Study On The Mechanism Of Triptolide-induced Apoptosis And Autophagy In Cancer Cells

Triptolide is a diterpene lactone extracted from Chinese medicinal herb leigongteng(Tripterygium wilfordii Hook F.) which has been used to treat inflammatory and immunological diseases for centuries. As the major active component in leigongteng, triptolide has attracted high attention for its multiple activities, especially for its anti-tumor effect. Triptolide has shown a broad spectrum of anti-tumor effect and cytotoxicity on many kinds of cancers. However, the mechanism of triptolide-induced apoptosis in Human Papillomavirus(HPV) positive cancer cells and triptolide-induced autophagy are not deeply investigated. In the present study, we investigated the mechanism of triptolide-induced apoptosis in HPV positive cancer cells and triptolide-induced autophagy, as well as the effect of autophagy on triptolide cytotoxicity using the techniques of molecular biology, cell biology and xenograft mouse model. The results are showing as follows:1. The effect of triptolide on HPV positive cancer cells was studied on laryngocarcinoma cells HEp-2(HPV18 positive). The results show that triptolide inhibits the proliferation, migration and clonegenic of HEp-2 cells. Meanwhile, triptolide induces apoptosis both through death receptor pathways and mitochondrial pathway in HEp-2 cells.2. Triptolide enhances the anti-tumor effect of radiation in laryngocarcinoma cells in vitro.3. We next detected the expression of pro-apoptosis protein with triptolide treatment to further study mechanism of triptolide-induced apoptosis using the methods of real time PCR, immunoblot and immunoflorescence. The results suggest that triptolide induces a p53-dependent apoptosis in HEp-2 cells. Triptolide induces the accumulation of p53 is mainly through two ways in HEp-2 cells: on the one hand, triptolide triggers DNA damage and induces p53 mRNA transcription; on the other hand, triptolide inhibits the expression of ubiquitin E3 ligase E6/E6 AP and disturbs their interaction with p53, which result in the block of proteasome-mediated p53 degradation. Then, activated p53 subsequently up-regulates the expression downstream pro-apoptosis gene and promotes cell apoptosis.4. The effect of triptolide on tumor cell autophagy was detected using the immunoblot and immunoflorescence methods. The results show that triptolide could induce autophagy in laryngocarcinoma cells HEp-2 and prostate cancer cells PC-3, LNCaP and C4-2.5. Using the methods of immunoblot and immunoflorescence, we investigated the mechanism of triptolide-induced autophagy in cancer cells. The results show that triptolide inhibits the expression of ubiquitin modification enzymes and chaperons, result in ER(Endoplasmic Reticulum) stress which further causes calcium to be released from the ER into the cytoplasm. The high cellular calcium level stimulates CaMKKβ to phosphorylate AMPKα at Thr172. Activated AMPK inhibits mTOR activity through phosphorylating TSC2 and raptor. Inactive mTOR no longer suppresses the ULK1 complex. Activated AMPK also directly phosphorylates ULK1 and Beclin 1, resulting in activation of the ULK1 complex and the Class III PI3 K complex. Activation of these complexes leads to the induction of autophagosome formation.6. We finally investigate the effect of autophagy on the anti-tumor activity of triptolide bothin vitro and in vivo. The results show that inhibition of autophagy with autophagy inhibitors significantly promotes triptolide-induced apoptosis in vitro. Meanwhile, combination use of triptolide and autophagy inhibitors also more significantly reduces the tumor weights and volumes compared to triptolide-only treatment group in xenografted mouse model. These results suggest that inhibition of autophagy promote the anti-tumor effect of triptolide.Collectively, our results suggested that triptolide inhibits the proliferation and induces apoptosis of HPV positive cancer cells. As well, triptolide also enhances the anti-cancer effect of radiation. Triptolide induces HPV positive cancer cells apoptosis mainly through promoting p53 transcription and inhibiting E6/E6 AP mediated p53 degradation. Furthermore, we also found that triptolide induces autophagy in cancer cells. Triptolide triggers ER Stress Response, then promotes the ER calcium release in cancer cells. The accumulation of calcium in cytoplasm promotes CaMKKβ mediated AMPK activation. Subsequently, activated AMPK activates the downstream pathway and triggers autophagy in cancer cells. Finally, it is also proved that inhibition of autophagy enhances the anti-cancer effect of triptolide both in vitro and in vivo, which implies that the combination use of triptolide with autophagy inhibitors may be a more effective therapeutics for cancer.