昆布多糖通过死亡受体-Caspasey依赖通路诱导人结肠癌LoVo细胞掉网作用的研究
摘要
昆布多糖(Laminarin)是昆布属、海带属等植物中含有的一种多糖类成分。前期研究发现,昆布多糖能抑制人结肠癌LoVo细胞的生长,在体外具有抗肿瘤作用。本研究在前期工作的基础上,以DR4. DR5/TRAIL途径为切入点,考察Caspase依赖通路,系统地研究昆布多糖诱导LoVo细胞凋亡的机制,为今后昆布多糖作为抗癌新药的研发及其临床应用提供理论和实验依据。
采用Western Blot法检测昆布多糖诱导的LoVo细胞中凋亡相关蛋白DR4. DR5和TRAIL蛋白的表达情况,结果表明,昆布多糖作用于LoVo细胞24h后能够显著上调LoVo细胞DR4. DR5和TRAIL蛋白的表达,400.800.1600μg/mL剂量组的相对蛋白表达量分别为DR4:107.57%、115.77%.117.35%; DR5:106.31%.134.02%. 142.34%; TRAIL:106.96%.124.3%.173.37%,呈一定的剂量依赖性,与阴性对照组比较差异具有统计学意义(P<0.05)。
前期研究还发现,昆布多糖可以通过线粒体通路诱导LoVo细胞凋亡,由于Bid是死亡受体通路与线粒体通路联系的关键因子,因此进一步采用、Vestern Blot法检测昆布多糖对人结肠癌LoVo细胞内Bid. tBid蛋白表达的影响,结果表明,昆布多糖作用于LoVo细胞24h后能够显著上调LoVo细胞Bid. tBid蛋白的表达,400、800、1600μg/mL剂量组的相对蛋白表达量分别为Bid:144.25%.215.01%.307.1%; tBid: 229.58%.272.48%.299.72%,呈一定的剂量依赖性,与阴性对照组比较差异具有统计学意义(P<0.05)。
采用比色法检测了与细胞凋亡途径密切相关的Caspase-9. Caspase-6. Caspase-7,三个酶的活性。结果表明,昆布多糖对LoVo细胞作用24h后Caspase-9. Caspase-6. Caspase-7的相对活性升高,400.800.1600μg/mL剂量组的Caspase-9相对活性分别为117.28%.276.41%.387.06%, Caspase-6相对活性分别为137.04%.253.77%. 383.49%, Caspase-7相对活性分别为150.44%、342.44%、534.44%,与阴性对照组比较有显著性差异(P<0.01)。
综上所述,昆布多糖诱导人结肠癌LoVo细胞发生凋亡的机制可能是：(1)通过上调DR4. DR5/TRAIL的表达启动死亡受体途径,促进pro-Caspase-8发生自身裂解成熟为有催化活性的Caspase-8,进而直接激活下游效应因子Caspase-3. Caspase-6. Caspase-7,导致细胞发生凋亡；(2)活化的Caspase-8切割Bid,其片断tBid可促使线粒体内Cyt-c向胞浆的释放,进一步激活Caspase-9,激活下游的Caspase-3. Caspase-6. Caspase-7,导致细胞发生凋亡。
Laminarin is a polysaccharide composition contained in the genus Laminaria and other plants. The previous studies showed that Laminarin could inhibit the growth of human colon cancer LoVo cells, and had the effect of anti-tumor in vitro.Based on the previous studies, caspase dependent pathway and the apoptosis mechanism of LoVo cells induced by Laminarin were studied, using DR4, DR5/TRAIL pathway as the starting point. And this study can provide considerable guidance and evidence for the research and development and clinical application of anti-cancer effect of Laminarin.
Detected by Western Blot laminarin-induced apoptosis in LoVo cells associated protein DR4, DR5 and TRAIL expression, the results show that, dose group of 400,800,1600μ/mL of laminarin in LoVo cells after 24h LoVo cells can significantly increase DR4, DR5, and TRAIL protein expression, the relative protein expression were DR4:107.57%,115.77%,117.35%; DR5:106.31%,134.02%,142.34%; TRAIL:106.96%,124.3%,173.37%, and was a dose-dependent, with the negative control group, a statistically significant difference (P<0.05).
In addition, this experiment detected the protein of Bid which was a key elements that contacted death receptor with mitochondrion, Western Blot results showed that:dose group of 400,800,1600μg/mL of laminarin in LoVo cells after 24h LoVo cells can significantly increase Bid, tBid protein expression, respectively, relative protein expression Bid:144.25%,215.01%, 307.1%; tBid:229.58%,272.48%,299.72%, and a dose-dependent, with the negative control group, a statistically significant difference (P<0.05).
We used colorimetric assay with the closely related apoptosis pathway, Caspase-9, Caspase-6, Caspase-7, three enzyme activity. The results showed that concentrations of dose group of 400,800,1600μg/mL of laminarin on LoVo cells 24h after the Caspase-9, Caspase-6, Caspase-7 of the relative activity with the increase of drug concentration increased, Caspase-9 relative activity were 117.28%,276.41%,387.06%, Caspase-6 relative activity were 137.04%, 253.77%,383.49%, Caspase-7 relative activity were 150.44%,342.44%,534.44%, with the negative control group were significantly differences (P＜0.01).
Therefore, the mechanism laminarin induced colon cancer LoVo cell to apoptosis is that: (1)Starting the way of death acceptor by raising DR4, DR5/TRAIL, promoting pro-Caspase-8 split to catalytic activity Caspase-8, and then activating the next effector Caspase-3, Caspase-6, Caspase-7, to lead to apoptosis. (2) Activated Caspase-8 can incise Bid whose part can promote the releasing of Cyt-c from mitochondria, activate Caspase-9, and Caspase-3,Caspase-6, Caspase-7 further to apoptosis.
采用Western Blot法检测昆布多糖诱导的LoVo细胞中凋亡相关蛋白DR4. DR5和TRAIL蛋白的表达情况,结果表明,昆布多糖作用于LoVo细胞24h后能够显著上调LoVo细胞DR4. DR5和TRAIL蛋白的表达,400.800.1600μg/mL剂量组的相对蛋白表达量分别为DR4:107.57%、115.77%.117.35%; DR5:106.31%.134.02%. 142.34%; TRAIL:106.96%.124.3%.173.37%,呈一定的剂量依赖性,与阴性对照组比较差异具有统计学意义(P<0.05)。
前期研究还发现,昆布多糖可以通过线粒体通路诱导LoVo细胞凋亡,由于Bid是死亡受体通路与线粒体通路联系的关键因子,因此进一步采用、Vestern Blot法检测昆布多糖对人结肠癌LoVo细胞内Bid. tBid蛋白表达的影响,结果表明,昆布多糖作用于LoVo细胞24h后能够显著上调LoVo细胞Bid. tBid蛋白的表达,400、800、1600μg/mL剂量组的相对蛋白表达量分别为Bid:144.25%.215.01%.307.1%; tBid: 229.58%.272.48%.299.72%,呈一定的剂量依赖性,与阴性对照组比较差异具有统计学意义(P<0.05)。
采用比色法检测了与细胞凋亡途径密切相关的Caspase-9. Caspase-6. Caspase-7,三个酶的活性。结果表明,昆布多糖对LoVo细胞作用24h后Caspase-9. Caspase-6. Caspase-7的相对活性升高,400.800.1600μg/mL剂量组的Caspase-9相对活性分别为117.28%.276.41%.387.06%, Caspase-6相对活性分别为137.04%.253.77%. 383.49%, Caspase-7相对活性分别为150.44%、342.44%、534.44%,与阴性对照组比较有显著性差异(P<0.01)。
综上所述,昆布多糖诱导人结肠癌LoVo细胞发生凋亡的机制可能是：(1)通过上调DR4. DR5/TRAIL的表达启动死亡受体途径,促进pro-Caspase-8发生自身裂解成熟为有催化活性的Caspase-8,进而直接激活下游效应因子Caspase-3. Caspase-6. Caspase-7,导致细胞发生凋亡；(2)活化的Caspase-8切割Bid,其片断tBid可促使线粒体内Cyt-c向胞浆的释放,进一步激活Caspase-9,激活下游的Caspase-3. Caspase-6. Caspase-7,导致细胞发生凋亡。
Laminarin is a polysaccharide composition contained in the genus Laminaria and other plants. The previous studies showed that Laminarin could inhibit the growth of human colon cancer LoVo cells, and had the effect of anti-tumor in vitro.Based on the previous studies, caspase dependent pathway and the apoptosis mechanism of LoVo cells induced by Laminarin were studied, using DR4, DR5/TRAIL pathway as the starting point. And this study can provide considerable guidance and evidence for the research and development and clinical application of anti-cancer effect of Laminarin.
Detected by Western Blot laminarin-induced apoptosis in LoVo cells associated protein DR4, DR5 and TRAIL expression, the results show that, dose group of 400,800,1600μ/mL of laminarin in LoVo cells after 24h LoVo cells can significantly increase DR4, DR5, and TRAIL protein expression, the relative protein expression were DR4:107.57%,115.77%,117.35%; DR5:106.31%,134.02%,142.34%; TRAIL:106.96%,124.3%,173.37%, and was a dose-dependent, with the negative control group, a statistically significant difference (P<0.05).
In addition, this experiment detected the protein of Bid which was a key elements that contacted death receptor with mitochondrion, Western Blot results showed that:dose group of 400,800,1600μg/mL of laminarin in LoVo cells after 24h LoVo cells can significantly increase Bid, tBid protein expression, respectively, relative protein expression Bid:144.25%,215.01%, 307.1%; tBid:229.58%,272.48%,299.72%, and a dose-dependent, with the negative control group, a statistically significant difference (P<0.05).
We used colorimetric assay with the closely related apoptosis pathway, Caspase-9, Caspase-6, Caspase-7, three enzyme activity. The results showed that concentrations of dose group of 400,800,1600μg/mL of laminarin on LoVo cells 24h after the Caspase-9, Caspase-6, Caspase-7 of the relative activity with the increase of drug concentration increased, Caspase-9 relative activity were 117.28%,276.41%,387.06%, Caspase-6 relative activity were 137.04%, 253.77%,383.49%, Caspase-7 relative activity were 150.44%,342.44%,534.44%, with the negative control group were significantly differences (P＜0.01).
Therefore, the mechanism laminarin induced colon cancer LoVo cell to apoptosis is that: (1)Starting the way of death acceptor by raising DR4, DR5/TRAIL, promoting pro-Caspase-8 split to catalytic activity Caspase-8, and then activating the next effector Caspase-3, Caspase-6, Caspase-7, to lead to apoptosis. (2) Activated Caspase-8 can incise Bid whose part can promote the releasing of Cyt-c from mitochondria, activate Caspase-9, and Caspase-3,Caspase-6, Caspase-7 further to apoptosis.
引文
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