茶多酚抑制HMGB1介导的自噬增强膀胱癌化疗敏感性的作用及其机制的研究

The biological characteristics of bladder cancer is easy to relapse. There is an urgent need for new therapeutics with high efficiency and low toxicity. While bladder infusion of chemotherapy drugs such as epirubicin is a common approach for bladder cancer，single drug based therapy is often ineffective. TNF-related apoptosis-inducing ligand (TRAIL) is a promising new anti-cancer drugs, but has not been tested in bladder cancer. Autophagy is a cellular self-protecting mechanism, inhibition of which usually enhances the effects of chemotherapy. In our preliminary studies, we found epirubicin and TRAIL induce autophagy in bladder cancer cells, and suppressing autophagy significantly improves anticancer activity. Because tea polyphenols are able to inhibit autophagy, we hypothesize that activation of autophagy protects cancer cells and reduce the anti-cancer activity of epirubicin or TRAIL in bladder cancer cells, and the combination of tea polyphenols and epirubicin or TRAIL will inhibit autophagy and thus improve therapeutic effect. In this project, by combining in vitro and in vivo studies, we will investigate the role and mechanism of autophagy in bladder infusion chemotherapy; and determine the anticancer effect of the combination of tea polyphenols and epirubicin or TRAIL used in bladder infusion. The successful completion of this study will not only prove TRAIL as a new drug for bladder cancer, but also determine the usefulness of combination of tea polyphenols and epirubicin or TRAIL for the treatment of bladder cancer.

膀胱癌的特点是术后易复发。膀胱单一药物灌注化疗往往难以达到满意的疗效。探索高效低毒的治疗措施十分必要。自噬是一种细胞自我保护机制，抑制自噬常常能显著增强放化疗的效果。我们的前期工作首次发现表柔比星（EPI）及TNF相关凋亡诱导配体(TRAIL)在膀胱癌中可以诱导自噬；而抑制自噬可显著提高癌细胞对EPI及TRAIL的敏感性，具有抗癌活性的茶多酚通过抑制HMGB1而抑制自噬。因此我们设想：抗癌药物EPI或TRAIL诱导膀胱癌细胞凋亡，但同时诱导的保护性自噬降低其抗癌活性。而联合应用茶多酚和EPI或TRAIL，不仅协同诱导凋亡，还可以抑制保护性自噬以增强疗效。本项目拟通过体内外实验研究联合应用茶多酚和EPI或 TRAIL的治疗效果以及自噬对膀胱化疗药物的增效分子机制。本研究的顺利完成，不仅探明TRAIL作为抗膀胱癌新药的机制，更将为联合应用茶多酚及EPI或TRAIL治疗膀胱癌提供理论和实验依据。

本课题研究结果显示：膀胱癌灌注化疗药表柔比星（EPI）在诱导膀胱癌细胞株T24和BIU-87细胞凋亡的同时诱导保护性自噬活化，应用茶多酚（TP）或氯喹（CQ）均可抑制EPI介导的自噬活化并增强EPI诱导的膀胱癌细胞凋亡，进一步提高其抗癌疗效。机制研究发现，MAPK/JNK通路参与调控EPI诱导的自噬活化，应用TP可阻断EPI诱导的JNK通路激活且可增强膀胱癌细胞对EPI的化疗敏感性。同时，构建人膀胱癌裸鼠移植瘤模型，体内实验结果表明EPI可诱导人膀胱癌裸鼠移植瘤自噬的活化，该过程可被TP显著抑制；在EPI抑制裸鼠移植瘤的生长情况下，TP联合EPI处理能进一步增强EPI体内抗癌活性。此外，作为膀胱癌一线化疗药的吉西他滨（GEM）和顺铂（DDP）也可诱导膀胱癌细胞凋亡和保护性自噬的产生，其诱导自噬活化的机制与MAPK通路密切相关。我们还发现了核转录因子高迁移率族蛋白1（HMGB1）在膀胱癌和癌旁正常组织间的表达存在差异，吉西他滨能促进HMGB1的释放，利用小干扰RNA（siRNA）敲低HMGB1表达后可抑制吉西他滨诱导的保护性自噬，并增强吉西他滨的促凋亡作用。此外，干扰HMGB1也可抑制吉西他滨诱导的JNK和ERK磷酸化，表明HMGB1-JNK/ERK通路参与吉西他滨诱导的保护性自噬活化。本研究为药物联合治疗和以信号分子为靶点开发临床新的药物治疗膀胱癌提供了新的理论依据，对逆转膀胱癌耐药提供新的思路，对增强膀胱癌的化疗敏感性有一定的临床指导价值。