甘草素对替莫唑胺诱导胶质瘤细胞凋亡的增敏作用及机制研究

发布时间：2018-05-05 07:47

本文选题：替莫唑胺 + 雌激素受体β激动剂　；参考：《吉林大学》2016年博士论文

【摘要】：研究背景:替莫唑胺(Temozolomide,TMZ)是一种新型咪唑四嗪类烷化剂,在生理环境可以通过非酶途径快速转化为活性化合物,并通过诱导DNA甲基化来促进肿瘤细胞凋亡。TMZ吸收迅速,易于透过血脑屏障,并具有良好的抗胶质瘤活性,因此在中枢神经系统肿瘤特别是恶性胶质瘤化疗中得到了广泛的应用。然而肿瘤细胞对TMZ的耐药性经常发生,是临床上化疗失败的主要原因。因此,如何解决TMZ耐药、增加恶性胶质瘤细胞对TMZ的敏感性已成为神经外科临床医生及研究人员的最新关注热点问题。雌激素受体β是一种与神经细胞发育和分化有关的多功能蛋白,具备抑癌基因活性,其表达水平的变化与神经胶质瘤的发生发展有着密切的关系。甘草素(Liquiritigenin,Liq)是存在于豆科植物甘草(Glycyrrhiza uralensis Fisch)中的二氢黄酮单体化合物,是一种天然的雌激素受体β(estrogen receptor,ERβ)激动剂。前期试验发现Liq能抑制多种肿瘤如乳腺癌、宫颈癌的增殖,同时具备增加化疗药物的敏感性,预防肿瘤复发的功效。本课题拟从体内和体外两个方面观察Liq对TMZ抗神经胶质瘤治疗效果的影响,探讨Liq、TMZ联合使用在临床应用方面的优越性;同时深入研究Liq增加肿瘤细胞对TMZ敏感性的分子基础,为Liq在胶质瘤治疗方面的重要作用提供理论基础和实验依据。方法:研究分体内、体外两个部分。体外研究我们选用了三种人神经胶质瘤细胞株:对TMZ低敏感性的U138,对TMZ高敏感性的U251以及TMZ耐药细胞株T98G,采用MTT法、流式细胞术法检测不同浓度的Liq单独使用或者与TMZ联用在体外对胶质瘤细胞增殖、凋亡和细胞周期的影响;采用Western Blot方法检测Liq和TMZ作用后ERβ、MGMT、Caspase-3、Akt、p-AKT、P70S6K、p-P70S6K等凋亡因子和信号分子蛋白表达水平及磷酸化水平的变化;采用RNA干扰的方法沉默ERβ的表达,明确Liq对PI3K/AKT/m TOR信号通路影响的机制;联合使用PI3K/AKT/m TOR通路抑制剂和激活剂,进一步探讨Liq对该通路的调控作用以及促进TMZ敏感性的分子机制。体内研究采用裸鼠胶质瘤模型作为研究对象,在裸鼠左腹皮下接种U138胶质瘤细胞(106/100μl)后,分为四组:对照组(DMSO),TMZ组(50 mg/kg/d),Liq组(30 mg/kg/d)以及联合组(TMZ 50 mg/kg/d+Liq 30 mg/kg/d),每日进行腹腔注射,观察各组裸鼠肿瘤生长状况,于接种移植瘤的30日处死裸鼠,在体内水平研究Liq对ERβ表达的影响以及对TMZ抗肿瘤效果的协同作用。结果:一、体外实验:胶质瘤细胞U138经过不同浓度的Liq(10、20、40、80、160和320μM)处理72 h后,细胞活性测试结果显示Liq对U138有显著的抑制作用,且伴随浓度的增加其抑制作用逐渐增强,呈现剂量依赖性效应。其中80μM的Liq既可有效抑制U138细胞的活性,但又不会导致细胞的过度死亡,因此我们选用该浓度的Liq进行后续研究。该浓度的Liq与不同浓度的TMZ(25、50、100、200、400和800μM)联合使用后,可以显著增加TMZ对U138增殖的抑制作用。当100μM TMZ与80μM的Liq联合处理72h后,U138肿瘤细胞存活率仅为50.36%,显著低于单独使用TMZ时肿瘤细胞的存活率(90.33%,P0.001),证明Liq可有效增强肿瘤细胞对TMZ的敏感性。流式细胞术细胞周期分析结果显示,与TMZ单独使用相比,联合使用Liq可进一步增强TMZ诱导的S期阻滞(64.63%vs 40.43%,P0.001),有效地抑制细胞进入增殖期。Annexin V/PI双染凋亡分析表明Liq和TMZ联合使用可以显著增加U138胶质瘤细胞凋亡率(27.5%vs 18.6%,P0.001),增加凋亡标志物Caspase-3剪切体的形成。Liq与TMZ的协同作用在另外两种胶质瘤细胞U251和T98G中也得到了验证。Western Blot检测PI3K/AKT/m TOR通路的结果表明,Liq可以激活ERβ,降低AKT和P70S6K磷酸化水平,从而抑制PI3K/AKT/m TOR信号通路的活性,但对肿瘤耐药基因MGMT并没有直接的影响。通过RNA干扰沉默ERβ表达后,Liq丧失了对PI3K/AKT/m TOR通路的调控作用,说明Liq是通过激活ERβ来调节PI3K/AKT/m TOR通路活性的。PI3K/AKT/m TOR通路的抑制剂XL765可以明显提高细胞对TMZ的敏感性,提示PI3K/AKT/m TOR通路的激活对神经胶质瘤细胞具有保护作用,从而拮抗TMZ的作用。联合使用PI3K/AKT/m TOR激活剂IGF-1可以拮抗Liq介导的TMZ毒性,提示Liq是通过抑制PI3K/AKT/m TOR来发挥作用的。二、体内实验:结果显示,对照组肿瘤平均体积为1.84±0.13cm3,单独使用TMZ组肿瘤平均体积为0.93±0.11cm3,单独使用Liq组肿瘤平均体积为1.34±0.15cm3,而联合使用TMZ和Liq组肿瘤平均体积为0.36±0.04cm3。此结果与体外细胞实验结果相符,联合应用Liq和TMZ可以显著抑制肿瘤生长,提高肿瘤对TMZ的敏感性,差异具有统计学意义(P0.05)。同时,Western Blot结果证明,Liq可以在体内增加肿瘤细胞ERβ的表达量,推断Liq可能在体内通过激活ERβ及相关信号通路来达到抑制肿瘤生长,提高肿瘤细胞对TMZ敏感性的作用。实验结论:1.Liq可抑制胶质瘤细胞(U138)增殖并明显促进其凋亡,可显著增强肿瘤细胞对TMZ的敏感性;2.Liq通过特异性激活ERβ来抑制PI3K/AKT/m TOR信号通路的活性;3.Liq对TMZ的增敏功能依赖于其对PI3K/AKT/m TOR通路的抑制;4.体内联合使用Liq和TMZ处理荷瘤小鼠发现Liq显著增加肿瘤细胞对TMZ的敏感性。以上实验结果提示Liq通过激活ERβ抑制PI3K/AKT/m TOR来促进TMZ治疗胶质瘤的效应。同时本研究也为其他ERβ激动剂可以作为一种有效的化疗药物用于神经胶质瘤的常规联合治疗提供了依据和线索。
[Abstract]:Background: Temozolomide (TMZ) is a new imidazole four alkyl alkylating agent, which can be transformed into active compound quickly by non enzyme pathway in physiological environment. By inducing DNA methylation to promote the rapid absorption of apoptosis.TMZ in tumor cells, it is easy to penetrate the blood brain barrier and has good antiglioma activity. The armature nervous system tumor, especially the malignant glioma, has been widely used in chemotherapy. However, the drug resistance of the tumor cells to TMZ often occurs, which is the main reason for the failure of the clinical chemotherapy. Therefore, how to solve the TMZ resistance and increase the sensitivity of malignant glioma cells to TMZ has become the latest in the Department of Neurosurgery clinicians and researchers The estrogen receptor beta is a multifunctional protein related to the development and differentiation of nerve cells and has the activity of tumor suppressor gene. The change of the expression level is closely related to the occurrence and development of glioma. Liquiritigenin (Liq) is found in the glycyrrhiza Glycyrrhiza (Glycyrrhiza uralensis Fisch). The two hydrogen flavone monomer compound is a natural estrogen receptor beta (estrogen receptor, ER beta) agonist. Earlier experiments showed that Liq could inhibit the proliferation of various tumors such as breast and cervix cancer, and also have the efficacy of increasing the sensitivity of chemotherapy drugs and preventing the recurrence of tumor. This subject is to observe the Liq to TMZ from the body and in vitro, in the body and in vitro. The effect of anti neuroglioma treatment, to explore the advantages of Liq, TMZ combined use in clinical application, and to further study the molecular basis of Liq to increase the sensitivity of tumor cells to TMZ, and to provide theoretical basis and experimental basis for the important role of Liq in the treatment of glioma. Methods: the study in the body and in vitro two parts. We selected three human glioma cell lines: TMZ low sensitivity U138, TMZ Gao Min sensitive U251 and TMZ resistant cell strain T98G, using MTT method, flow cytometry to detect the effects of the different concentrations of Liq alone or with TMZ on the proliferation, apoptosis and cell cycle of glioma cells in vitro; Western Blo. T was used to detect the changes in ER beta, MGMT, Caspase-3, Akt, p-AKT, P70S6K, p-P70S6K, and the level of protein expression and phosphorylation of signal molecules, such as ER beta, Akt, p-AKT, P70S6K, p-P70S6K, and the expression of ER beta by RNA interference. To further explore the regulatory effect of Liq on the pathway and the molecular mechanism to promote the sensitivity of TMZ, in vivo study of nude mice glioma model was used as the research object. After subcutaneous inoculation of U138 glioma cells (106/100 Mu L) in the left abdomen of nude mice, four groups were divided: the control group (DMSO), the TMZ group (50 mg/kg/d), the Liq group (30 mg/kg/d) and the TMZ 5 (TMZ 5). 0 mg/kg/d+Liq 30 mg/kg/d), daily intraperitoneal injection was carried out to observe the tumor growth status of nude mice in each group. The effects of Liq on the expression of ER beta and the synergistic effect on the anti tumor effect of TMZ were studied in vivo at 30 days after inoculation. Results: 1. In vitro experiment: the U138 cells were treated with different concentrations of Liq (10,20,40,80160) in vitro. After 320 M) treatment of 72 h, the results of cell activity test showed that Liq had a significant inhibitory effect on U138, and with the increase of concentration, the inhibitory effect gradually increased and showed a dose dependent effect. The Liq of 80 mu M could effectively inhibit the activity of U138 cells, but did not lead to excessive cell death. Therefore, we choose the Liq of this concentration. After the combined use of the concentration of Liq and different concentrations of TMZ (25,50100200400 and 800 u M), the inhibitory effect of TMZ on U138 proliferation was significantly increased. The survival rate of the tumor cells was only 50.36% when the 100 M TMZ and 80 micron Liq were combined with 72h, significantly lower than the survival rate of the tumor cells (90.33%, 01) Liq could effectively enhance the sensitivity of tumor cells to TMZ. Flow cytometry cell cycle analysis showed that combined use of Liq could further enhance TMZ induced S phase block (64.63%vs 40.43%, P0.001) compared with TMZ alone, and effectively inhibit the cell entering the proliferative.Annexin V/PI double staining apoptosis analysis showed that Liq and TMZ were combined. The use can significantly increase the apoptosis rate of U138 glioma cells (27.5%vs 18.6%, P0.001), and the synergistic effect of.Liq and TMZ on the formation of Caspase-3 shear body,.Liq and TMZ, in the other glioma cells, U251 and T98G, also verified the.Western Blot detection of PI3K/AKT/m TOR pathway. The phosphorylation level of 0S6K inhibits the activity of the PI3K/AKT/m TOR signaling pathway, but does not have a direct effect on the tumor resistance gene MGMT. After RNA interference silencing ER beta expression, Liq loses its regulatory role in the TOR pathway of PI3K/AKT/m, indicating that Liq is a pathway that regulates the activity of PI3K/AKT/m pathway activity by activating ER beta. The inhibitor XL765 can significantly increase the sensitivity of the cell to TMZ, suggesting that the activation of the PI3K/AKT/m TOR pathway can protect the glioma cells and antagonize the role of TMZ. The combination of PI3K/AKT/m TOR activator IGF-1 can antagonize the toxicity of Liq mediated TMZ, suggesting that Liq is used to inhibit PI3K/AKT/m to play a role. Two, body The results showed that the average volume of tumor in the control group was 1.84 0.13cm3, the average volume of the tumor in the TMZ group was 0.93 + 0.11cm3, the average volume of the tumor in the group Liq was 1.34 + 0.15cm3, and the average volume of the tumor in the group of TMZ and Liq was 0.36 + 0.04cm3., and the results were in accordance with the results of the cell experiment in vitro. The combination of Liq and TMZ was used. It can significantly inhibit tumor growth and increase the sensitivity of tumor to TMZ, and the difference is statistically significant (P0.05). At the same time, Western Blot results show that Liq can increase the expression of ER beta in tumor cells in vivo. It is concluded that Liq may inhibit the growth of tumor by activating ER beta and related signaling pathways in vivo, and improve the sensitivity of tumor cells to TMZ. Inductive effect. Experimental conclusion: 1.Liq can inhibit the proliferation of glioma cell (U138) and obviously promote its apoptosis, which can significantly enhance the sensitivity of tumor cells to TMZ; 2.Liq inhibits the activity of PI3K/AKT/m TOR signaling via specific activation of ER beta; 3.Liq's sensitizing function to TMZ depends on its inhibition of PI3K/AKT/m TOR pathway; 4. in vivo The tumor bearing mice treated with Liq and TMZ found that Liq significantly increased the sensitivity of the tumor cells to TMZ. These results suggest that Liq promotes the effect of TMZ on the treatment of glioma by activating ER beta to inhibit PI3K/AKT/m TOR. The present study also provides other ER beta agonists as an effective chemotherapeutic agent for neuroglioma. Combined treatment provides a basis and clues.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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