熊果酸通过ROS/AMPK/STAT3/COX-2途径抑制胃癌侵袭转移

Cyclooxygenase-2 (COX-2) plays important roles in the carcinogenesis, invasion and metastasis of gastric cancer. Our previous study has found that ursolic acid induced apoptosis of gastric cancer cells by down-regulation of COX-2 expression (Int J Pharm. 2013, 441:261-268). However, further studies are required to elucidate the molecular mechanism by which ursolic acid inhibits COX-2 expression in gastric cancer. The results of our preliminary experiments showed that ursolic acid induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) by triggering the generation of reactive oxygen species (ROS), which in turn inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation and COX-2 expression. Moreover, the reduction of STAT3 phosphorylation and COX-2 expression were blocked by N-acetyl-L-cysteine (NAC, a ROS scavenger) and compound C (an AMPK inhibitor). Therefore, we proposed a scientific hypothesis: ursolic acid inhibits cell proliferation, invasion and metastasis of gastric cancer may be mediated by down-regulation of COX-2 expression through ROS/AMPK/STAT3 signaling pathway. To validate this scientific hypothesis, the present project was undertaken to investigate the molecular mechanisms of ursolic acid on the COX-2 gene expression using a broad range of techniques including RNA interference (RNAi), gene overexpression and western blot assay. The soft agar assay, wound closure assay, and tumorigenicity assay in nude mice were performed to further confirm that ursolic acid inhibits COX-2-mediated invasion and metastasis of gastric cancer through ROS/AMPK/STAT3 pathway. The results of the study would provide a new potential pharmacological target for the prevention and treatment of gastric cancer metastasis.

COX-2在胃癌发生、浸润和转移中发挥重要作用。我们前期研究发现，熊果酸通过下调COX-2表达诱导胃癌细胞凋亡(Int J Pharm 2013, 441:261-268)。但是，熊果酸抑制COX-2表达的分子机制有待进一步探讨。预实验发现，熊果酸促进胃癌细胞内ROS生成并激活AMPK，抗氧化剂和AMPK抑制剂逆转熊果酸抑制STAT3磷酸化和COX-2表达的作用。据此我们提出科学假说：熊果酸可能通过ROS/AMPK/STAT3信号通路抑制COX-2表达，从而抑制胃癌细胞增殖、侵袭和转移。为验证这一科学假说，本项目拟利用RNAi、基因过表达和Western blot等手段，探讨熊果酸抑制COX-2表达的分子机制；利用软琼脂分析、细胞划痕实验和裸鼠原位移植胃癌模型，进一步证实熊果酸通过ROS/AMPK/STAT3通路抑制COX-2介导的胃癌侵袭和转移，为临床防治胃癌转移提供新的潜在的药物靶点。

环氧化酶-2（Cyclooxygenase-2, COX-2）在胃癌的发生、侵袭转移中发挥重要的作用。我们的前期研究发现，熊果酸能通过下调COX-2的表达诱导胃癌细胞凋亡，但熊果酸抑制COX-2表达的分子机制尚未完全明确。因此，本项目探讨熊果酸抑制胃癌细胞COX-2表达的分子机制，进一步研究熊果酸在防治胃癌侵袭转移中的作用。.本项目包括以下五方面的研究：.1.研究熊果酸抑制胃癌细胞COX-2表达及其相关分子机制，结果显示熊果酸浓度与时间依赖性抑制胃癌细胞COX-2表达，熊果酸通过ROS/AMPK/STAT3信号转导通路抑制胃癌细胞COX-2表达。.2.研究熊果酸和COX-2表达对胃癌细胞增殖的影响，结果显示熊果酸可能通过ROS/AMPK/STAT3信号转导通路下调COX-2表达而抑制胃癌细胞增殖。.3.研究熊果酸载药纳米微粒（ursolic acid nanoparticles, UA-NPs）对肝癌细胞增殖的影响，结果显示UA-NPs较熊果酸单体能更有效地抑制肝癌细胞的增殖以及裸鼠移植瘤的生长。.4.研究联合熊果酸与紫杉醇对胃癌细胞的增殖的影响，结果显示熊果酸与紫杉醇之间存在协同作用，通过抑制COX-2、PCNA、Bcl-2的表达，增加Bax的表达抑制胃癌细胞增殖并诱导其凋亡。.5.结合上述研究结果，探讨汉防己碱/紫杉醇明胶载药纳米微粒对胃癌细胞增殖的影响，结果显示汉防己碱/紫杉醇明胶载药纳米微粒能抑制胃癌细胞的增殖与侵袭能力。