盐霉素抑制胃癌血管生成与增强化疗敏感性的作用及机制研究

The majority of advanced gastric cancer are resistant to anticancer drugs and show unsatisfactory responsiveness to conventional chemotherapy drugs on patient survival rate. Therefore, the control of such chemoresistance underscores the need for new strategies to develop effective treatments for gastric cancer. At present, VEGFR2 and AXL-mediated signaling pathways have being novel and potential targets for molecular-targeted biotherapy and drug development. In our earlier studies, our findings showed that AXL expression in patients resistant to chemotherapy was significantly higher in the case-control studies. When stratifying the studies by the pathological variables, the depth of invasion, lymph node metastasis, vascular invasion, and TNM stage provided significant prognostic information. Our results indicate that the increased levels of AXL protein appear to mark chemoresistance and may be converted from candidate to the routine clinical setting for clinicians to predict the outcome of single patient with gastric carcinoma. Based on previous results, we screened inhibitors from the FDA approved drug libraries and found that the compound, salinomycin, strongly inhibited cell proliferation, migration and capillary-like tube formation of primarily cultured human umbilical vascular endothelial cells (HUVECs) in vitro in a dose-dependent manner, and dramatically reduced oxygen-triggered neovascularization in mouse cornea in vivo. Additionally, western blot analysis also revealed that salinomycin suppressed phosphorylation and activation of VEGFR2 and AXL kinase, with IC50 of 0.1μmol/L, implying that salinomycin may be one of efficient tumor angiogenesis inhibitor. Therefore, through working on different platforms that are in vitro, ex vivo and in vivo models, we systematically investigated the biological activities of the compound on tumor angiogenesis, and its role in the improved clinical anti-cancer properties of IGFR1 inhibitors OSI-906 and BMS-754807. These findings may pave a way to better reveal the novel functions of salinomycin in tumor angiogenesis and its molecular basis for the anticancer action, and thus provide a scientific basis for gene targeted therapy.

化疗耐药成为影响胃癌患者预后的瓶颈。研究表明：VEGF/VEGFR2和GAS6/AXL信号途径是介导肿瘤复发和化疗耐药的重要靶标。通过基因芯片、免疫组化、Western blot及RT-PCR技术检测分析，发现化疗耐药的胃癌组织中AXL表达显著高于化疗敏感的胃癌组织及正常胃黏膜对照，提示AXL很可能参与化疗耐药，与患者存活率密切相关。项目前期初步证明盐霉素能有效阻断内皮细胞中VEGFR2/AXL的活化，并显著抑制体外VEGF诱导的血管内皮细胞增殖、迁移、分化、成管和切断体内氧诱导视网膜病变模型中新生血管的生成，提示盐霉素很可能具有抗血管生成的作用；课题组拟通过多层次、多水平的试验项目研究盐霉素抑制肿瘤血管新生和增强化疗药物IGFR抑制剂的抗癌疗效及分子机制，以期阐明盐霉素的抗癌新功能和抗肿瘤新机制，为肿瘤个体化治疗提供科学依据，为研制具有我国自主知识产权的肿瘤生物治疗药物奠定理论基础。

抗肿瘤血管新生已成为继手术、化疗、放疗后的第四种治疗肿瘤的方法。其中，VEGF/VEGFR2信号途径是介导胃癌血管生成和肿瘤生长的重要靶标。现有研究表明聚醚类抗生素盐霉素在预防和控制肿瘤方面显示良好的效果，可有效抑制多种人肿瘤干细胞的生长，然而盐霉素是否可以靶向血管新生抑制胃癌生长，国内外相关报道鲜见。本研究发现盐霉素不仅能够显著地抑制血管内皮细胞HUVECs的增殖、迁移以及管状结构形成，还可以在体内基质凝胶植入模型（试验）中显著抑制VEGF诱导的新生血管生成。此外，研究发现盐霉素对肿瘤细胞也有一定的杀伤作用，可促发胃癌细胞（SGC-7901）的凋亡，然而其对人正常胃黏膜细胞（GES-1）却没有明显的毒性作用。运用分子对接软件，发现盐霉素与VEGFR2的活性口袋结合，从而影响VEGF与VEGFR2的结合。盐霉素能够剂量时间依赖性地阻断内皮细胞生长因子受体2（VEGFR2）介导的STAT3信号途径，包括抑制STAT3的磷酸化、二聚化入核以及血管新生/生长相关基因的转录和表达，如Bcl2，Bcl-xL和VEGF等发生过程。在胃癌细胞SGC-7901裸鼠荷瘤模型中，连续28天腹腔分别注射剂量为3mg/kg/d和 5mg/kg/d 的盐霉素，可以显著抑制胃癌肿瘤的体积、重量和血管生成。通过免疫组化及蛋白印迹技术，发现该化合物能降低肿瘤组织中VEGF、p-VEGFR2、p-STAT3、Ki-67蛋白以及血管内皮细胞标记物CD31的表达，促进凋亡蛋白Caspase-3的表达。以上结果说明：盐霉素能够通过抑制VEGF/VEGFR2介导的STAT3通路抑制胃癌血管生成，从而抑制肿瘤的生长，盐霉素很可能是一种新型抗肿瘤药物。