缺糖增加Bcl-2抑制剂S1诱导人宫颈癌细胞凋亡敏感性的实验研究

发布时间：2018-05-02 00:48

本文选题：缺糖/EBSS + Bcl-2抑制剂　；参考：《吉林大学》2014年硕士论文

【摘要】：恶性肿瘤细胞能量代谢异常，出现能量代谢机制重建，“有氧糖酵解”（亦称之为“Warburg效应”）为显著特征，表现为葡萄糖摄取量增高，糖酵解增加，胞外乳酸聚积。糖酵解途径为肿瘤细胞提供ATP，还为肿瘤细胞的生存、侵袭提供优势。众多学者对恶性肿瘤糖酵解特点和机制进行了大量的研究，力求找到治疗恶性肿瘤新途径。近年来，探索糖剥夺或阻断糖酵解途径的策略正备受关注。目前认为，Bcl-2家族蛋白在凋亡通路调节中发挥关键的作用。研究表明，缺糖引起的凋亡与Bcl-2家族蛋白调控密切相关。葡萄糖缺乏时，糖原合成酶激酶3β（glycogen synthesis kinase3β，GSK3β）磷酸化Bcl-2家族抗凋亡蛋白Mcl-1，并靶向Mcl-1使之通过蛋白酶体途径降解。缺糖促使激活单磷酸腺苷活化蛋白激酶（Adenosine monophosphate(AMP)-activated protein kinase，AMPK），诱导Bim转录，也可造AMPK依赖性哺乳动物雷帕霉素靶蛋白（mammalian target ofrapamycin，mTOR）失活，抑制Mcl-1转录。此外，缺糖还可通过AMPK引起肿瘤抑制基因p53的转录和蛋白稳定，激活某些Bcl-2家族促凋亡蛋白如Bax、Puma和Noxa的转录。还有实验结果显示，缺糖还促使细胞激活ER应激，调节Bcl-2家族蛋白活性和转录。糖代谢可能参与了Bcl-2家族蛋白在凋亡通路的调控，糖分解代谢在转录水平和转录后水平调控Bcl-2家族蛋白的功能。 BH3-only蛋白与Bcl-2家族抗凋亡蛋白特异性结合抑制其发挥作用。Bcl-2家族蛋白都具有BH3结构域，抗凋亡蛋白和促凋亡蛋白正是通过这个共同的结构域形成异源二聚体，相互制约。BH3-only蛋白通过干扰或者促成这些二聚体和多聚体的形成和稳定，调控细胞生存和凋亡。Bcl-2在肿瘤细胞往往高表达，使细胞逃逸凋亡。Bcl-2抑制剂S1是分子靶向治疗药物，通过模拟BH3-only蛋白，双靶点抑制Bcl-2和Mcl-1，激活Bax/Bak，实现诱导肿瘤细胞凋亡。课题组前期研究表明，在黑色素瘤B16细胞、卵巢癌细胞SKOV3和神经胶质瘤U251细胞中，S1能有效地诱导细胞凋亡。同时缺糖和S1都能诱导内质网应激和自噬，这种适应性反应对癌细胞可能有保护作用，影响癌细胞对缺糖和S1诱导凋亡的敏感性，抑制这种适应性反应，可能为肿瘤治疗提供了靶点。目前发现，肿瘤细胞的治疗是通过多靶点、多因素、多环节的调控，一个基因突变不足以使细胞死亡。所以当药物作用于一个靶点时，往往不足以达到使癌细胞致死的杀伤力，只有共同作用于多个靶点时，才有可能达到更好的治疗效果。为了使S1更有效的发挥作用，我们的研究根据肿瘤细胞高度依赖葡萄糖生存的特点，采用Earle's平衡盐缓冲液（EBSS）模拟缺糖环境，和S1共同作用Hela细胞，发现缺糖增强了S1诱导Hela细胞凋亡敏感性。同时测得EBSS和S1共同作用诱导Hela细胞发生内质网应激和自噬程度加重，我们在细胞中加入自噬抑制剂，结果显示，Hela细胞凋亡的敏感性进一步增强。说明自噬在缺糖和S1对Hela细胞的损伤的过程中具有一定的保护作用。方法（1）MTT法检测EBSS和S1作用的Hela细胞生存率。（2）倒置显微镜观察EBSS和S1作用的Hela细胞生长状态。（3）Hoechst33342染色观察EBSS和S1作用的Hela细胞核凋亡情况。（4）Western blot方法检测线粒体凋亡相关蛋白Cyto C、Caspase-3、PARP-1，Bcl-2家族蛋白，以及对Bcl-2家族蛋白有调控作用的因子mTOR、p70S6K、p53的变化；检测内质网应激标志性蛋白PDI、GRP78蛋白表达变化以及内质网应激相关蛋白PERK、CHOP、IRE1、JNK等表达变化；检测自噬相关蛋白LC3、Atg12-Atg5、P62、Beclin1表达变化。（5）共聚焦显微镜观察细胞内质网应激标志性蛋白GRP78荧光强度变化；观察细胞自噬标志性蛋白LC3点状聚集变化。（6）LysoTracker染色，共聚焦显微镜观察EBSS和S1作用的Hela细胞中溶酶体的变化。结果（1）缺糖增强S1对Hela细胞生长的抑制作用。 MTT检测结果表明EBSS（缺糖）和S1都能降低Hela细胞的生存率，随着EBSS作用时间延长，S1剂量增加及作用时间延长，生存率逐渐降低；同时倒置显微镜下观察细胞，细胞密度降低，细胞收缩变圆。当EBSS和S1共同作用Hela细胞时，结果显示，与单独作用相比，存活率显著下降，细胞密度明显降低。说明缺糖增强S1对Hela细胞生长的抑制作用。（2）缺糖增强S1诱导的Hela细胞凋亡。 Hoechst33342染色观察到EBSS和S1都分别引起Hela细胞核发生固缩、碎裂，染色增强，二者共同作用后，比单独作用变化更加显著。Western blot结果显示，EBSS和S1作用的Hela细胞与对照组相比，凋亡相关蛋白Cyto C、Caspase-3、PARP-1表达显著增加，Bcl-2家族促凋亡蛋白Bax、Bim、Noxa明显增加，抗凋亡蛋白Bcl-2、Mcl-1显著减少；对Bcl-2家族蛋白有调控作用的因子mTOR、p70S6K磷酸化程度降低，p53表达增加。当EBSS和S1二者共同作用后，比单独作用变化更明显。 EBSS和S1分别单独作用能够抑制抗凋亡蛋白Bcl-2、Mcl-1的蛋白表达和活性，促进促凋亡蛋白Bax、Bim、Noxa的表达和活化，从而诱导Hela细胞内的Cyto C释放以及Caspase-3、PARP-1活化，导致Hela细胞凋亡。当EBSS和S1共同作用后，Hela细胞凋亡增加。（3）缺糖增强S1诱导的Hela细胞内质网应激。激光共聚焦显微镜观察内质网应激标志蛋白GRP78荧光表达变化。与对照组相比，EBSS和S1单独作用的Hela细胞中GRP78的荧光强度明显增加，，联合作用后，比单独作用变化更明显。Western blot检测，EBSS和S1分别单独作用能够诱导Hela细胞内质网应激标志性蛋白GRP78、PDI表达上调，说明缺糖和S1均能引起内质网应激。进一步Western blot检测表明，内质网应激相关蛋白p-PERK、IRE1表达上调，未折叠蛋白反应（unfolded protein response，UPR）信号通路下游蛋白p-eIF2α、ATF4、CHOP、p-JNK表达增加，说明在Hela细胞中PERK通路和IRE1通路被活化。当EBSS和S1联合作用后，上述内质网应激反应相关蛋白表达变化程度增强，可能影响Bcl-2家族蛋白的表达和活性，调控凋亡。（4）缺糖增强S1诱导的Hela细胞自噬。内质网应激在诱导细胞凋亡的同时，也诱导细胞的自噬。激光共聚焦显微镜观察自噬标志蛋白LC3荧光表达变化，与对照组相比，EBSS和S1单独作用的Hela细胞中LC3点状聚集都明显增多；联合作用后，程度加重。LysoTracker染色，观察到EBSS和S1单独作用的Hela细胞中溶酶体有一定程度增加；联合作用后，与单独作用组相比，溶酶体显著增加。Western blot检测，EBSS和S1分别单独作用明显促进Hela细胞自噬相关蛋白表达变化，P62表达减少，LC3-Ⅱ蛋白水平增加，表示有自噬发生；联合作用与单独作用相比蛋白表达有更加显著的变化，说明自噬程度加强。（5）自噬抑制剂阻断缺糖和S1共同作用诱导的Hela细胞自噬，进一步增强细胞凋亡。 MTT结果表明，自噬抑制剂进一步加强了EBSS和S1共同作用对Hela细胞增殖的抑制作用；LysoTracker染色，观察到CQ使EBSS和S1共同作用的Hela细胞中溶酶体有一定程度减少；Western blot结果显示，自噬相关蛋白表达发生变化，凋亡相关蛋白表达增加，表明自噬被抑制，细胞凋亡敏感性增强。结论缺糖能够明显抑制Hela细胞的生长，并通过调节Bcl-2家族蛋白，诱导细胞凋亡；Bcl-2抑制剂S1双靶点抑制Bcl-2和Mcl-1，诱导凋亡。两者共同作用，缺糖增加了S1诱导Hela细胞凋亡的敏感性，抑制自噬，凋亡程度进一步增加。
[Abstract]:The energy metabolism of malignant tumor cells is abnormal, and the energy metabolism mechanism is rebuilt. "Aerobic glycolysis" (also called "Warburg effect") is characterized by high glucose uptake, increased glycolysis, and extracellular lactate accumulation. Glycolytic pathway provides ATP for tumor cells, and also provides advantages for tumor cells to survive and invasion. Many scholars have done a lot of research on the characteristics and mechanisms of glycolysis for malignant tumors, and strive to find new ways to treat malignant tumors. In recent years, the strategy of exploring sugar deprivation or blocking glycolysis is being paid much attention.
It is believed that Bcl-2 family proteins play a key role in the regulation of apoptosis pathway. Studies have shown that the apoptosis induced by glucose deficiency is closely related to the regulation of Bcl-2 family proteins. When glucose deficiency, the glycogen synthetase kinase 3 beta (glycogen synthesis kinase3 beta, GSK3 beta) phosphorylates the Bcl-2 family anti apoptotic protein Mcl-1, and targets Mcl-1 to pass eggs Glucose deficiency promotes activation of Adenosine monophosphate (AMP) -activated protein kinase, AMPK, induces Bim transcription, and can also make the AMPK dependent mammal rapamycin target protein (mammalian target ofrapamycin,) deactivation and inhibits transcription. The transcriptional and protein stability of the tumor suppressor gene p53 activates the transcription of some Bcl-2 family Pro apoptotic proteins such as Bax, Puma and Noxa. Experimental results show that glucose deficiency also activates ER stress and regulates the activity and transcription of Bcl-2 family proteins. Sugar metabolism may be involved in the regulation of Bcl-2 family proteins in the apoptotic pathway and glycometabolism Transcriptional and post transcriptional levels regulate the function of Bcl-2 family proteins.
The specific binding of BH3-only protein to the anti apoptotic protein of the Bcl-2 family inhibits the action of the.Bcl-2 family proteins with the BH3 domain. The anti apoptotic protein and the apoptotic protein are the formation of the allogeneic two polymer through this common domain, which restricts the formation of.BH3-only proteins by interfering or contributing to the formation of these two polymers and polymers. And the stability, regulation of cell survival and apoptosis of.Bcl-2 in tumor cells often high expression, make cells escape apoptosis.Bcl-2 inhibitor S1 is a molecular targeting therapy drug, through the simulation of BH3-only protein, double target inhibition of Bcl-2 and Mcl-1, activation of Bax/Bak, to induce tumor cell apoptosis. S1 can effectively induce apoptosis in nested cancer cells SKOV3 and glioma U251 cells.
At the same time, both glucose deficiency and S1 can induce endoplasmic reticulum stress and autophagy. This adaptive response may have protective effects on cancer cells, which may affect the sensitivity of cancer cells to glucose deficiency and S1 induced apoptosis, and inhibit this adaptive response, which may provide targets for cancer treatment.
At present, it is found that the treatment of tumor cells is through multiple targets, multiple factors and multiple links. One gene mutation is not enough to cause the death of the cells. So when the drug acts on a target, it is often not enough to kill the killing of cancer cells. It is possible to achieve better therapeutic effect only when the target is used together in multiple targets. In order to make S1 more effective, our study was based on the high dependence of the tumor cells on glucose survival, using the Earle's balanced salt buffer solution (EBSS) to simulate the glucose deficiency environment and the co action of S1 to Hela cells. It was found that glucose deficiency enhanced the S1 induced apoptosis sensitivity of Hela cells. Meanwhile, the common action of EBSS and S1 induced Hela cells to induce the occurrence of Hela cells. Endoplasmic reticulum stress and autophagy are aggravated, and autophagic inhibitors are added to the cells. The results show that the sensitivity of Hela cell apoptosis is further enhanced. It shows that autophagy has a protective effect in the process of impaired glucose and S1's damage to Hela cells.
Method
(1) MTT assay was used to detect the Hela cell survival rate of EBSS and S1.
(2) the growth state of Hela cells treated with EBSS and S1 was observed by inverted microscope.
(3) Hoechst33342 staining was used to observe the apoptosis of Hela cells treated by EBSS and S1.
(4) Western blot method was used to detect the mitochondrial apoptosis related protein Cyto C, Caspase-3, PARP-1, Bcl-2 family proteins, and the changes in mTOR, p70S6K, p53, the regulation of Bcl-2 family proteins, and the expression of endoplasmic reticulum stress marker proteins and endoplasmic reticulum stress related proteins. The expression of autophagy related proteins LC3, Atg12-Atg5, P62 and Beclin1 was detected.
(5) confocal microscopy was used to observe the changes of the fluorescence intensity of the endoplasmic reticulum stress marker protein GRP78, and to observe the change of dot aggregation of the autophagy marker protein LC3.
(6) LysoTracker staining and confocal microscopy were used to observe the changes of lysosomes in EBSS and S1 Hela cells.
Result
(1) glucose deprivation enhances the inhibitory effect of S1 on the growth of Hela cells.
The results of MTT test showed that both EBSS (sugar deficiency) and S1 could reduce the survival rate of Hela cells. With the prolongation of the time of EBSS action, the increase of S1 dose and the prolongation of the action time and the gradual decrease of the survival rate. At the same time, the cell density decreased and the cell contraction became round under the inverted microscope. When EBSS and S1 co acted on Hela cells, the results showed, and the results showed alone. Compared with the control group, the survival rate decreased significantly and cell density decreased significantly, indicating that lack of sugar enhanced the inhibitory effect of S1 on the growth of Hela cells.
(2) the lack of sugar enhanced the apoptosis of Hela cells induced by S1.
Hoechst33342 staining showed that both EBSS and S1 caused Hela nucleus contraction, fragmentation, and staining enhancement. After the combination of the two, the.Western blot results showed that the Hela cells acting on EBSS and S1 were compared with the control group, and the apoptosis related protein Cyto C, Caspase-3, expression increased significantly. Apoptotic protein Bax, Bim, Noxa were significantly increased, anti apoptotic protein Bcl-2, Mcl-1 significantly decreased, and Bcl-2 family protein has a regulated factor mTOR, p70S6K phosphorylation level decreased, p53 expression increased. When EBSS and S1 two together, more obvious than the individual action.
EBSS and S1 separately inhibit the expression and activity of anti apoptotic protein Bcl-2, Mcl-1, promote the expression and activation of apoptotic protein Bax, Bim, Noxa, and induce Cyto C release in Hela cells and Caspase-3, PARP-1 activation, resulting in apoptosis.
(3) glucose deprivation enhances the endoplasmic reticulum stress induced by S1 in Hela cells.
The fluorescence intensity of endoplasmic reticulum stress marker protein GRP78 was observed by laser confocal microscopy. Compared with the control group, the fluorescence intensity of GRP78 was significantly increased in the Hela cells of EBSS and S1 alone. After the combination, the.Western blot detection was more obvious than the single action change. The individual action of EBSS and S1 could induce the endoplasmic reticulum of Hela cells respectively. The expression of GRP78 and PDI was up-regulated, indicating that both glucose deficiency and S1 could cause endoplasmic reticulum stress. Further Western blot detection showed that the expression of endoplasmic reticulum stress related protein p-PERK, IRE1 expression up, and unfolded protein reaction (unfolded protein response, UPR) PERK pathway and IRE1 pathway are activated in the cell. When the EBSS and S1 are combined, the expression of the stress response related proteins in the endoplasmic reticulum is enhanced, which may affect the expression and activity of Bcl-2 family proteins and regulate the apoptosis.
(4) glucose deficiency enhanced autophagy induced by S1 in Hela cells.
Endoplasmic reticulum stress also induced autophagy at the same time of inducing cell apoptosis. Laser confocal microscopy observed the changes in autophagy LC3 fluorescent expression. Compared with the control group, the LC3 point aggregation of LC3 in the Hela cells of EBSS and S1 was significantly increased; the degree of.LysoTracker staining was aggravated and EBSS and S1 single were observed after the combined action. The lysosomes in the only Hela cells increased to a certain extent. After the combined action, the lysosomes significantly increased the.Western blot detection compared with the single action group. The separate action of EBSS and S1 promoted the expression of autophagy related protein in Hela cells, the expression of P62 decreased, and the level of LC3- II protein increased, indicating the occurrence of autophagy; combined action. Compared with the single action, the protein expression had a more significant change, indicating that the degree of autophagy was strengthened.
(5) autophagy inhibitor blocked the autophagy of Hela cells induced by the combination of glucose and S1, and further enhanced cell apoptosis.
MTT results showed that the autophagy inhibitor further enhanced the inhibitory effect of EBSS and S1 on the proliferation of Hela cells. LysoTracker staining showed that the lysosomes in Hela cells with the joint action of EBSS and S1 decreased to a certain extent, and Western blot results showed that the expression of autophagy related egg white was changed and the expression of apoptosis related proteins increased. Addition showed that autophagy was inhibited and the sensitivity of apoptosis increased.
conclusion
Sugar deficiency can obviously inhibit the growth of Hela cells and induce apoptosis by regulating Bcl-2 family proteins. The double target of Bcl-2 inhibitor S1 inhibits Bcl-2 and Mcl-1 and induces apoptosis. Both the two effects can increase the sensitivity of S1 induced apoptosis of Hela cells, inhibit autophagy and increase the degree of death.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【共引文献】