齐墩果酸皂苷OAS-10抑制A1和A3型腺苷受体介导的胶质瘤细胞增殖的分子机理

Invasive human glioma cell can't be completely removed by surgery. It's difficult to cure and vulnerable to relapse. Our group have isolated many saponins with anti-glioma activity from medicinal plants. Of these saponins, OAS-10 had the strongest activity. OAS-10 can combine with A1 and A3 receptors, respectively, in rat glioma tissue. What's more, OAS-10 induced apoptosis in a variety of glioma cell lines, significantly lowered levels of VEGF expression and inhibited the proliferation of glioma in nude mice. However, its specific mechanism of action is unclear. Recent studies have shown that adenosine concentrations significantly increased in glioma with hypoxic microenvironment. Saponin at low concentrations can combine with A1R and interfere with the formation of adenosine-A1R-G protein complex. A3R has similar amino acid sequences with A1R and they both present on the tumor cell membrane. Therefore, we speculate that OAS-10 may promote tumor cell apoptosis or inhibit the angiogenesis and proliferation of glioma by combination with A1R and A3R, respectively. These studies will contribute to firstly reveal the molecular mechanism of anti-tumor effect based on the direct targets of OAS-10.

脑胶质瘤常呈浸润性生长，手术无法彻底切除，难以治愈、极易复发。本课题组从药用植物中分离的多种皂苷类成分具有抗胶质瘤活性，其中活性最强的齐墩果酸皂苷OAS-10能分别与大鼠脑胶质瘤组织的A1和A3型腺苷受体结合，诱导多种胶质瘤细胞凋亡；降低裸鼠VEGF表达水平，并显著抑制裸鼠胶质瘤的增殖。但其具体作用机制尚不清楚。新近研究表明，胶质瘤缺氧微环境中腺苷浓度明显增加；低浓度皂苷可与A1R结合、干扰腺苷-A1R-G蛋白复合物形成。A3R与A1R氨基酸序列相似度高，均存在于肿瘤细胞膜上。因此我们推测，OAS-10可分别与A1R和A3R结合，从而促进肿瘤细胞发生凋亡、抑制胶质瘤血管生成和细胞增殖。本课题将详细评价OAS-10对A1R和A3R的抑制作用、及通过A1R和A3R介导的抑制胶质瘤增殖作用及分子机制研究。阐明上述问题，将有助于我们首次基于直接作用靶点、揭示皂苷类成分抗肿瘤作用分子机制。

胶质瘤是脑组织发病率最高的肿瘤，常呈浸润性生长，具有高复发率、高死亡率和低治愈率的特点。脑胶质瘤原位治疗效果明确，但其具体机制尚未完全阐明。本课题组通过RT-PCR实验，筛选OAS-10对脑胶质瘤细胞作用的基因靶点，明确药物与A1型和A3型腺苷受体的结合力。同时，针对人源ADORA3 mRNA的序列构建了3种不同的RNAi慢病毒并感染U251细胞，进行了干涉效率的验证。通过体外细胞实验，阐述了OAS-10通过腺苷受体介导的促胶质瘤凋亡和抑制其增殖的调控作用及分子机制，探索了低氧诱导时腺苷受体在胶质瘤增殖中的重要调控作用。我们的研究结果表明，与空白对照组相比，OAS-10只使A3型腺苷受体发生了显著下调。构建慢病毒实验中，只有3号干涉病毒使ADORA3在RNA水平下调了55.7%。体外细胞实验中，细胞转染后活力与正常组相比无差异。机制研究中，OAS-10不能通过调节HIF-1α和VEGF的蛋白表达而对U251细胞产生抑制作用，也无通过A3AR失活损坏HIF-1α表达作用。OAS-10没有抑制U251细胞的增殖，诱导细胞周期阻滞和凋亡的作用。OAS-10也不可能通过下调p44/42 MAPK和Akt通路来调节与腺苷受体的相互作用。同时，课题组进行了皂苷类成分的拓展实验。这些研究为其他皂苷类成分抗胶质瘤作用的探索提供了借鉴并奠定了一定的工作基础，为脑胶质瘤的治疗提供了实验依据。本研究有望为脑胶质瘤防治药物的开发提供新思路和策略。