丹酚酸A通过抑制GRP78分泌调控微环境逆转肿瘤耐药的分子机制

Salvianolic acid A is an important bioactive ingredient of Salvia miltiorrhiza Bunge, which has been shown to reverse drug resistance of tumor significantly, but the target and molecular mechanisms remain unclear, restricting its application in cancer treatment. GRP78 is a tumor-specific secreted protein that modifies the microenvironment and makes tumor cells resistant to drug intervention. Previous study found that salvianolic acid A could directly bind to GRP78 and inhibit the secretion of GRP78 from tumor cells. The experiments in vivo showed that salvianolic acid A could enhance the sensitivity of tumor-bearing nude mice to 5-FU and oxaliplatin, however this effect of salvianolic acid A could be attenuated by injection of secreted GRP78 into the tumor. It is speculated accordingly that salvianolic acid A may inhibit GRP78 secretion and block its impact on the microenvironment, thereby reversing drug resistance of tumor. This project intends to use molecular docking and genetic engineering methods to explore the specific binding site of salvianolic acid A and GRP78. The molecular mechanism of salvianolic acid A binding to GRP78 to inhibit its secretion was elucidated using fluorescence tracing technology combined with protein ubiquitination analysis. The successful implementation of this project will clarify the novel mechanism of salvianolic acid A reversing tumor drug resistance and lay a theoretical basis for its clinical application in tumor chemotherapy and provide new ideas and directions for drug development based on tumor microenvironment.

丹酚酸A是丹参的重要活性成分，有研究表明丹酚酸A有显著的逆转肿瘤耐药作用，但作用靶点和分子机制尚不清楚，制约了其在肿瘤治疗中的应用。GRP78是肿瘤特异性分泌蛋白，它能改造微环境，帮助肿瘤细胞抵抗药物干预。本项目前期研究发现：丹酚酸A能与GRP78直接结合，并抑制肿瘤细胞中GRP78分泌；体内实验结果显示，丹酚酸A能增强荷瘤裸鼠对5-FU和奥沙利铂的敏感性，而向瘤内注射分泌型GRP78则减弱了丹酚酸A的这一作用。据此推测，丹酚酸A可能通过抑制GRP78的分泌，阻断其对微环境的影响，从而逆转肿瘤耐药。本项目拟采用分子对接和基因工程手段探究丹酚酸A与GRP78结合的具体位点；采用荧光示踪技术并结合蛋白泛素化分析阐明丹酚酸A结合GRP78后抑制其分泌的分子机制。本项目的顺利实施将阐明丹酚酸A逆转肿瘤耐药的新机制，为其在肿瘤化疗的临床应用提供理论依据，为基于肿瘤微环境的药物开发提供新的思路和方向。

丹酚酸A（SAA）是丹参的重要活性成分，有研究表明SAA有显著的逆转肿瘤耐药作用，但作用靶点和分子机制尚不清楚，制约了其在肿瘤治疗中的应用。GRP78是肿瘤特异性分泌蛋白，它能改造微环境，帮助肿瘤细胞抵抗药物干预。本项目通过荧光光谱和表面等离子共振技术检测发现SAA与GRP78能够形成较强的非共价结合，通过构建GRP78的截短体发现，SAA结合于GRP78的C端结构域。计算机分子模拟对接结果显示，C端第633位的赖氨酸能与SAA分子形成氢键，突变K633位点后GRP78与SAA的相互作用消失。进一步研究发现，SAA并不影响肿瘤细胞中外泌体的形成，而是抑制了GRP78与去泛素化酶USP8的相互作用，从而导致GRP78 K633位点的单泛素化难以清除，使得本该进入外泌体的GRP78进入了溶酶体降解途径。小鼠体内实验结果显示，SAA与5-FU和奥沙利铂联合使用时能够显著干预小鼠肿瘤的生长，并降低血清中GRP78的含量，降低肿瘤中CD31（内皮细胞）、CD206（肿瘤相关巨噬细胞）和α-SMA（成纤维细胞）的表达，然而对GRP78的633位赖氨酸进行突变后，SAA改善微环境提高化疗效率的作用显著减弱。上述研究成果表明SAA通过抑制肿瘤细胞分泌GRP78改善了肿瘤微环境，从而发挥提高化疗效率，逆转肿瘤耐药的作用。