AG490增强HER2阳性乳腺癌细胞对曲妥珠单抗敏感性的实验研究
发布时间：2018-07-29 12:44
【摘要】：背景与目的乳腺癌是女性常见的恶性肿瘤之一。乳腺癌的发生与多种癌基因激活或抑癌基因失活有关。尤其是一些与细胞生长及内分泌相关的基因,如HER2、ERK、ERa等。因此,目前在临床治疗中使用了许多靶向这些特异性基因的药物,如赫赛汀、他莫西芬等,取得了较好的疗效。但随着这些药物的广泛使用,耐药性逐渐成为限制其疗效的关键因素。赫赛汀是靶向HER2的一类临床一线治疗用药,它可与乳腺癌细胞表面的HER2受体特异性结合,从而阻止HER2受体发生二聚化而激活,进而抑制了其介导的下游信号通路,产生抗瘤效应。但赫赛汀单独用药的客观反应率并不高,只有12%-34%,且近50%的患者在使用赫赛汀的初期就会产生耐药性,因此,深入阐明乳腺癌细胞对曲妥珠单抗耐药的相关机制并采取针对性的措施成为目前乳腺癌治疗中的重要科学问题。由于曲妥珠单抗所封闭的靶分子HER2为乳腺癌细胞表面的一类受体,该受体激活后可引起细胞内多条信号通路的活化,包括Ras/MAPK信号通路、PI3K/AKT/mTOR信号通路、JAK/STAT信号通路以及PLC-y信号通路等。这些信号通路的异常和乳腺癌的增殖、生长等密切相关。因此,曲妥珠单抗治疗过程所产生的耐药性也往往和相关信号通路异常密切相关。研究表明,HER2信号通路中任一环节发生改变,都可能导致曲妥珠单抗治疗抵抗。目前已发现的抵抗机制包括：HER家族受体与胰岛素样生长因子1受体(insu-lin-like growth factor 1 receptor, IGF-1R)信号增加；HER2截短突变体p95-HER2的累积；PI3K/AKT/mTOR信号通路的异常活化。除了这些可能导致HER2阳性乳腺癌细胞对曲妥珠单抗治疗抵抗的机制外,JAK/STAT通路的激活也可能是引起抵抗的另一重要机制,但目前相关报道很少。JAK是一类非受体型酪氨酸激酶,该家族包括JAK1、JAK2、JAK3和TYK2四个成员。JAK激活后可以磷酸化STAT而使后者活化,从而构成JAK/STAT信号通路。JAK/STAT信号通路在多种肿瘤组织中均存在过度激活的现象。在乳腺癌组织中,总的STAT3与磷酸化STAT3的表达水平均显著高于正常乳腺组织,且与肿瘤的侵袭转移程度密切相关。但STAT3的激活是否与乳腺癌细胞对曲妥珠单抗的治疗抵抗有关目前尚未见报道。因此,本研究拟首先以HER2阳性的乳腺癌细胞为研究对象,观察曲妥珠单抗处理过程中STAT3的激活情况,并利用基因沉默技术及特异性抑制剂联合曲妥珠单抗处理来观察细胞的反应,从而判断STAT3的激活在乳腺癌细胞耐受曲妥珠单抗中的作用,以期为进一步的临床治疗提供理论基础与科学依据。AG490是目前研究中常用的一种JAK/STAT通路特异性抑制剂。AG490可以竞争性结合酪氨酸激酶JAK2,从而抑制其活性。AG490抑制JAK的激活后,导致STAT3磷酸化被抑制,造成STAT3不能入核与相关DNA元件结合,从而抑制肿瘤细胞的增殖。当前,AG490已成为临床治疗多种肿瘤的抗癌药物,但AG490能否增敏曲妥珠单抗尚不清楚。另外,由于激活的STAT3能上调抗凋亡基因Bcl-xL、Bcl-2 与 Mcl-1的表达,激活促血管生成生长因子VEGF的表达,还能上调细胞周期调节蛋白cyclins D1/D2以及促细胞增殖蛋白c-myc与Survivin的表达,并且有下调促凋亡基因p53表达的作用。尤其最近研究发现,抑癌基因SARI也可能参与了JAK/STAT信号介导的肿瘤发生。SARI又称为BATF2,在正常人组织中表达水平较高,而在相应的肿瘤细胞中表达水平降低。外源性表达SARI可以抑制肿瘤细胞的增殖,而过表达SARI却对正常细胞无明显的增殖抑制效应。但SARI抗癌的分子机制还没完全清楚,特别是调控SARI表达的上游信号还不清楚。AG490作用后是否可通过激活SARI来杀伤乳腺癌细胞也值得进一步探讨。方法通过CCK-8实验测定不同浓度的曲妥珠单抗对HER2阳性细胞SK-BR3增殖的抑制作用,通过台盼蓝染色实验测定不同浓度的曲妥珠单抗对HER2阳性细胞SK-BR3死亡的影响,通过克隆形成实验测定不同浓度的曲妥珠单抗对HER2阳性细胞SK-BR3克隆形成能力的影响,通过免疫印迹实验检测曲妥珠单抗作用后SK-BR3细胞中STAT3的激活情况,然后通过siRNA沉默SK-BR3细胞中的STAT3,免疫印迹实验确定沉默效果,然后通过CCK-8实验和台盼蓝实验观察沉默STAT3后曲妥珠单抗对SK-BR3细胞增殖与死亡的影响。再进一步使用STAT3抑制剂AG490单独或与曲妥珠单抗联合处理SK-BR3细胞,通过CCK-8实验和台盼蓝实验观察不同处理对细胞增殖与死亡的影响。然后采用相同的策略,通过CCK-8实验测定不同浓度的AG490分别对HER2阳性与HER2阴性细胞增殖的抑制作用,通过台盼蓝染色实验测定不同浓度的AG490分别对HER2阳性与HER2阴性细胞死亡的影响,通过克隆形成实验测定不同浓度的AG490分别对HER2阳性与HER2阴性细胞克隆形成能力的影响,通过免疫印迹实验检测AG490作用后细胞中SARI的激活情况,然后通过siRNA沉默细胞中的SARI,免疫印迹实验确定沉默效果,然后通过CCK-8实验和台盼蓝实验观察沉默SARI后AG490对细胞增殖与死亡的影响。通过生物信息方法预测SARI基因启动子区中存在的STAT3结合位点,并采用PCR方法从乳腺癌细胞中扩增SARI基因的启动子区,并针对性的扩增包含或不包含STAT3结合位点的片段,将各片段插入报告基因载体pGL3中,然后转染细胞,观察AG490处理前后荧光素酶活性变化情况,从而确定AG490是否能够通过上调SARI的转录来增强其表达并抗癌。结果曲妥珠单抗能以剂量依赖的方式抑制SK-BR3细胞的增殖,0.25、0.5、1.0、2.0,4.0 mg/ml曲妥珠单抗的细胞相对增殖率分别为76.22%、72.97%、61.08%、42.16%和23.78%。曲妥珠单抗也能以剂量依赖的方式促进SK-BR3细胞死亡0.25、0.5、1.0、2.0、4.0 m//ml曲妥珠单抗相对应的细胞死亡率分别为：10.35+1.66%、18.37+2.49%、31.97±3.08%、46.78±5.67%、63.77±7.81%。与对照组相比,曲妥珠单抗还能以剂量依赖的方式抑制SK-BR3细胞的克隆形成,0.25、0.5、1.0、2.0、4.0 mg/ml曲妥珠单抗相对应的细胞克隆形成率分别为：64.79+8.38%、35.21±13.29%、26.03±10.79%、16.29±10.34%、6.18±6.06%。曲妥珠单抗还可以浓度依赖性地激活SK-BR3细胞中的STAT3信号通路,体现为磷酸化STAT3水平显著升高。用特异性siRNA沉默SK-BR3细胞中的STAT3后,CCK-8实验分析发现可以显著增强曲妥珠单抗对SK-BR3细胞的增殖抑制率,台盼蓝实验分析发现可以显著增加曲妥珠单抗对SK-BR3细胞的死亡率。CCK-8实验结果表明,与AG490单独处理组或曲妥珠单抗单独处理组相比,AG490联合曲妥珠单抗可以显著增加SK-BR3细胞的增殖抑制率；台盼蓝实验结果也表明,与AG490单独处理组或曲妥珠单抗单独处理组相比,AG490联合曲妥珠单抗可以显著增加SK-BR3细胞的死亡率。本研究结果进一步证明,抑制STAT3信号通路确实可以增加曲妥珠单抗对SK-BR3细胞的杀伤率。AG490能以剂量依赖的方式抑制SK-BR3细胞与MDA-MB-231细胞的增殖,AG490在两种细胞中的IC50值分别为43.281 μM与28.327μM。 AG490也能以剂量依赖的方式促进SK-BR3细胞与MDA-MB-231细胞死亡,SK-BR3细胞的对照组死亡率为1.02+0.24%,AG490处理组按照浓度梯度升高(25、50、100 μM)关系相对应的死亡率分别为：5.78±1.67%、16.89+2.67%、31.08±4.65%；而在MDA-MB-231细胞,对照组死亡率为1.77+0.66%,AG490处理组按照浓度梯度升高(25、50、100 μM)关系相对应的死亡率分别为：8.05+2.05%、27.26±2.89%、44.18±4.98%。与对照组相比,AG490还能以剂量依赖的方式抑制SK-BR3细胞与MDA-MB-231细胞的克隆形成。在SK-BR3细胞,对照组与实验组(依浓度升高)的细胞克隆数分别为：559±52、361+38、206±29、117±15个；在MDA-MB-231细胞,对照组与实验组(依浓度升高)的细胞克隆数分别为：389±42、211+23、149±17、68±13个。定量PCR检测发现,随AG490处理浓度的升高,SK-BR3与MDA-MB-231细胞中SARI的mRNA水平均显著升高；Western blot分析也进一步证实,AG490可以剂量依赖的方式增强SK-BR3与MDA-MB-231细胞中SARI蛋白的表达水平。沉默SARI后AG490对MDA-MB-231细胞的增殖抑制效应降低。生物信息学分析发现,在位于-1787到-1797位置存在1个经典的STAT3结合位点。将包含SARI启动子区不同大小的两个片段(-1到-2000bp,-1到-1700bp)分别克隆到报告质粒pGL3-basic中,并与对照质粒同时转染MDA-MB-231细胞后进行荧光素酶报告基因检测,发现不含STAT3结合位点的-1到-1700bp片段在AG490处理前后荧光素酶活性无明显改变,而包含该结合位点的-1到-2000bp片段则在AG490处理后荧光素酶活性显著增高。这一结果说明AG490作用后可通过增加SARI启动子活性而增强SARI的转录来提高其表达,这一过程可能与AG490对STAT3的抑制及STAT3对SARI启动子活性的抑制相关。结论1.曲妥珠单抗可以剂量依赖性地抑制HER2阳性乳腺癌细胞的增殖、促进其死亡、并抑制其细胞克隆的形成。2.曲妥珠单抗处理导致HER2阳性乳腺癌细胞中STAT3的激活,沉默STAT3或抑制STAT3的活性均能增强HER2阳性乳腺癌细胞对曲妥珠单抗的敏感性。3.AG490可以剂量依赖性地分别抑制HER2阳性与HER2阴性乳腺癌细胞的增殖、促进其死亡、并抑制其细胞克隆的形成。4.AG490处理导致HER2阳性与HER2阴性乳腺癌细胞中SARI表达上调,沉默SARI后能降低乳腺癌细胞对AG490的敏感性。5.SARI基因的启动子区存在STAT3的结合位点,不含该结合位点的启动子片段被AG490激活的能力要显著低于野生型启动子的受激活能力。意义1.STAT3的激活是导致HER2阳性乳腺癌细胞对曲妥珠单抗耐受的重要机制。2.AG490能有效增强HER2阳性乳腺癌细胞对曲妥珠单抗的敏感性。3.AG490杀伤乳腺癌细胞与其对抑癌基因SARI的转录上调作用相关。
[Abstract]:Background and objective breast cancer is one of the most common malignant tumors in women. The occurrence of breast cancer is related to a variety of oncogene activation or inactivation of tumor suppressor genes. Especially, some genes related to cell growth and endocrinology, such as HER2, ERK, and ERa, have been used in clinical therapy, such as Hertz, such as Hertz. But with the extensive use of these drugs, drug resistance has gradually become a key factor in limiting its efficacy. Herceptin is a class of clinical therapy targeted to HER2, which can specifically bind to the HER2 receptor on the surface of breast cancer cells to prevent the activation of the dimerization of HER2 receptors. It also inhibits the downstream signaling pathway and produces antitumor effects. But the objective response rate of Herceptin alone is not high, only 12%-34%, and nearly 50% of the patients will have resistance in the early stages of Herceptin. Therefore, the mechanism of breast cancer cell resistance to trastuzumab is clarified and the targeted measures are taken. Since the target molecule HER2 closed by trastuzumab is a kind of receptor on the surface of breast cancer cells, the receptor activates the activation of multiple signal pathways in the cells, including the Ras/MAPK signaling pathway, PI3K/AKT/mTOR signaling pathway, JAK/STAT signaling pathway and PLC-y signaling pathway. The abnormalities of these signaling pathways are closely related to the proliferation and growth of breast cancer. Therefore, drug resistance produced by the therapy of trastuzumab is often closely related to abnormal signal pathways. Studies have shown that any link in the HER2 signaling pathway may lead to the resistance of trastuzumab, which has been found at present. Resistance mechanisms include the increase of the HER family receptor and the insulin like growth factor 1 receptor (insu-lin-like growth factor 1 receptor, IGF-1R), the accumulation of HER2 truncated mutant p95-HER2, and the abnormal activation of the PI3K/AKT/mTOR signaling pathway. In addition to these, these may lead to the resistance of the HER2 positive breast cancer cells to the resistance to trumpet monoclonal antibodies. In addition, the activation of the JAK/STAT pathway may also be another important mechanism for resistance, but it is rarely reported that.JAK is a class of non receptor tyrosine kinase, which includes four members of JAK1, JAK2, JAK3 and TYK2 that can be activated by phosphorylated STAT after.JAK activation, thus forming the JAK/STAT signaling pathway in the.JAK/STAT signaling pathway. There is a phenomenon of excessive activation in various tumor tissues. In breast cancer, the total expression of STAT3 and phosphorylated STAT3 is significantly higher than that of normal breast tissue, and it is closely related to the degree of invasion and metastasis of the tumor. But the activation of STAT3 is not reported to be related to the resistance of breast cancer cells to the treatment of trastuzumab. Therefore, we should first study the HER2 positive breast cancer cells, observe the activation of STAT3 during the treatment of trastuzumab, and use the gene silencing technique and the specific inhibitor combined with trastuzumab to observe the cell response, so as to determine the activation of STAT3 in the tolerance of breast cancer cells to the resistance of trastuzumab. The role of.AG490 is to provide theoretical basis and scientific basis for further clinical treatment. A JAK/STAT pathway specific inhibitor,.AG490, is a competitive binding tyrosine kinase JAK2, which inhibits the activation of JAK by inhibiting its active.AG490, resulting in the inhibition of STAT3 phosphorylation, resulting in the failure of STAT3 to enter the nucleus. Related DNA elements are combined to inhibit the proliferation of tumor cells. Currently, AG490 has become an anticancer drug in the clinical treatment of various tumors. But it is not clear whether AG490 can sensitized to curfdrop monoclonal antibody. In addition, the activation of STAT3 can increase the expression of anti apoptotic gene Bcl-xL, Bcl-2 and Mcl-1, and activate the expression of angiogenic growth factor VEGF. It can increase the expression of cell cycle regulation protein cyclins D1/D2 and cell proliferation promoting protein c-myc and Survivin, and down regulation of p53 expression of apoptosis promoting gene. In particular, recent studies have found that tumor suppressor gene SARI may also participate in JAK/STAT signal mediated tumor.SARI, known as BATF2, in normal human tissues. High levels of expression in the corresponding tumor cells decrease. Exogenous expression of SARI can inhibit the proliferation of tumor cells, but overexpression of SARI has no obvious proliferation inhibition effect on normal cells. However, the molecular mechanism of SARI is not completely clear, especially if the upstream signal regulating the expression of SARI is not clear if the effect of.AG490 action is not clear. To activate SARI to kill the breast cancer cells is also worth further exploring. Methods the inhibitory effect of different concentrations of trastuzumab on the proliferation of SK-BR3 in HER2 positive cells was measured by CCK-8 experiment. The effect of different concentration of trastuzumab on the SK-BR3 death of HER2 positive cells was determined by trypan blue staining. The effect of different concentration of trastuzumab on the SK-BR3 clone formation ability of HER2 positive cells was determined. The activation of STAT3 in SK-BR3 cells after the action of trastuzumab was detected by Western blot, and then STAT3 in SK-BR3 cells was silenced by siRNA and immunoblotting was used to determine the silencing effect, and then through CCK-8 experiment and trypan blue. The effect of trastuzumab on the proliferation and death of SK-BR3 cells after STAT3 was observed. Further use of STAT3 inhibitor AG490 alone or with trastuzumab to treat SK-BR3 cells, the effects of different treatments on cell proliferation and death were observed by the CCK-8 experiment and trypan blue experiment. Then the same strategy was adopted by the CCK-8 experiment. The inhibitory effects of different concentrations of AG490 on the proliferation of HER2 positive and HER2 negative cells were measured, and the effects of AG490 on HER2 positive and HER2 negative cell death were measured by trypan blue assay, and the cloning ability of AG490 with different concentrations of AG490 to determine the ability of HER2 positive and HER2 negative cells to be cloned respectively. The activation of SARI in cells after AG490 action was detected by immunoblotting, and then the effect of silence was determined by SARI in siRNA silencing cells and Western blot test. Then the effects of AG490 on cell proliferation and death after silent SARI were observed by CCK-8 experiment and trypan blue experiment. SARI gene was predicted by biological information method. The STAT3 binding site in the promoter region was initiated and the promoter region of the SARI gene was amplified from the breast cancer cells by PCR method, and the fragments contained or not included in the STAT3 binding site were amplified and inserted into the report gene carrier pGL3, and then transfected to the cells. The changes of luciferase activity before and after AG490 treatment were observed, and the changes of the luciferase activity before and after AG490 treatment were observed. To determine whether AG490 can increase its expression and anticancer by up regulation of SARI, trastuzumab can inhibit the proliferation of SK-BR3 cells in a dose-dependent manner, and the relative proliferation rates of 0.25,0.5,1.0,2.0,4.0 mg/ml trastuzumab are 76.22%, 72.97%, 61.08%, 42.16% and 23.78%., as well as dose of trastuzumab. The relative mortality of SK-BR3 cell death 0.25,0.5,1.0,2.0,4.0 m//ml trastuzumab is: 10.35+1.66%, 18.37+2.49%, 31.97 + 3.08%, 46.78 + 5.67%, 63.77 + 7.81%., compared with the control group. The clones of SK-BR3 cells can be inhibited in a dose-dependent manner, 0.25,0.5,1.0,2.0,4.0 M The relative cell clone formation rates of g/ml trastuzumab are: 64.79+8.38%, 35.21 + 13.29%, 26.03 + 10.79%, 16.29 + 10.34%, 6.18 + 6.06%., which can also activate STAT3 signaling pathway in SK-BR3 cells in a concentration dependent manner, reflecting a significant increase in the level of phosphorylated STAT3. ST in SK-BR3 cells with specific siRNA After AT3, CCK-8 experimental analysis showed that the inhibitory rate of trastuzumab to SK-BR3 cells could be significantly enhanced. Trypan blue experimental analysis found that the death rate of trastuzumab to SK-BR3 cells could be significantly increased by.CCK-8 experimental results, and compared with AG490 alone or trastuzumab alone, AG490 combined with trastuzumab. The proliferation inhibition rate of SK-BR3 cells was significantly increased; trypan blue experimental results also showed that AG490 combined with trastuzumab could significantly increase the mortality of SK-BR3 cells compared with the single treatment group of AG490 alone or trastuzumab. The results of this study further demonstrated that the inhibition of the STAT3 signaling pathway could indeed increase the number of trastuzolones. The anti SK-BR3 cell killing rate.AG490 can inhibit the proliferation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The IC50 values of AG490 in two cells are respectively 43.281 M and 28.327 u M. AG490, respectively, to promote the death of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The mortality of the control group of the SK-BR3 cells is the same. 4%, the relative mortality of AG490 treatment group was 5.78 + 1.67%, 16.89+2.67%, 31.08 + 4.65% according to the increase of concentration gradient (25,50100 M), while in MDA-MB-231 cells, the mortality of the control group was 1.77+0.66%, and the mortality rate of AG490 treatment group in accordance with the increase of concentration gradient (25,50100 u M) was 8.05+2.05%, 27.26 +. 2.89%, 44.18 + 4.98%., compared with the control group, AG490 could inhibit the clone formation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. In SK-BR3 cells, the number of cell clones in the control group and the experimental group were 559 + 52361+38206 + 29117 + 15, respectively, in the MDA-MB-231 cells, the control group and the experimental group. The number of cell clones was 389 + 42211+23149 + 17,68 + 13 respectively. Quantitative PCR detection showed that the mRNA level of SARI in SK-BR3 and MDA-MB-231 cells increased significantly with the increase of AG490 concentration, and Western blot analysis further confirmed that AG490 could be used to enhance the SK-BR3 and protein in the cells in dose-dependent manner. The inhibitory effect of AG490 on the proliferation of MDA-MB-231 cells after SARI was reduced. Bioinformatics analysis found that there were 1 classical STAT3 binding sites in the position from -1787 to -1797. The two fragments containing the different sizes of the SARI promoter region (-1 to -2000bp, -1 to -1700bp) were cloned into the reported plasmid pGL3-basic, respectively. The luciferase reporter gene was detected after transfection of MDA-MB-231 cells with the control plasmid, and there was no significant change in the luciferase activity of the -1 to -1700bp fragments without the STAT3 binding site before and after AG490 treatment, while the -1 to -2000bp fragment containing the binding site increased significantly after the AG490 treatment. After the action of AG490, the expression of SARI can be enhanced by increasing the activity of SARI promoter, which may be related to the inhibition of AG490 to STAT3 and the inhibition of STAT3 on the activity of SARI promoter. Conclusion 1. trastuzumab can inhibit the proliferation of HER2 positive breast cancer cells in a dose-dependent manner, promote its death, and inhibit it. The formation of cell clones.2. trastuzumab leads to the activation of STAT3 in HER2 positive breast cancer cells. Silence STAT3 or inhibition of STAT3 activity can enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab..3.AG490 can inhibit the proliferation of HER2 positive and HER2 negative breast cancer cells in a dose-dependent manner and promote their death. .4.AG490 treatment led to the formation of SARI expression in HER2 positive and HER2 negative breast cancer cells, and the silence SARI could reduce the binding site of STAT3 in the promoter region of the breast cancer cell to AG490 sensitivity.5.SARI gene, and the ability to activate the promoter fragment without the binding site was significant. The activation of 1.STAT3 is an important mechanism for the tolerance of HER2 positive breast cancer cells to trastuzumab,.2.AG490 can effectively enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab, and the.3.AG490 killing of breast cancer cells is related to the up regulation of the tumor suppressor gene SARI.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
[Abstract]:Background and objective breast cancer is one of the most common malignant tumors in women. The occurrence of breast cancer is related to a variety of oncogene activation or inactivation of tumor suppressor genes. Especially, some genes related to cell growth and endocrinology, such as HER2, ERK, and ERa, have been used in clinical therapy, such as Hertz, such as Hertz. But with the extensive use of these drugs, drug resistance has gradually become a key factor in limiting its efficacy. Herceptin is a class of clinical therapy targeted to HER2, which can specifically bind to the HER2 receptor on the surface of breast cancer cells to prevent the activation of the dimerization of HER2 receptors. It also inhibits the downstream signaling pathway and produces antitumor effects. But the objective response rate of Herceptin alone is not high, only 12%-34%, and nearly 50% of the patients will have resistance in the early stages of Herceptin. Therefore, the mechanism of breast cancer cell resistance to trastuzumab is clarified and the targeted measures are taken. Since the target molecule HER2 closed by trastuzumab is a kind of receptor on the surface of breast cancer cells, the receptor activates the activation of multiple signal pathways in the cells, including the Ras/MAPK signaling pathway, PI3K/AKT/mTOR signaling pathway, JAK/STAT signaling pathway and PLC-y signaling pathway. The abnormalities of these signaling pathways are closely related to the proliferation and growth of breast cancer. Therefore, drug resistance produced by the therapy of trastuzumab is often closely related to abnormal signal pathways. Studies have shown that any link in the HER2 signaling pathway may lead to the resistance of trastuzumab, which has been found at present. Resistance mechanisms include the increase of the HER family receptor and the insulin like growth factor 1 receptor (insu-lin-like growth factor 1 receptor, IGF-1R), the accumulation of HER2 truncated mutant p95-HER2, and the abnormal activation of the PI3K/AKT/mTOR signaling pathway. In addition to these, these may lead to the resistance of the HER2 positive breast cancer cells to the resistance to trumpet monoclonal antibodies. In addition, the activation of the JAK/STAT pathway may also be another important mechanism for resistance, but it is rarely reported that.JAK is a class of non receptor tyrosine kinase, which includes four members of JAK1, JAK2, JAK3 and TYK2 that can be activated by phosphorylated STAT after.JAK activation, thus forming the JAK/STAT signaling pathway in the.JAK/STAT signaling pathway. There is a phenomenon of excessive activation in various tumor tissues. In breast cancer, the total expression of STAT3 and phosphorylated STAT3 is significantly higher than that of normal breast tissue, and it is closely related to the degree of invasion and metastasis of the tumor. But the activation of STAT3 is not reported to be related to the resistance of breast cancer cells to the treatment of trastuzumab. Therefore, we should first study the HER2 positive breast cancer cells, observe the activation of STAT3 during the treatment of trastuzumab, and use the gene silencing technique and the specific inhibitor combined with trastuzumab to observe the cell response, so as to determine the activation of STAT3 in the tolerance of breast cancer cells to the resistance of trastuzumab. The role of.AG490 is to provide theoretical basis and scientific basis for further clinical treatment. A JAK/STAT pathway specific inhibitor,.AG490, is a competitive binding tyrosine kinase JAK2, which inhibits the activation of JAK by inhibiting its active.AG490, resulting in the inhibition of STAT3 phosphorylation, resulting in the failure of STAT3 to enter the nucleus. Related DNA elements are combined to inhibit the proliferation of tumor cells. Currently, AG490 has become an anticancer drug in the clinical treatment of various tumors. But it is not clear whether AG490 can sensitized to curfdrop monoclonal antibody. In addition, the activation of STAT3 can increase the expression of anti apoptotic gene Bcl-xL, Bcl-2 and Mcl-1, and activate the expression of angiogenic growth factor VEGF. It can increase the expression of cell cycle regulation protein cyclins D1/D2 and cell proliferation promoting protein c-myc and Survivin, and down regulation of p53 expression of apoptosis promoting gene. In particular, recent studies have found that tumor suppressor gene SARI may also participate in JAK/STAT signal mediated tumor.SARI, known as BATF2, in normal human tissues. High levels of expression in the corresponding tumor cells decrease. Exogenous expression of SARI can inhibit the proliferation of tumor cells, but overexpression of SARI has no obvious proliferation inhibition effect on normal cells. However, the molecular mechanism of SARI is not completely clear, especially if the upstream signal regulating the expression of SARI is not clear if the effect of.AG490 action is not clear. To activate SARI to kill the breast cancer cells is also worth further exploring. Methods the inhibitory effect of different concentrations of trastuzumab on the proliferation of SK-BR3 in HER2 positive cells was measured by CCK-8 experiment. The effect of different concentration of trastuzumab on the SK-BR3 death of HER2 positive cells was determined by trypan blue staining. The effect of different concentration of trastuzumab on the SK-BR3 clone formation ability of HER2 positive cells was determined. The activation of STAT3 in SK-BR3 cells after the action of trastuzumab was detected by Western blot, and then STAT3 in SK-BR3 cells was silenced by siRNA and immunoblotting was used to determine the silencing effect, and then through CCK-8 experiment and trypan blue. The effect of trastuzumab on the proliferation and death of SK-BR3 cells after STAT3 was observed. Further use of STAT3 inhibitor AG490 alone or with trastuzumab to treat SK-BR3 cells, the effects of different treatments on cell proliferation and death were observed by the CCK-8 experiment and trypan blue experiment. Then the same strategy was adopted by the CCK-8 experiment. The inhibitory effects of different concentrations of AG490 on the proliferation of HER2 positive and HER2 negative cells were measured, and the effects of AG490 on HER2 positive and HER2 negative cell death were measured by trypan blue assay, and the cloning ability of AG490 with different concentrations of AG490 to determine the ability of HER2 positive and HER2 negative cells to be cloned respectively. The activation of SARI in cells after AG490 action was detected by immunoblotting, and then the effect of silence was determined by SARI in siRNA silencing cells and Western blot test. Then the effects of AG490 on cell proliferation and death after silent SARI were observed by CCK-8 experiment and trypan blue experiment. SARI gene was predicted by biological information method. The STAT3 binding site in the promoter region was initiated and the promoter region of the SARI gene was amplified from the breast cancer cells by PCR method, and the fragments contained or not included in the STAT3 binding site were amplified and inserted into the report gene carrier pGL3, and then transfected to the cells. The changes of luciferase activity before and after AG490 treatment were observed, and the changes of the luciferase activity before and after AG490 treatment were observed. To determine whether AG490 can increase its expression and anticancer by up regulation of SARI, trastuzumab can inhibit the proliferation of SK-BR3 cells in a dose-dependent manner, and the relative proliferation rates of 0.25,0.5,1.0,2.0,4.0 mg/ml trastuzumab are 76.22%, 72.97%, 61.08%, 42.16% and 23.78%., as well as dose of trastuzumab. The relative mortality of SK-BR3 cell death 0.25,0.5,1.0,2.0,4.0 m//ml trastuzumab is: 10.35+1.66%, 18.37+2.49%, 31.97 + 3.08%, 46.78 + 5.67%, 63.77 + 7.81%., compared with the control group. The clones of SK-BR3 cells can be inhibited in a dose-dependent manner, 0.25,0.5,1.0,2.0,4.0 M The relative cell clone formation rates of g/ml trastuzumab are: 64.79+8.38%, 35.21 + 13.29%, 26.03 + 10.79%, 16.29 + 10.34%, 6.18 + 6.06%., which can also activate STAT3 signaling pathway in SK-BR3 cells in a concentration dependent manner, reflecting a significant increase in the level of phosphorylated STAT3. ST in SK-BR3 cells with specific siRNA After AT3, CCK-8 experimental analysis showed that the inhibitory rate of trastuzumab to SK-BR3 cells could be significantly enhanced. Trypan blue experimental analysis found that the death rate of trastuzumab to SK-BR3 cells could be significantly increased by.CCK-8 experimental results, and compared with AG490 alone or trastuzumab alone, AG490 combined with trastuzumab. The proliferation inhibition rate of SK-BR3 cells was significantly increased; trypan blue experimental results also showed that AG490 combined with trastuzumab could significantly increase the mortality of SK-BR3 cells compared with the single treatment group of AG490 alone or trastuzumab. The results of this study further demonstrated that the inhibition of the STAT3 signaling pathway could indeed increase the number of trastuzolones. The anti SK-BR3 cell killing rate.AG490 can inhibit the proliferation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The IC50 values of AG490 in two cells are respectively 43.281 M and 28.327 u M. AG490, respectively, to promote the death of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. The mortality of the control group of the SK-BR3 cells is the same. 4%, the relative mortality of AG490 treatment group was 5.78 + 1.67%, 16.89+2.67%, 31.08 + 4.65% according to the increase of concentration gradient (25,50100 M), while in MDA-MB-231 cells, the mortality of the control group was 1.77+0.66%, and the mortality rate of AG490 treatment group in accordance with the increase of concentration gradient (25,50100 u M) was 8.05+2.05%, 27.26 +. 2.89%, 44.18 + 4.98%., compared with the control group, AG490 could inhibit the clone formation of SK-BR3 cells and MDA-MB-231 cells in a dose dependent manner. In SK-BR3 cells, the number of cell clones in the control group and the experimental group were 559 + 52361+38206 + 29117 + 15, respectively, in the MDA-MB-231 cells, the control group and the experimental group. The number of cell clones was 389 + 42211+23149 + 17,68 + 13 respectively. Quantitative PCR detection showed that the mRNA level of SARI in SK-BR3 and MDA-MB-231 cells increased significantly with the increase of AG490 concentration, and Western blot analysis further confirmed that AG490 could be used to enhance the SK-BR3 and protein in the cells in dose-dependent manner. The inhibitory effect of AG490 on the proliferation of MDA-MB-231 cells after SARI was reduced. Bioinformatics analysis found that there were 1 classical STAT3 binding sites in the position from -1787 to -1797. The two fragments containing the different sizes of the SARI promoter region (-1 to -2000bp, -1 to -1700bp) were cloned into the reported plasmid pGL3-basic, respectively. The luciferase reporter gene was detected after transfection of MDA-MB-231 cells with the control plasmid, and there was no significant change in the luciferase activity of the -1 to -1700bp fragments without the STAT3 binding site before and after AG490 treatment, while the -1 to -2000bp fragment containing the binding site increased significantly after the AG490 treatment. After the action of AG490, the expression of SARI can be enhanced by increasing the activity of SARI promoter, which may be related to the inhibition of AG490 to STAT3 and the inhibition of STAT3 on the activity of SARI promoter. Conclusion 1. trastuzumab can inhibit the proliferation of HER2 positive breast cancer cells in a dose-dependent manner, promote its death, and inhibit it. The formation of cell clones.2. trastuzumab leads to the activation of STAT3 in HER2 positive breast cancer cells. Silence STAT3 or inhibition of STAT3 activity can enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab..3.AG490 can inhibit the proliferation of HER2 positive and HER2 negative breast cancer cells in a dose-dependent manner and promote their death. .4.AG490 treatment led to the formation of SARI expression in HER2 positive and HER2 negative breast cancer cells, and the silence SARI could reduce the binding site of STAT3 in the promoter region of the breast cancer cell to AG490 sensitivity.5.SARI gene, and the ability to activate the promoter fragment without the binding site was significant. The activation of 1.STAT3 is an important mechanism for the tolerance of HER2 positive breast cancer cells to trastuzumab,.2.AG490 can effectively enhance the sensitivity of HER2 positive breast cancer cells to trastuzumab, and the.3.AG490 killing of breast cancer cells is related to the up regulation of the tumor suppressor gene SARI.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
【参考文献】
相关期刊论文 前1条
1 路钊;郑少鹏;牛静;贾弘y，
本文编号：2152710
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