摘要
目的：
研究三肽化合物酪丝缬肽(Tyroservatide,YSV)在体内外的抗肺癌作用,初步探讨其影响人肺癌细胞组蛋白乙酰化修饰,阻抑细胞周期进程,促进肿瘤细胞凋亡的可能机制。
方法：
1.应用MTS法和中空纤维测定法观察YSV对人肺癌NCI-H292细胞体外和体内生长的影响。
2.应用Annexin V/PI双染法、DNA Ladder检测方法观察YSV对人肺癌NCI-H292细胞的凋亡诱导作用；并以流式细胞术观察YSV对人肺癌NCI-H292细胞的细胞周期的影响。
3.应用实时荧光定量PCR和Western Blot方法观察YSV对人肺癌NCI-H292细胞周期蛋白依赖激酶抑制因子p21、p27的mRNA和蛋白表达的影响。
4.应用Western Blot方法,观察YSV对人肺癌NCI-H292细胞组蛋白乙酰化H3、H4水平的影响；应用荧光检测技术和分光光度法分别观察YSV对人肺癌NCI-H292细胞的组蛋白去乙酰化酶(HDAC)和组蛋白乙酰化酶(HAT)活性的影响。
5.应用染色质免疫沉淀(ChIP)方法,观察YSV对人肺癌NCI-H292细胞的细胞周期蛋白依赖激酶抑制因子p21、p27启动子相关组蛋白乙酰化程度的影响。
结果：
1.YSV在体外(0.2～3.2mg/ml)和体内(160～320μg/kg/d)均能够明显抑制人肺癌NCI-H292细胞的增殖(P<0.05)。
2. YSV (0.8,1.6mg/ml)能够诱导人肺癌NCI-H292细胞凋亡,使凋亡细胞数明显增多,形成明显的DNA Ladder;能够阻抑人肺癌NCI-H292细胞的细胞周期进程,使S期细胞百分比明显增高,G2/M期百分比明显降低(P<0.05)。
3.YSV能够上调人肺癌NCI-H292细胞的细胞周期蛋白依赖激酶抑制因子p21、p27的表达。
4.YSV能够上调人肺癌NCI-H292细胞组蛋白H3和H4的乙酰化水平；抑制组蛋白去乙酰化酶的活性,对组蛋白乙酰化酶的活性未见明显影响。
5.YSV能够诱导人肺癌NCI-H292细胞的细胞周期蛋白依赖激酶抑制因子p21、p27基因启动子区域组蛋白乙酰化水平增加,激活p21、p27基因的转录,诱导细胞周期阻滞。
结论：
YSV能够明显抑制人肺癌NCI-H292细胞的体内外增殖,阻抑肿瘤细胞的细胞周期,诱导凋亡。其作用机制是通过抑制人肺癌NCI-H292细胞组蛋白去乙酰化酶的活性,增加细胞周期蛋白依赖激酶抑制因子p21、p27基因启动子区域组蛋白H3、H4的乙酰化水平,促进p21、p27基因转录的活化和表达,进而影响细胞周期进程,使细胞阻滞于S期,抑制细胞增殖,诱导细胞调亡。
Objective:
To explore the anticancer effect of tripeptide tyroservatide(YSV) on human lung carcinoma NCI-H292 cells and to approach its possible mechanism by affecting histone acetylation, arresting cell cycle and inducing apoptosis.
Methods:
1. The lung carcinoma cell line NCI-H292 was used to explore the effect of YSV on cell proliferation. In vitro effect was assayed by MTS method, and in vivo effect was assay by hollow fiber assay.
2. The apoptosis-inducing function of YSV on human lung carcinoma NCI-H292 cells was assayed by AnnexinV/PI, DNA Ladder. The effect of YSV on cell cycle of human lung carcinoma NCI-H292 cells was assayed by FCM method.
3. The effect of YSV on p21, p27 mRNA and protein expression in human lung carcinoma NCI-H292 cells were assayed by Real-time PCR and Western Blot methods.
4. Western Blot was used to observed the effects of YSV on acetylated histones H3 and H4 expression in human lung carcinoma NCI-H292 cells; the activity of histone deacetylase (HDAC) in human lung carcinoma NCIH292 cells was assayed by fluorescence detection methods; the activity of histone acetylase (HAT) in human lung carcinoma NCI-H292 cells was assayed by spectrophotometer.
5. Chromatin immunoprecipitation assay (ChIP) was used to observed the effects of YSV on histone acetylation of some regions of promoters of p21WAF1 and p27KIP1 gene in lung carcinoma NIC-H292.
Results:
1. YSV (0.2～3.2mg/ml in vitro and 160～320μg/kg/d in vivo) can remarkably inhibit growth of human lung carcinoma cells NCI-H292 (P<0.05).
2. YSV (0.8,1.6mg/ml) could induce the apoptosis in NCIH292 cells:apoptotic cells amount in sample treated with YSV were more than that of control group; the DNA Ladders were detected in NCI-H292 cells treated with YSV. YSV could stop the cell cycle of tumor cells by remarkably increasing the percentage of S phase cells (P<0.05), decreasing the percentage of G2/M phase cells.
3. YSV could increase the mRNA and protein expression of CDKI p21 and p27 in human lung carcinoma NCI-H292 cells (P<0.05).
4. YSV can increase acetylated histones H3 and H4 expression (P<0.05), remarkably inhibit the HDAC activity, have no obvious effects on the HAT activity.
5. YSV can cause an accumulation of acetylated histone H3 and H4 in p21WAF1 and p27KIP1 gene promoters associated region of chromation in lung carcinoma NIC-H292, activate the transcriptions of p21 and p27 gene, induce cell cycle arrest.
Conclusion:
YSV can inhibit the growth of lung carcinoma cells in vivo and in vitro, stop the cell cycle of tumor cells, and induce those cells apoptosis. The mechanism of its anticancer effect is as follows:YSV can inhibit HDAC activity, increase acetylated histones H3 and H4 expression, cause an accumulation of acetylated histone H3 and H4 in p21WAF1 and p27KIP1 gene associated region of chromation, then promote p21 and p27 expression, and so cell cycle of tumor cell can be affected. YSV can make NCI-H292 cells blocked in S phase, inhibit cell proliferation, and induce the apoptosis of tumor cell.
引文
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