双环醇对人肝癌细胞HepG2的周期抑制及自噬诱导作用的研究

发布时间：2019-05-14 03:07

【摘要】：肝癌是全球第五大肿瘤疾病,具有极高的发病率及致死率。目前虽然在临床上主要采取化学疗法、切除法、肝移植、免疫疗法等多种方法治疗肝癌,但是在发达国家,肝癌疾病的生存率仅为3-5%,这促使人们不得不继续寻找新的治疗肝癌疾病的方法。双环醇(4,4’-二甲氧基-5,6,5’,6’-双(亚甲二氧基)-2-羟甲基-2’-甲氧羰基联苯)为抗肝炎化学合成新药,在临床上已被广泛应用于治疗HBV感染。此外,一些研究证明双环醇还具有提高肝功能、保护肝脏免受化学物毒害的作用；同时也有研究发现双环醇可以诱导肝癌细胞中热休克蛋白HSP27/70的表达,从而抑制细胞线粒体途径的凋亡。但是,对双环醇的抗肿瘤效果以及双环醇在肿瘤细胞周期与自噬方面功能的研究仍少有涉及,而相关文献中对双环醇作用后的癌细胞进行高通量筛选差异表达基因等初步研究表明双环醇具有作为抗癌药物的潜能。因此本研究重点探索了双环醇对HepG2细胞的毒性作用,并且在体外和体内环境探究了双环醇对肝癌细胞作用的分子机制。结果如下：1.双环醇显著抑制HepG2细胞增殖,而对正常肝细胞LO2的毒性很低。MTT实验表明,双环醇对HepG2的IC50值为LO2细胞的三分之一。而通过流式细胞术研究发现,双环醇对HepG2细胞活性的抑制作用主要源于其能够抑制细胞周期以及诱导细胞产生自噬,而在双环醇作用下的HepG2的凋亡水平没有发生变化。在细胞周期方面,双环醇能够诱导HepG2细胞发生时间和浓度梯度依赖的G1期细胞阻滞。在细胞自噬方面,经过双环醇处理后,细胞内自噬标志蛋白LC3Ⅰ向LC3 Ⅱ转化。另外,通过荧光显微镜对GFP-RFP-LC3转染细胞的观察和透射电镜直接观察显示,细胞内自噬小体和自噬溶酶体的数量在双环醇诱导处理后显著增加。这些结果说明双环醇通过抑制细胞周期和诱导细胞自噬,来抑制HepG2的细胞活性。2.双环醇同时抑制PI3K/AKT通路和Ras/Raf/MEK/ERK通路。通过western blotting方法,证明双环醇影响了G1期调控相关蛋白的活性。双环醇能够诱导p21CIP1和p27KIP1表达,抑制CDK2-cyclinE和CDK4-cyclinD复合体水平,将Rb蛋白去磷酸化导致细胞周期阻滞于G1期,同时本课题还发现双环醇通过抑制mTOR蛋白活性诱导了细胞自噬。通过进一步研究周期与自噬相关上游信号通路,发现双环醇显著抑制了AKT蛋白的磷酸化,下调了PI3K/AKT通路。转染AKTcDNA使双环醇对肿瘤细胞的周期抑制和自噬诱导效果降低,而转染AKT siRNA或加入化学抑制剂LY294002分别使双环醇的抑瘤作用加强。这些结果表明AKT在双环醇抑制肿瘤细胞活性过程中发挥重要的作用。另外,Ras/Raf/MEK/ERK通路也被双环醇所抑制。转染ERK siRNA或加入化学抑制剂PD98059也分别使双环醇周期抑制及自噬诱导效果增强。以上结果说明双环醇的细胞毒性效果与PI3K/AKT通路和Ras/Raf/MEK/ERK通路有密切关系。3.通过裸鼠移植瘤模型研究双环醇在体内环境的作用。利用腋下移植方法将HepG2细胞移植于裸鼠,通过口服给药双环醇与索拉非尼。结果表明,双环醇减小了小鼠体内的肿瘤体积并且降低了肿瘤的重量,同时对小鼠不产生毒性。HE染色和TUNEL染色方法表明在肿瘤组织中双环醇引起了肿瘤细胞一定程度的死亡,而在肝组织中没有发现类似现象。对肿瘤组织进行western blotting和免疫组化实验,发现双环醇降低了肿瘤组织内的AKT和ERK磷酸化水平,抑制了蛋白活性。综上所述,双环醇对于肿瘤细胞的抑制增殖作用来源于细胞周期抑制与细胞自噬。在体外和体内环境中,双环醇的毒性与其对PI3K/AKT通路和Ras/Raf/MEK/ERK通路的抑制作用有关。这些结果使人们对双环醇的药物效果有了更深的理解,为发展新的抗肿瘤药物奠定了一定的理论基础。
[Abstract]:Hepatocellular carcinoma is the fifth largest tumor in the world and has a very high morbidity and mortality. Although a variety of methods such as chemotherapy, excision, liver transplantation, and immunotherapy have been used to treat the liver cancer, the survival rate of the liver cancer is only 3-5% in the developed countries, which has prompted the people to continue to find new methods of treating the liver cancer. Bicyclol (4,4 '-dimethoxy-5,6,5',6 '-bis (methylenedioxy) -2-hydroxymethyl-2'-methoxyphenyl) is a new drug for anti-hepatitis chemical synthesis, and has been widely used in the treatment of HBV infection. In addition, some studies have shown that the bicyclol also has the function of improving the liver function, It is also found that the double-ring alcohol can induce the expression of the heat shock protein HSP27/70 in the liver cancer cell, thereby inhibiting the apoptosis of the mitochondrial pathway of the cell. The anti-tumor effect of the double-ring alcohol and the study of the function of the double-ring alcohol in the cell cycle and autophagy of the tumor are still rare, The preliminary study on the high-throughput screening of differentially expressed genes in the cancer cells after the double-ring alcohol in the relevant literature shows that the double-ring alcohol has the potential as an anti-cancer drug, and therefore, the research focuses on the toxic effect of the bicyclol on the HepG2 cells, And the molecular mechanism of the double-ring alcohol on the action of the liver cancer cells is explored in the in vitro and in vivo environment, and the results are as follows:1. the double-ring alcohol can obviously inhibit the proliferation of the HepG2 cells, and the toxicity to the normal liver cells LO2 is very low. The IC50 value of bicyclol on HepG2 cells is one-third of the LO2 cell, and by flow cytometry it is found that the inhibitory effect of bicyclol on the activity of HepG2 cells is mainly due to its ability to inhibit cell cycle and to induce autophagy of the cells, and the level of apoptosis of HepG2 cells under the action of the bicyclol is not changed. In the aspect of the cell cycle, the double-ring alcohol can induce the G1 phase cell block which is dependent on the time and the concentration gradient of the HepG2 cells, In addition, the observation of GFP-RFP-LC3 transfected cells and the direct observation of transmission electron microscope (TEM) of GFP-RFP-LC3 were observed by fluorescence microscope. The number of autophagy bodies and autophagy lysosomes in the cells increased significantly after the double-ring alcohol induction treatment. These results indicated that the double-ring alcohol inhibited the cell activity of HepG2 by inhibiting the cell cycle and the autophagy of the cells.2. The bicyclol inhibited the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway at the same time. The double-ring alcohol can induce the expression of p21CIP1 and p27KIP1, inhibit the level of CDK2-cyclinE and CDK4-cyclinD complex, and dephosphorylation of Rb protein results in cell cycle arrest in G1 phase. At the same time, it was found that the double-ring alcohol induced autophagy of the cell by inhibiting the activity of mTOR protein. By further studying the upstream signal path of the cycle and autophagy, it was found that the double-ring alcohol significantly inhibited the phosphorylation of the AKT protein. The PI3K/ AKT pathway was downregulated. The effect of Bicyclol on the cycle inhibition and autophagy of the tumor cells was reduced by the transfection of AKTcDNA. The results show that AKT plays an important role in the inhibition of tumor cell activity. The results indicated that the cytotoxicity of the double-ring alcohol was closely related to the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway. The effect of the alcohol in the in vivo environment is that the HepG2 cells are transplanted to a nude mouse by an underarm transplantation method, and the bicyclol and sorafenib are administered by oral administration. The results show that the bicyclol reduces the tumor volume in the mice and reduces the weight of the tumor, The method of HE staining and TUNEL staining showed that the double-ring alcohol in the tumor tissue caused a certain degree of death of the tumor cells, and no similar phenomenon was found in the liver tissue. Western blotting and immunohistochemistry were performed on the tumor tissues. In conclusion, the inhibition and proliferation of the bicyclol on the tumor cells is derived from cell cycle inhibition and autophagy. In vitro and in vivo environment, The toxicity of the double-ring alcohol is related to the inhibition of the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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