI, characteristic features of apoptosis, such as chromatin condensation, nuclear fragmentation and disappearance of surface microvillia were detected by morphologic study in cells at 48 hours. But at 24 hours, there was no apoptotic phenomenon, Instead, lots of autophagosomes with double-membrane structure were detected in cytosol, which contained portions of the cytosol, mitochondria and ER. Special biological staining with AO and MDC revealed formation of acid vesicular organelles (AVOs). By using confocal microscopy and Western Blot, recruitment of processed microtubule-associated protein 1 light chain 3-â…¡(MAP-LC3-â…¡) to autophagosomes was observed and the amount of LC3-â…¡was increased.3-MA, a specific autophagy inhibitor, could reverse ORI-induced accumulation of MDC and augment of LC3-II. ORI could upregulate P21 expression, which was blocked by inhibition of autophagy.Conclusions:These findings indicated that autophagy occurred before the onset of apoptosis in ORI-treated PC-3 and LNCaP human prostate cancer cells, and this autophagy-inducing activity was related to the level of P21. Our studies provide experimental evidence for the use of ORI in clinic.