血通灵冲剂抗肿瘤侵袭转移相关机制研究

【作者】 姚蓝；

【导师】 崔征； 李在宪；

【作者基本信息】 沈阳药科大学 ， 生药学， 2008， 硕士

【摘要】 金属蛋白酶-9是肿瘤细胞侵袭和转移中起关键作用的酶。本文探讨中药传统方剂血通灵抑制PMA诱导人乳腺癌细胞(MCF-7)金属蛋白酶-9表达的作用机制。研究中发现,血通灵的乙酸乙酯提取物(HE:Hyul-Tong-Ryung EtOAt extract,根据药物韩文名发音的首写子母和提取溶剂的首写子母组合而成)在低剂量条件下选择性的显著抑制不同癌细胞及正常细胞中金属蛋白酶活性的表达,同时在细胞侵袭实验中发现,该提取物能显著抑制豆蔻酰佛波醇乙酯(PMA)诱导人乳腺癌细胞侵入人工基底膜的能力。蛋白印迹实验(Western blot)发现,HE抑制MMP-9的蛋白表达,提示HE的活性作用靶点可能位于MMP-9基因转录调控部位。RT-PCR及启动子活性分析实验表明,此推测的正确性。Western blot和电泳迁移率(EMSA)实验进一步对人乳腺癌细胞信号通路检测中发现,激活蛋白(AP-1)转录因子的异源二聚体c-Jun磷酸化在给药后显著下调,因此阻止了AP-1的激活,相关文献报导指出,在AP-1的上游信号通路中丝裂原活化蛋白激酶(MAPKs)家族的ERK1/2,JNK,p38,在MMP-9表达调中,发挥重要作用。实验表明,在PMA诱导下的MCF-7细胞中,ERK1/2和部分JNK对MMP-9表达起决定作用,与此同时,HE通过抑制MAPKs信号通路中的调控蛋白因子ERK1/2,部分抑制JNK,阻断了下游AP-1转录因子的激活,抑制金属蛋白酶的表达,发挥其潜在抗肿瘤细胞的侵袭和转移作用。更多还原

【Abstract】 The expression of matrix metalloproteinase-9(MMP-9) has been implicated in the invasion and metastasis of cancer cells. We concentrated on the mechanism of inhibitory effect of traditional Chinese formula Hyul-Tong-Ryung EtOAt extract (HE) on the PMA-induced MMP-9 secretion and expression in the human cell lines.Here we found that in zymography assay, HE inhibits the PMA-induced MMP-9 induction in different human cell lines in the non-toxicity concentration. Based on this reason, HE drastically decreased the invasion ability of PMA-induced MCF-7 cells. In order to find out if the inhibitory effect is dues to the amount of protein expression, Western blot was used to detect the MMP-9 protein expression. The results show the same trend as zymograpgy test .These results suggested that the selective inhibitory effect of HE on the PMA induced MMP-9 secretion is correlated with the synthesis of MMP-9 protein.Furturemore as evidenced by MMP-9 promoter, RT-PCR and electrophoretic mobility shift (EMSA) assays, HE significantly and specifically inhibited MMP-9 gene expression by blocking PMA-simulated activation of activator protein-1 (AP-1) which is through the phosphorylated c-Jun the heterodimer protein of AP-1.Study about the regulation of MMP-9 transcription and expression indicated that, gene expression could be activated by MAPK signal transduction pathways including ERK1/2, p38, and JNK, which are the upstream modulators of AP-1 and NF-kB. Our data showed that the activation of ERK1/2 and JNK play the major role in regulating the MMP-9 expression. At the same time, HE dramatically suppressed the phosphorylation of ERK1/2 and partial of JNK, which suggested that HE suppressed PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7.更多还原