替莫唑胺联合吩噻嗪类药物对人脑胶质瘤的体外抗肿瘤作用和机制研究

In vitro anti-tumor activities and mechanisms of phenothiazines in combined treatment with temozolomide in human glioma cell lines

本研究主要探究替莫唑胺分别与氯丙嗪、奋乃静联合使用后对人脑胶质瘤的体外抗肿瘤作用及其作用机制。通过MTT法检测氯丙嗪、奋乃静和替莫唑胺单独及联合使用后对肿瘤细胞的生长抑制作用;通过集落形成、细胞凋亡、周期分布、活性氧(ROS)产生及线粒体膜电位(JC-1)检测实验,比较单独及联合用药的抗肿瘤效果;通过免疫荧光实验检测p53基因的表达水平,通过流式细胞术和共聚焦成像比较不同给药方式下肿瘤细胞的自噬情况,探究氯丙嗪协同替莫唑胺的抗肿瘤作用机制;通过免疫荧光实验检测蛋白磷酸酶2A(PP2A)、蛋白磷酸酶2A抑制剂(CIP2A)和原癌基因(c-Myc)蛋白的表达水平,通过细胞流式检测细胞中肿瘤干细胞标记物(CD44、CD133)和乙醛脱氢酶(ALDH)的表达水平,从而探究奋乃静和替莫唑胺联合使用的抗肿瘤作用机制。结果显示,与单独使用替莫唑胺相比,联合用药72 h后替莫唑胺对U87和U251细胞的半数抑制浓度(IC50)均降低,并且提高了替莫唑胺诱导细胞凋亡和周期阻滞的能力。此外,氯丙嗪和替莫唑胺联用通过激活胶质瘤细胞中p53基因的相关通路诱导细胞自噬的增加;奋乃静和替莫唑胺联用提高了胶质瘤细胞对替莫唑胺的敏感性,其作用机制可能与抑制CIP2A/PP2A/c-Myc信号通路有关。综上所述,替莫唑胺分别与氯丙嗪、奋乃静联合使用具有协同的抗肿瘤效果,为人脑胶质瘤的治疗提供新策略。

In the study,to explore the anti-tumor effects and mechanisms of chlorpromazine(CPZ)and perphenazine(PPZ)combined with temozolomide(TMZ)on human glioma cell lines,we performed MTT assays to determine the growth inhibitory rate of CPZ,PPZ and TMZ in mono and combined treatments.The anti-tumor effects of CPZ and PPZ alone or in combination with TMZ were determined by colony formation,cell apoptosis,cell cycle arrest,reactive oxygen species(ROS)production and mitochondrial membrane potential(MMP)detection(JC-1).The expression level of p53 was detected by immunofluorescence assay.Furthermore,autophagy under different administrations was detected by flow cytometry and confocal imaging to explore the anti-tumor mechanism of CPZ and TMZ.Protein phosphatase 2A(PP2A),cancerous inhibitor of protein phosphatase 2A(CIP2A)and proto-oncogene protein(c-Myc)were detected by immunofluorescence assay,tumor stem cell markers(CD44,CD133)and aldehyde dehydrogenase(ALDH)were detected by flow cytometry to explore the anti-tumor mechanism of PPZ and TMZ.The results showed that after 72 h treatments of combinations,the values of half maximal inhibitory concentration(IC50)of TMZ on U87 and U251 cells were reduced,and the ability of TMZ to induce apoptosis and cycle arrest was improved.In addition,the combination of CPZ and TMZ could induce an increased effect of autophagy via activating the relevant pathway of p53 gene in glioma cells.The combination of PPZ and TMZ increased the sensitivity of glioma cells to TMZ,and the underlying mechanism might be related to the inhibition of CIP2A/PP2A/c-Myc signaling pathway.In conclusion,CPZ and PPZ combined with TMZ,showed the significant synergistic effects in cancer treatment,which are the novel and potential therapeutic regimens providing a new treatment strategy for human glioma.