微囊藻毒素LR对人肝癌细胞SMMC-7721的作用研究
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摘要
研究背景：微囊藻毒素(Microcystin,MC)是一类广泛分布于全世界水体环境中的具有较强毒性作用的藻类毒素,微囊藻毒素主要作用于动物肝脏,而随着研究的不断深入,MC对其它器官的毒性作用也被不断揭示包括肾脏毒性,生殖毒性以及神经毒性等。MC具有很强的促癌作用,与蛋白磷酸酶2A(protein phosphatase2A, PP2A)的结合并对该酶活性的抑制是微囊藻毒素的重要毒性作用机制之一。PP2A不仅在正常细胞中扮演着重要角色,对于肿瘤细胞的增殖,生长,迁移等过程也具有重要的影响,目前已经有许多肿瘤药物的开发正是基于其对PP2A抑制进而抑制肿瘤的生长和增殖,而MC对肿瘤细胞的影响的研究还比较少。基于以上原因,我们选取人肝癌细胞SMMC-7721来研究MCLR对于的作用。
研究方法：采用人肝癌细胞株SMMC-7721,应用流式细胞仪、免疫印迹技术,免疫荧光法以及免疫共沉淀技术从细胞周期、细胞凋亡、细胞骨架以及骨架相关蛋白的表达和修饰等方面进行MC-LR对于肿瘤细胞的作用的初步研究。
研究结果：在本实验浓度和时间作用下,SMMC-7721的细胞周期和细胞凋亡没有明显的变化,但是MC-LR引起了细胞骨架的明显重构；MC-LR能够进入细胞并促进了PP2A-C亚基的磷酸化修饰、PP2A调节蛋白α4的表达以及α4与PP2A-C亚基的结合；MC-LR作用下,SMMC-7721细胞PP2A的活性受到了明显的抑制；MC-LR引起了多种活性受PP2A调节的骨架相关蛋白包括HSP27,VASP,Cofilin的磷酸化水平升高以及Rac1的激活状态。
研究结论：MC-LR能够进入SMMC-7721细胞并通过影响PP2A-C亚基的磷酸化水平、α4的表达以及a4与PP2A-C亚基的结合等多种途径抑制了细胞内PP2A的活性；MC-LR对于SMMC-7721细胞的周期和凋亡没有明显的改变,但是细胞骨架发生了重构,其机制是MC-LR抑制PP2A活性进而影响了受PP2A调节的骨架相关蛋白的功能所引起的。
研究意义及展望：本研究相关结论不仅有助于探讨MC-LR对肿瘤细胞的影响机制,也为进一步研究MC-LR是否可能通过影响肿瘤细胞骨架系统而影响细胞的粘附、迁移等一系列细胞功能进而探讨将其作为潜在的肿瘤治疗药物提供了重要依据。
Background:Microcystin (MC) is one of the strong toxic algal toxins which distributed widely around the world, which was considered to target liver in the past. With the further research of MC carried out, the toxic effects of MC to other organs are continued to be revealed,such as the toxicity to kidney, reproductive system, neuron andso on. At the same time, MC is also considered as a potent tumor initiator. Binding with PP2A and then inducing the inhibition of PP2A activity is considered as one of the most important mechanisms of MC's toxicity. Furthermore, PP2A not only plays important role in normal cells, but also has important implications in the process of tumor cells'proliferation, growth and migration. Now, there are many tumor drugs are developed based on inducing the inhibition of PP2A and thereby inhibiting the growth and proliferation of tumors. Therefore we are very interested in the research about the effects of MC-LR on the tumor cells,then,we chose the SMMC-7721cells to research the effects of MCLR on it.
Methods:The liver cancer cell SMMC-7721cells was selectedto study the MC-LR's effects on the cell cycle,apoptosis,cytoskeleton and the cytoskeleton associated proteins expression and modification by using flow cytometry immunoblotting,immunofluorescence and immunoprecipitation.
Results:MC-LR could enter into the cells and then inhibited the activity of PP2A,induced the phosphorylation of PP2A-C subunit and the expression of a4, increasing the binding between a4and PP2A.The treatment of MC-LR did not change the SMMC-7721cells' cell cycle and apoptosis, but under the same treatment, there are significant changes in the cytoskeletons of SMMC-7721cells,including actin and tubulin;Furthermore, MC-LR induced the changes of some cytoskeleton associate proteins, including the phosphorylation of HSP27,VASP, cofilin and the activation of Rac1.
Conclusion:MC-LR could enter into the SMMC-7721cells and combine with PP2A, it inhibited the activity of PP2A by the way of inducing the phosphorylation of PP2A-C subunit, the expression of a4and the binding between a4and PP2A;MC-LR did not induce the change of cell cycle and apoptosis, but changed the cytoskeleton of the cells;the changes of the cytoskeleton-associate proteins regulated by PP2A contribute to the changes of cytoskeleton.
Significance and prospective:Our study not only has its important meaning in discovering the mechanisms of MC-LR's effects on cancer cells,but also provide the reference for the future works:Whether MC-LR could affect the cancer cells' adhesion, migration and others through leading to the changes of cytoskeleton, and the possibility of developing MC-LR to be an anti-cancer drug.
研究方法：采用人肝癌细胞株SMMC-7721,应用流式细胞仪、免疫印迹技术,免疫荧光法以及免疫共沉淀技术从细胞周期、细胞凋亡、细胞骨架以及骨架相关蛋白的表达和修饰等方面进行MC-LR对于肿瘤细胞的作用的初步研究。
研究结果：在本实验浓度和时间作用下,SMMC-7721的细胞周期和细胞凋亡没有明显的变化,但是MC-LR引起了细胞骨架的明显重构；MC-LR能够进入细胞并促进了PP2A-C亚基的磷酸化修饰、PP2A调节蛋白α4的表达以及α4与PP2A-C亚基的结合；MC-LR作用下,SMMC-7721细胞PP2A的活性受到了明显的抑制；MC-LR引起了多种活性受PP2A调节的骨架相关蛋白包括HSP27,VASP,Cofilin的磷酸化水平升高以及Rac1的激活状态。
研究结论：MC-LR能够进入SMMC-7721细胞并通过影响PP2A-C亚基的磷酸化水平、α4的表达以及a4与PP2A-C亚基的结合等多种途径抑制了细胞内PP2A的活性；MC-LR对于SMMC-7721细胞的周期和凋亡没有明显的改变,但是细胞骨架发生了重构,其机制是MC-LR抑制PP2A活性进而影响了受PP2A调节的骨架相关蛋白的功能所引起的。
研究意义及展望：本研究相关结论不仅有助于探讨MC-LR对肿瘤细胞的影响机制,也为进一步研究MC-LR是否可能通过影响肿瘤细胞骨架系统而影响细胞的粘附、迁移等一系列细胞功能进而探讨将其作为潜在的肿瘤治疗药物提供了重要依据。
Background:Microcystin (MC) is one of the strong toxic algal toxins which distributed widely around the world, which was considered to target liver in the past. With the further research of MC carried out, the toxic effects of MC to other organs are continued to be revealed,such as the toxicity to kidney, reproductive system, neuron andso on. At the same time, MC is also considered as a potent tumor initiator. Binding with PP2A and then inducing the inhibition of PP2A activity is considered as one of the most important mechanisms of MC's toxicity. Furthermore, PP2A not only plays important role in normal cells, but also has important implications in the process of tumor cells'proliferation, growth and migration. Now, there are many tumor drugs are developed based on inducing the inhibition of PP2A and thereby inhibiting the growth and proliferation of tumors. Therefore we are very interested in the research about the effects of MC-LR on the tumor cells,then,we chose the SMMC-7721cells to research the effects of MCLR on it.
Methods:The liver cancer cell SMMC-7721cells was selectedto study the MC-LR's effects on the cell cycle,apoptosis,cytoskeleton and the cytoskeleton associated proteins expression and modification by using flow cytometry immunoblotting,immunofluorescence and immunoprecipitation.
Results:MC-LR could enter into the cells and then inhibited the activity of PP2A,induced the phosphorylation of PP2A-C subunit and the expression of a4, increasing the binding between a4and PP2A.The treatment of MC-LR did not change the SMMC-7721cells' cell cycle and apoptosis, but under the same treatment, there are significant changes in the cytoskeletons of SMMC-7721cells,including actin and tubulin;Furthermore, MC-LR induced the changes of some cytoskeleton associate proteins, including the phosphorylation of HSP27,VASP, cofilin and the activation of Rac1.
Conclusion:MC-LR could enter into the SMMC-7721cells and combine with PP2A, it inhibited the activity of PP2A by the way of inducing the phosphorylation of PP2A-C subunit, the expression of a4and the binding between a4and PP2A;MC-LR did not induce the change of cell cycle and apoptosis, but changed the cytoskeleton of the cells;the changes of the cytoskeleton-associate proteins regulated by PP2A contribute to the changes of cytoskeleton.
Significance and prospective:Our study not only has its important meaning in discovering the mechanisms of MC-LR's effects on cancer cells,but also provide the reference for the future works:Whether MC-LR could affect the cancer cells' adhesion, migration and others through leading to the changes of cytoskeleton, and the possibility of developing MC-LR to be an anti-cancer drug.
引文
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