Genistein通过蛋白激酶/FLT4/MMP2通路抑制结肠癌转移的机制研究

Genistein belongs to natural isoflavones, which can inhibit the tyrosine kinase activity, and has strong anti-tumor effect and lower toxicity. In the previous study, we found that genistein could inhibit the invasion and metastasis of colon cancer cells in vivo, and its downstream metastasis-associated genes were screened and it was found that FLT4 and MMP2 expression levels were decreased after genistein treatment while Akt, p38 MAPK and ERK1/2 were inhibited at the phosphorylation level. FLT4 is a vascular endothelial growth factor receptor, and it has been reported that its activation could promote p38 MAPK pathway, and MAPK has also been reported to promote FLT4 and MMP2 expression. Therefore, we hypothesize that genistein could inhibit FLT4 and MMP2 expression through inhibition of protein kinase phosphorylation, thereby inhibiting colon cancer cell metastasis, and FLT4 could regulate MMP2 and metastasis by p38 MAPK pathway. The project intends to illustrate the mechanism of genistein inhibition of colon cancer metastasis by the network composed of protein kinase, FLT4 and MMP2, and lay the theoretical foundation for the clinical application of this small molecule compounds.

Genistein为天然异黄酮类物质，可以抑制酪氨酸激酶活性，具有较强的抗肿瘤作用和较低的毒副作用。我们在前期研究中发现，genistein可以在体内外抑制结肠癌细胞的侵袭转移。对其下游转移相关基因进行筛选，发现FLT4和MMP2在genistein处理后表达水平降低，同时Akt、p38 MAPK及ERK1/2的磷酸化水平受到抑制。FLT4 为血管内皮生长因子受体，有报道指出其活化可以激活p38 MAPK通路，而MAPK也被报道可以促进FLT4及MMP2表达。因此我们推断genistein通过抑制蛋白激酶磷酸化抑制FLT4和MMP2表达，进而抑制结肠癌细胞侵袭转移，且FLT4可通过p38 MAPK通路调控MMP2表达和结肠癌细胞侵袭转移。本项目拟通过体内外实验阐明genistein通过蛋白激酶、FLT4及MMP2通路抑制结肠癌转移的机制，为该小分子化合物的临床应用奠定理论基础。

前期在genistein抑制结肠癌转移的研究中发现, genistein在不抑制肿瘤细胞生长的剂量下,可以抑制结肠癌细胞在体内和体外的侵袭、愈合以及肿瘤转移相关分子MMP2和FLT4的表达, 同时genistein可以对Akt、ERK1/2、p38 MAPK的磷酸化产生明显抑制。然而这些信号通路又是如何影响MMP2和FLT4的表达并进而影响结肠癌细胞的转移侵袭能力却不得而知。在本项研究中，通过基因芯片筛选、qPCR及Western Blot技术验证了genistein能够抑制细胞中FLT4和MMP2的表达，并且用组织芯片技术进一步验证了FLT4和肿瘤转移的相关性。通过慢病毒技术构建FLT4稳定过表达及下调的结肠癌细胞系，通过Transwell和划痕实验，证实通过敲减FLT4基因可以抑制肿瘤细胞的侵袭和愈合。通过免疫印迹技术发现，FLT4的敲减不但能造成了肿瘤侵袭相关分子MMP2的下调，还能造成磷酸激酶ERK1/2的上调。最后通过ERK1/2磷酸酶抑制剂和Transwell实验证实，抑制ERK1/2磷酸化可以恢复被敲减FLT4的结肠癌细胞系的侵袭，说明ERK1/2磷酸化是FLT4调控肿瘤细胞侵袭的重要分子。既往FLT4所涉及的通路在结肠癌中尚不明确,本研究对其具体信号通路的阐明有助于拓展对结肠癌转移机制的认识。