中药雷丸蛋白pPeOp抑胃癌作用中JAK/STAT3信号通路负性调控的机制研究

Gastric carcinoma is a commonly occurred malignant tumor in clinic, which has been treated by surgery excision combined with chemotherapy. However, the efficacy of such treatment is not acceptable unless an antitumor drug with strong targeting activity can be employed. In our preliminary study, we found that the pPeOp protein in traditional Chinese medicine Omphalia lapidescens has significant anti-tumor effect against gastric carcinoma with strong selective activity. The tumor inhibition rate of pPeOp on tumor-bearing nude mice has attained more than 55%. The gene microarray analysis showed that this protein has an outstanding effect on JAK/STAT signaling pathway. Since pPeOp cannot penetrate through the cytomembrane, we speculate that its anti-tumor effect should be related to the JAK/STAT signaling pathway. This study plans to activate or block the JAK/STAT signaling to analyze the effect of pPeOp on this pathway from various aspects using the methods of TUNEL, immunohistochemistry, flow-cytometer, cell-based impedance sensing, western blot, and RT-PCR. During this study, it is expected to clarify the relevance between the selectivity of pPeOp on gastric carcinoma and the JAK/STAT signaling pathway, thereby providing basis for new targeting drug development against gastric carcinoma.

胃癌是临床常见恶性肿瘤，多以手术切除辅以化疗，但疗效仍不够理想，若提高抗肿瘤药物的靶向性，将明显提高疗效。本项目前期研究发现，临床抗肿瘤辅助中药雷丸所含的pPeOp蛋白对人胃癌细胞具有显著的选择抑制作用，对荷瘤裸鼠的抑瘤率达55%以上。基因芯片实验结果显示，该蛋白对JAK/STAT信号通路的影响尤为显著。鉴于pPeOp蛋白不能透过细胞膜，我们推测其抑瘤作用可能与JAK/STAT信号通路有关。本项目拟从整体到组织、细胞及分子等多个层次，通过激动和阻断（抑制剂和RNAi）JAK/STAT信号通路，应用TUNEL、免疫组化、免疫荧光、流式细胞技术、细胞阻抗分析、western blot和RT-qPCR等多种技术方法，观察分析pPeOp蛋白对JAK/STAT信号通路的具体影响环节及影响程度，以期初步明确pPeOp蛋白抑制胃癌与该通路的相关性，为进一步研究开发新型靶向抗胃癌药物提供依据。

胃癌是全球最常见的消化系统恶性肿瘤之一。随着研究的不断深入，逐渐发现JAK-STAT3信号通路与肿瘤细胞生长、增殖和分化密切相关，成为抗肿瘤药物开发的新靶标。前期研究发现雷丸pPeOp蛋白作用于胃癌细胞后，可能与细胞表面的蛋白受体结合，发挥调控JAK-STAT3信号通路的作用，使肿瘤细胞不能持续生长，达到抑制肿瘤的目的。本课题通过RTCA和细胞活性实验，证明雷丸pPeOp蛋白可显著抑制胃癌细胞增殖和活性，且与作用剂量呈正相关。通过流式细胞技术和Western blot实验证明，雷丸pPeOp蛋白能显著诱导细胞凋亡、抑制细胞迁移，并将胃癌细胞阻滞于S期以延缓细胞周期，且细胞周期阻滞作用可能与真核生物解旋酶（MCM2-7复合物）的形成有关。进一步研究发现，JAK-STAT3信号通路激动剂IL-6可增强雷丸pPeOp蛋白的抗肿瘤作用，JAK-STAT3信号通路抑制剂NSC74859可减弱pPeOp蛋白的抗肿瘤作用。裸鼠荷瘤实验表明，雷丸pPeOp蛋白可显著抑制STAT3干扰型胃癌细胞MC-4在裸鼠体内增殖，且裸鼠成瘤情况与STAT3存在一定关系。综上所述，JAK-STAT3信号通路可能是雷丸pPeOp蛋白发挥抑胃癌作用的重要信号通路。