丙戊酸通过干扰DNA损伤修复通路抑制肿瘤细胞生长分子机制的研究

发布时间：2018-04-26 07:34

本文选题：丙戊酸 + 羟基脲　；参考：《山东大学》2016年硕士论文

【摘要】：目的乳腺癌作为一种女性常见的恶性肿瘤,已成为世界妇女健康的共同威胁。化学药物一直是临床肿瘤的主要治疗方法,但化疗药物自身的副作用以及长期应用后肿瘤细胞的抗药性使其临床应用受到了极大地限制。因此,临床上迫切需要一种新型的抗肿瘤药物或更有效的治疗策略用于肿瘤患者的治疗。近些年来,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors, HDACi)被广泛的研究,并已经作为一种新型的抗肿瘤细胞生长药物进入临床试验研究阶段。我们和其他研究小组的实验结果都证明组蛋白去乙酰化酶抑制剂中一种代表性药物,丙戊酸(Valproic acid, VPA),可抑制乳腺癌细胞的生长,但其抗肿瘤作用的机制目前还不十分清楚。此外,我们课题组既往研究还证明DNA复制蛋白A2高度磷酸化(RPA2-p)-介导的同源重组(Homologous Recombination, HR)通路可特异地参与羟基脲(hydroxyurea, HU)-诱导的DNA复制阻滞的修复过程。为此,本课题拟重点探讨VPA是否通过干扰RPA2-p介导的HR修复通路而造成DNA复制障碍,进而达到抑制肿瘤细胞生长的作用；更为重要的是,HU也是临床上常用的抗肿瘤药物,为指导VPA与HU联合用药治疗肿瘤患者提供有力的理论和实验依据。方法1.细胞克隆形成实验观察两种药物对乳腺癌细胞系MCF7细胞存活的影响单独VPA组用0.5mM的VPA作用MCF7细胞24或48h；单独HU组用2mMHU作用MCF7细胞18h；联合用药组用0.5mM的VPA预处理24或48h,换2mMHU和0.5mM VPA混合液作用MCF7细胞18h后,换新鲜培养基继续培养14天,计算克隆形成率。2.彗星实验检测两种药物对细胞核DNA的损伤情况用0.5mM VPA作用MCF7细胞24h、 2mMHU作用细胞18h或两药联合作用细胞,收集各组细胞制成单细胞悬液后进行彗星实验,观察细胞拖尾情况。3.DNA双链断裂标志物yH2AX焦点形成和HR机制中关键蛋白RPA2-p和Rad51表达水平的检测用0.5mM VPA作用MCF7细胞24h、2mMHU作用细胞18h或两药联合作用细胞。利用免疫荧光技术,将各组细胞固定后观察细胞核内yH2AX、 RPA2-p和Rad51焦点形成情况;再利用免疫印迹实验,收集各实验组细胞的蛋白裂解液检测RPA2-p和Rad51蛋白的表达水平。4.流式细胞仪检测两种药物对细胞DNA损伤后修复效率的影响首先对MCF7细胞进行转染,把带有标记绿色荧光蛋白的同源底物pDR-GFP转染入细胞内进行筛选,建立稳定表达同源底物pDR-GFP的MCF7细胞系。然后对含同源底物pDR-GFP的MCF7细胞分别进行单独0.5mM VPA作用24或48h、单独2mMHU作用18h或两药联合处理后,收集单细胞悬液,利用流式细胞术测定药物对细胞HR修复效率的影响。结果1.在HU-诱导的DNA复制阻滞中,VPA增加了细胞内DNA双链断裂损伤的蓄积免疫荧光实验结果显示,对照组细胞核内yH2AX焦点阳性率为10.52%。0.5mM VPA作用于MCF7细胞后,与对照组相比,细胞核内yH2AX焦点阳性率有增加趋势,但差异无统计学意义(P0.05)。而2mM HU单独作用MCF7细胞可引起细胞核内γH2AX焦点阳性率出现显著增加,一些阳性率比较高的细胞内γH2AX焦点融合成片,与对照组相比阳性率约升高3.5倍(P0.01)。两药联合作用后,细胞核γH2AX焦点阳性率与对照组相比增加近5倍(P0.01),分别是单独VPA或HU组阳性率的3.83或1.33倍。彗星实验结果显示,与对照组相比,单独0.5mM VPA或2mMHU作用细胞后均可引起彗星拖尾长度的增加(P0.01),且HU组彗星拖尾比VPA组要更长一些(P0.01)；VPA和HU联合作用后,彗星拖尾长度与对照组相比增加了74.44%(P0.01),同时也比单药组细胞的彗星拖尾长度长(P0.01)。2.在HU-介导的复制阻滞中,VPA抑制了同源重组修复分子机制免疫荧光实验结果显示,对照组带有γH2AX焦点的细胞阳性率为6.64%；单独0.5mM VPA或2mM HU作用组,yH2AX焦点阳性率分别降为9.19%或20.92%,各单药处理组细胞核内yH2AX焦点的阳性率比对照组仍旧要高(P0.01),而两药联合作用组的焦点阳性率更高,为34.92%。利用表达同源底物pDR-GFP的MCF7细胞,通过流式细胞术测定药物对细胞HR修复效率的影响。实验结果显示,对照组细胞HR修复效率为85×10-6；单独VPA作用后,细胞的HR修复效率降为65×10-5(P0.05)；单独HU作用,HR修复效率显著升高,上升为148×10-6(P0.01)；而VPA和HU两药联合作用后,与单独HU相比,细胞HR修复效率出现了明显的下降,为100×10-6(P0.01)。3.在HU-介导的复制阻滞中,VIA抑制了RPA2-p和Rad51的功能免疫印迹和免疫荧光实验结果均显示,未处理组和单独VPA处理组细胞均未检测到细胞核内RPA2-p的表达；HU作用于细胞后,细胞核内RPPA2-p表达和焦点阳性率显著增加；联合VPA作用后,与单独HU组相比,细胞核内RPA2-p的表达和焦点阳性率出现显著降低(P0.01)。蛋白质免疫印迹实验结果显示,各处理组细胞Rad51蛋白表达量基本一致,无显著性差异(P0.05)。但是,免疫荧光实验检测到对照组细胞核内Rad51焦点阳性率为24.74%; 0.5mMVPA处理MCF7细胞后,与对照组相比细胞核内Rad51焦点阳性率有一定程度降低(P0.01); 2mMHU作用18h后,细胞核内Rad51焦点显著升高；而2mMHU联合0.5mM VPA作用组细胞的Rad51焦点阳性率与HU组相比出现明显下降(P0.01),大约降到40%左右。Rad51和RPA2-p焦点不仅能够较好的共定位与细胞核内,而且各处理组细胞核内Rad51和RPA2-p焦点阳性率都非常接近。4.VPA能增加肿瘤细胞对HU的敏感性,两药联合对肿瘤细胞具有协同杀伤作用细胞克隆形成实验结果显示,与对照组相比,单独0.5mM的VPA作用MCF7细胞24或48h后,细胞存活率分别下降21.55%(P0.01)和30.17%(P0.01)；单独2mM的HU作用细胞18h后,细胞存活率下降61.21%(P0.01)；而2mMHU和0.5mM VPA联合作用细胞后,细胞存活率显著降低,约下降80%(P0.01)。进一步将实验结果进行校正后,与校正前趋势相一致。说明不但单独VPA或HU能抑制细胞生长,VPA和HU两药联合对MCF7细胞有协同杀伤作用。克隆形成实验结果进一步显示,将MCF7细胞内RPA2的磷酸化位点突变(muRPA2)后,在应答HU损伤时,与wtRPA2细胞相比,muRPA2细胞的存活率下降至42.58%(P0.01),提示灭活RPA2磷酸化通路可增加细胞对HU的敏感性；如果wtRPA2细胞经VPA预处理后,与未经VPA处理的细胞相比,细胞存活率下降了37.82%(P0.01),这一变化与muRPA2细胞对HU损伤应答有相同趋势；但是muRPA2细胞经VPA预处理后,与其无VPA处理组细胞相比,细胞存活率仅下降了15.69%(P0.01)。以上结果进一步确认VPA抑制细胞生长是通过干扰RPA2-p介导的DNA修复通路实现的。5.VPA与PARP抑制剂联合应用对肿瘤细胞具有协同杀伤作用克隆形成实验结果显示,VPA或ABT888作用于MCF7细胞后都可以造成细胞存活率的显著降低(P0.01)；两药联合作用组的细胞存活率下降更为显著。联合作用组的存活率不仅明显低于对照组(P0.01),而且相对于VPA或ABT888单独作用组也有显著的降低(P0.01),实验结果进一步确认VPA能够干扰HR修复通路。结论1.在DNA复制阻断条件下,安全剂量VPA可引起更多的DNA双链断裂损伤的蓄积。2.安全剂量VPA可通过干扰PA2p-Rad51介导的HR修复通路造成DNA复制障碍,从而抑制肿瘤细胞的生长。3.安全剂量VPA能增加肿瘤细胞对HU的敏感性,VPA和HU联合用药对抑制乳腺癌MCF7细胞的生长起到协同作用。
[Abstract]:Objective breast cancer, as a common malignant tumor of women, has become a common threat to the health of women in the world. Chemical drugs have always been the main treatment for clinical tumors. However, the side effects of chemotherapy drugs and the drug resistance of cancer cells after long-term application have greatly restricted their clinical application. Therefore, it is urgently needed in clinic. In recent years, histone deacetylase inhibitors (HDACi) has been widely studied and has been used as a new type of antitumor cell growth drug into clinical trials. The results of the group have proved that a representative drug of histone deacetylase inhibitor, Valproic acid (VPA), can inhibit the growth of breast cancer cells, but the mechanism of its anti-tumor effect is not yet very clear. In addition, our group has previously studied the high phosphorylation of DNA replicin A2 (RPA2-p) - mediate Homologous Recombination (HR) pathway can participate in the repair process of hydroxyurea (hydroxyurea, HU) - induced DNA replication. To this end, this topic intends to focus on whether VPA obstructs the growth of tumor cells by interfering with the HR repair pathway mediated by RPA2-p and thus to inhibit the growth of tumor cells. It is important that HU is also a clinically commonly used antitumor drug, providing a powerful theoretical and experimental basis for guiding the combination of VPA and HU in the treatment of cancer patients. Method 1. cell clone formation experiments were conducted to observe the effects of two drugs on the survival of MCF7 cells in breast cancer cell lines alone. The VPA action of 0.5mM was used in the VPA group of MCF7 cells 24 or 48h; a single HU group was used. The MCF7 cell 18h was treated with 2mMHU, the combination group pretreated 24 or 48h with VPA of 0.5mM, and the 2mMHU and 0.5mM VPA mixture acted on MCF7 cell 18h, and then the fresh medium continued to be cultured for 14 days. 18h or two drugs combined cells, collecting cells from each group and making a single cell suspension to conduct a comet experiment, observing the formation of the.3.DNA double strand break marker yH2AX focus and the detection of the key protein RPA2-p and Rad51 expression levels in the HR mechanism by using 0.5mM VPA as MCF7 cell 24h, 2mMHU acting cell 18h or two drugs. Cell. Using immunofluorescence technique to observe the formation of yH2AX, RPA2-p and Rad51 focal points in the nucleus after immobilization, and then use immunoblotting experiment to collect the expression level of RPA2-p and Rad51 protein in the cells of each experimental group by.4. flow cytometry to detect the effect of two kinds of drugs on the repair efficiency after the cell DNA damage. The MCF7 cells were transfected first, and the homologous substrate pDR-GFP with the labeled green fluorescent protein was transfected into the cell, and the MCF7 cell line that expressed the homologous substrate pDR-GFP was established. Then, the MCF7 cells containing the homologous substrate pDR-GFP were separately used as 0.5mM VPA for 24 or 48h, and the single 2mMHU action 18h or two drug combinations were used. After collecting single cell suspension and using flow cytometry to determine the effect of drug on cell HR repair efficiency. Results 1. in HU- induced DNA replication block, VPA increased the accumulation of DNA double strand breaks in cell immunofluorescence test results, and the positive rate of yH2AX focus in the nucleus was 10.52%.0.5mM VPA in MCF7 cells in the control group. Compared with the control group, the positive rate of yH2AX focus in the nucleus increased, but the difference was not statistically significant (P0.05), but the positive rate of 2mM HU alone in MCF7 cells could cause a significant increase in the positive rate of the gamma H2AX focus in the nucleus. Some positive rates of intracellular gamma H2AX focal points were fused into slices, and the positive rate was about 3.5 higher than that of the control group. P0.01. After the combination of two drugs, the positive rate of the nucleus gamma H2AX focus increased nearly 5 times compared with the control group (P0.01), which was 3.83 or 1.33 times the positive rate of the single VPA or HU group respectively. The comet experiment showed that compared with the control group, 0.5mM VPA or 2mMHU acting cells could cause the increase of the tail length of the comet (P0.01), and the HU group comet. The tail was longer than the VPA group (P0.01); after the combination of VPA and HU, the tail length of comets increased by 74.44% (P0.01) compared with the control group. Meanwhile, the length of the comet tail was longer than that of the single drug group (P0.01).2. in HU- mediated replication block, and VPA inhibited the results of the molecular mechanism immunofluorescence test of the homologous regroup and the control group. The positive rate of cells with gamma H2AX focus was 6.64%, and the positive rate of yH2AX focus decreased to 9.19% or 20.92% respectively in the group of 0.5mM VPA or 2mM HU, and the positive rate of yH2AX focus in the nucleus of each single drug treatment group was higher than that of the control group (P0.01), while the positive rate of the focal point in the two drug combination group was higher, and the expression of the homologous substrate pDR for 34.92%. was used for 34.92%.. The effect of -GFP MCF7 cells on the efficiency of HR repair was measured by flow cytometry. The experimental results showed that the efficiency of HR repair in the control group was 85 x 10-6, and the efficiency of HR repair was reduced to 65 * 10-5 (P0.05) after the action of VPA alone; the efficiency of HR repair was significantly increased and increased to 148 x 10-6 (P0.01) by the action of HU, while VPA and HU two were used. After the combined action, the HR repair efficiency of cells decreased significantly compared with the single HU, which was 100 * 10-6 (P0.01).3. in HU- mediated replication block. VIA inhibited the functional immunoblotting and immunofluorescence results of RPA2-p and Rad51, and the expression of RPA2-p in the untreated and isolated VPA cells was not detected. After the action of HU on the cells, the expression of RPPA2-p and the positive rate of the focal point in the nucleus increased significantly, and the expression of RPA2-p and the positive rate of the focus in the nucleus were significantly lower than that of the single HU group after the action of VPA (P0.01). The results of protein immunoblotting showed that the expression of Rad51 protein in the cells of each treatment group was basically the same, and there was no significant difference. P0.05. However, the immunofluorescence test showed that the positive rate of Rad51 focus in the nucleus of the control group was 24.74%. After 0.5mMVPA treatment of MCF7 cells, the positive rate of Rad51 focus in the nucleus was reduced to a certain degree (P0.01). After 2mMHU action 18h, the Rad51 focus in the nucleus increased significantly; 2mMHU combined with 0.5mM VPA group cells. The positive rate of Rad51 focus decreased significantly compared with that of the HU group (P0.01), which decreased to about 40%.Rad51 and RPA2-p focus not only in the co localization and in the nucleus, but also in the nucleus Rad51 and RPA2-p foci in each treatment group, which were very close to.4.VPA to increase the sensitivity of the tumor cells to HU. The two drugs combined with the tumor cells. Experimental results showed that the cell survival rate decreased by 21.55% (P0.01) and 30.17% (P0.01) after MCF7 cells of MCF7 cells 24 or 48h respectively compared with the control group, compared with the control group, and the cell survival rate decreased by 61.21% (P0.01) after 18h in 2mM alone, while 2mMHU and 0.5mM cells were combined to act on cells. The cell survival rate decreased significantly, about 80% (P0.01). Further, the experimental results were corrected, and the results were in accordance with the pre correction trend. It was shown that not only VPA or HU alone could inhibit cell growth, but the VPA and HU two drugs have synergistic killing effect on MCF7 cells. The cloning and formation of experimental results showed that the phosphorylation site of RPA2 in MCF7 cells was a step. After muRPA2, the survival rate of muRPA2 cells decreased to 42.58% (P0.01) in response to HU damage compared with wtRPA2 cells, suggesting that the inactivated RPA2 phosphorylation pathway increased the cell's sensitivity to HU; if wtRPA2 cells were pretreated with VPA, the cell survival rate decreased by 37.82% (P0.01) compared with those without VPA, and this change was associated with muRP. The response of A2 cells to HU damage response was the same, but the cell survival rate of muRPA2 cells was only 15.69% (P0.01) compared with those without VPA treatment group after VPA pretreatment. The results further confirmed that the growth of VPA suppressor cells was combined with.5.VPA and PARP inhibitors that interfered with RPA2-p mediated DNA repair pathway. The results of the synergistic killing cloning of the tumor cells showed that VPA or ABT888 could significantly decrease the cell survival rate (P0.01) after the action of MCF7 cells, and the cell survival rate of the two drug combined group decreased significantly. The survival rate of the combined group was not only lower than that of the control group (P0.01), but also relative to VPA or ABT88. 8 the single action group also had a significant decrease (P0.01). The experimental results further confirmed that VPA could interfere with the HR repair pathway. Conclusion under the condition of DNA replication, the safe dose VPA can cause more.2. safe dose of DNA double strand break damage, VPA can cause DNA replication obstacle by interfering PA2p-Rad51 mediated HR repair pathway, thus inhibiting the DNA replication disorder. The growth of.3., a safe dose of VPA, can increase the sensitivity of tumor cells to HU, and the combination of VPA and HU plays a synergistic role in inhibiting the growth of MCF7 cells in breast cancer.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.5

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