[Mechanism of autophagy inhibition for sensitizing HeLa cell apoptosis induced by trichosanthin]

This research investigated the effect and mechanism of trichosanthin(TCS) in inducing autophagy and apoptosis of HeLa cells in cervical cancer. Two-step chromatography was used to prepare TCS. MTT assay was used to detect the inhibition effect of TCS on the proliferation of HeLa cells. The formation of autophagic vesicles and apoptotic bodies in HeLa cells treated with TCS was observed under transmission electron microscopy, and green fluorescent protein(GFP)-microtubule-associated protein 1 light chain beta 3(LC3B) fusion protein location was observed under laser confocal microscopy. The expression of autophagy and apoptosis-related proteins was detected by Western blot. It was found that the prepared TCS had high purity and biological activity. TCS could inhibit the proliferation of HeLa cells significantly in a concentration-dependent and time-dependent manner. Obvious autophagic vesicles were observed after HeLa cells were treated with TCS for 12 h, and typical apoptotic bodies were formed after 48 h. Under laser confocal microscopy, LC3B protein was observed to shift from a diffuse distribution to a spotted aggregation distribution in the cytoplasm. Western blot results showed that the expression of autophagy-activating protein LC3BⅡ began to increase in HeLa cells after being treated with TCS for 12 h and increased with the increase in drug concentration. Apoptosis-related protein poly ADP-ribose polymerase(PARP) began to activate as cleaved PARP after being treated with TCS for 24 h. The combination of TCS and 3-methyladenine(3-MA) further promoted the activation of cleaved PARP. The results showed that TCS could significantly inhibit the growth of HeLa cells in cervical cancer. Moreover, TCS induced autophagy in HeLa cells earlier than apoptosis, and inhibiting autophagy could sensitize apoptosis of HeLa cells induced by TCS. This indicated that autophagy induced by TCS was a protective cell response, and TCS combined with autophagy inhibitors could enhance the anti-cervical cancer effect of TCS.