中药开口箭皂苷介导胃癌细胞释放HMGB1调控TAMs极化的抗胃癌机制研究

Tumor-associated macrophages (TAM) affect the chemotherapy and poor prognosis of cancer. Re-polarization TAM to an anti-tumor M1-like mode is a new strategy in oncology. Therefore, it is important to explore new drugs regulating the M2→M1 switch for the treatment of gastric cancer. Previous studies showed that tumor inhibition rate of saponin from Tupistra chinensis in nude mice is 54.9%. Second, among all these saponin from T. chinensis, T17 is the most activity new compound belong to spirostanol saponin. T17 induces the high-mobility group box 1 protein (HMGB1) translocation and release in gastric cancer cells. Last, the T17 conditioned medium (the medium of gastric cancer cells treated with T17) promotes M1 polarization of macrophages compared with DMSO conditioned medium. Due to the phenotype of macrophages is closely related to extracellular HMGB1, we speculate that the anti-gastric cancer activity of T17 may be related to its induction of HMGB1 release from gastric cancer cells and subsequent skews macrophage polarization. However, the specific mechanism is still unclear. In this study, HMGB1 wild type or HMGB1 shRNA gastric cancer cell and macrophage co-culture system, and HMGB1 wild type or HMGB1 shRNA gastric cancer cells bearing nude mice will be used. Cell biology and molecular biology detection will be applied to reveal the effect of HMGB1 on T17-induced apoptosis and autophagy of gastric cancer cells, to clarify the effect of TAM on the anti-gastric cancer activity of T17, to explore the regulatory mechanism of HMGB1 released by T17 treated gastric cancer cells on macrophage phenotype, and to elucidate the anti-gastric cancer mechanism of T17. This study provides an experimental basis for the treatment of gastric cancer with saponin from Tupistra chinensis. It is a prospective exploration to find the leading compounds that can interfere with TAM polarization from natural products.

肿瘤相关巨噬细胞（TAMs）影响癌症化疗效果及预后，靶向TAMs的M2→M1型重极化过程是抗肿瘤新策略，探寻调控此过程的新药对胃癌治疗意义重大。皂苷是中药开口箭有效成分之一，前期研究发现开口箭皂苷（TC）裸鼠抑瘤率达54.9%，抗肿瘤活性最优的开口箭皂苷T17诱导胃癌细胞凋亡及HMGB1的核质转移与释放，经其处理的胃癌细胞培养基促进巨噬细胞向M1型极化。巨噬细胞表型与HMGB1密切相关，由此推测T17抗胃癌活性与其介导凋亡细胞释放HMGB1并调控巨噬细胞极化有关，但具体机制不明。本项目拟采用HMGB1野生/低表达胃癌-巨噬细胞共培养模型及荷瘤裸鼠模型，从分子、细胞及整体水平明确TAMs对T17抗胃癌活性的影响，探究被动释放的HMGB1对巨噬细胞表型的调控及机制，并阐明T17抗胃癌机制。本研究为TC治疗胃癌提供实验依据，是从中药天然产物中寻找干预TAMs表型转化的先导化合物的前瞻性探索。

胃癌是世界上致死率排名第三的癌症，由于早期癌症无明显体征，初诊时多数病人已错过手术时机，而目前传统化疗手段对晚期胃癌的治疗效果并不理想，更有效的抗胃癌手段和药物的寻找仍具有很大挑战。最新研究表明肿瘤相关巨噬细胞（TAMs）影响胃癌化疗效果，化疗药物若是能够调控TAMs表型，在一定程度上可以增强化疗效果。本项目研究结果表明：（1）开口箭皂苷在体外、体内均具有潜在的抗胃癌活性，抑制裸鼠胃癌移植瘤生长却对裸鼠体重无明显影响。（2）开口箭皂苷T17通过诱导caspase依赖的，p53非依赖的凋亡促进胃癌细胞死亡。（3）开口箭皂苷诱导胃癌细胞自噬，自噬抑制剂的联合使用增强了其抗胃癌活性。（4）开口箭皂苷T17通过抑制HMGB1的表达及促进HMGB1核质转移诱导胃癌细胞凋亡和自噬，且该过程与p53状态无关。（5）经过PMA和IL-4的诱导，成功诱导出THP-1巨噬细胞和M2表型巨噬细胞，并构建了巨噬细胞-胃癌细胞共培养模型模拟TAMs；经开口箭皂苷T17的处理，共培养体系中的M2型巨噬细胞减少，HMGB1抑制剂的加入取消了该作用。此外，体内实验同样表明开口箭皂苷降低了巨噬细胞的浸润及M2型TAMs的表达。综上所述，我们的研究阐明了开口箭皂苷抑制胃癌细胞增殖的分子机制，揭示了HMGB1的抑制在开口箭皂苷诱导的胃癌细胞凋亡与自噬过程中的作用，表明中药开口箭皂苷的抗胃癌还与其促进HMGB1释放，降低M2型TAMs有关。本项目的完成为胃癌的中药天然产物治疗提供了新的研究思路，为胃癌的联合治疗药物研发提供了理论参考。