生育三烯酚调控上皮间质转化与抑制胃癌转移相关性研究

Epithelial-mesenchymal transition (EMT) is a critical role in progression, metastasis and drug resistance of cancer. Tocotrienols have a potential anticancer activity in vitro. The results from the applicant's preliminary work showed that tocotrienols inhibited adhesion, invasion and migration of human gastric cancer cell and the protein expression related to EMT markers - E-cadherin, et al. It indicated that regulation of EMT would be one of mechanisms about inhibition of metastasis of gastric cancer by tocotrienols. In current study, we will establish a EMT model of gastric cancer cell to determine the relationship between regulating EMT by tocotrienols and inhibition of tumor metastasis by in vitro and in vivo. In addition, we will further explore whether this effect of tocotrienols on regulating EMT is also related to TGF signal, miRNAs expression and inflammatory reaction. The mechanism of tocotrienols would be expounded in decreasing invasion and migration of cancer cell in this study. In a human gastric tumor xenograft model, we will determine the size, volume, vascular density of tumor and tumor infiltrated to surrounding tissues. We will also examine the tumor metastasis and expression of proteins in tumor tissues including E-cadherin, et al. These results will confirm the results from in vitro studies. Thus, our study will clear and improve the mechanisms of prevention and inhibition of tumor metastasis, and apply supporting evidences for development and utilization of tocotrienols in the future.

肿瘤细胞上皮间质转化（EMT）在肿瘤进程、转移和耐药性上起重要作用。生育三烯酚具有潜在的抗肿瘤作用，申请者前期研究表明，生育三烯酚抑制人胃癌细胞的黏附、侵袭和迁移与调节EMT标记物E-cadherin等蛋白表达有关。提示调控EMT可能是生育三烯酚抑制胃癌转移的机制之一。因此，本研究拟从离体和整体研究入手，通过建立人胃癌细胞EMT模型，探讨生育三烯酚调控肿瘤细胞EMT与抑制肿瘤转移的关系，明确这种调控作用是否与调节TGF信号转导途径、miRNAs分子表达和抑制炎症反应有关，阐明生育三烯酚降低肿瘤细胞侵袭、迁移能力的作用机制；通过建立接种人胃癌细胞动物模型，观察生育三烯酚对肿瘤形成、肿瘤血管密度、有无远处转移和对周围组织侵润等影响，并检测肿瘤组织E-cadherin等相关蛋白表达，验证离体试验研究结果。此项研究将明确并完善生育三烯酚预防肿瘤形成和抑制肿瘤转移的机制，为其合理开发提供理论依据。

生育三烯酚是在棕榈油、米糠中富含的异戊二烯类化合物，除具有抗氧化活性，还具有潜在的抗肿瘤特性。前期研究证实，生育三烯酚有抑制胃癌细胞增殖、诱导凋亡，抗血管生成以及降低肿瘤细胞迁移和侵袭的能力。鉴于肿瘤细胞上皮间质转化（epithelial-mesenchymal transition, EMT）在肿瘤进程、转移和耐药性上起重要作用。生育三烯酚抑制肿瘤转移作用，可能与其调控EMT有关。因此，利用离体和整体实验，研究了生育三烯酚调控EMT与抑制肿瘤转移的关系。研究结果表明：（1）10ng/mL TGF-β1作用SGC-7901细胞24h后，促进E-cadherin、 Vimentin 等相关蛋白表达，并诱导SGC-7901细胞发生EMT，增强肿瘤细胞的迁移和侵袭能力。（2）生育三烯酚通过上调肿瘤细胞E-cadherin和降低Vimentin蛋白表达，以及影响金属蛋白酶MMP2和MMP9外分泌作用，降低肿瘤细胞的迁移和侵袭作用。并通过影响COX-2的活性，调节其下游基质金属蛋白酶的活力、蛋白表达及侵袭能力，从而影响肿瘤转移。（3）生育三烯酚通过抑制ATM的磷酸化和胞浆中的定位，增强肿瘤细胞蛋白磷酸酶2A表达，影响NF-κB的表达，达到抑制肿瘤作用，并在整体研究中验证。（4）在离体和整体研究中，生育三烯酚均可通过抑制TGF-β表达，影响NF-κB的活性抑制影响下游转录因子Twist，调节E-cadherin、 Vimentin、 SPARC等EMT标志蛋白，影响肿瘤侵袭和转移作用。（5）生育三烯酚与5-Fu联合用药与单独用药相比对人胃癌细胞SGC-7901的增殖抑制及诱导凋亡有明显的增效作用，可能与抑制肿瘤细胞P-gp、MRP1表达有关，其机制有待进一步研究证实。以上结果显示，生育三烯酚可通过调节EMT等途径，影响人胃癌细胞增殖、迁移和侵袭作用，达到预防肿瘤作用。