新型三氟甲基喹唑啉衍生物通过PI3K/Akt信号通路抑制骨肉瘤的进展

Novel trifluoromethyl quinazoline derivatives inhibit osteosarcoma progression through the PI3K/Akt signaling pathway

关键词:  骨肉瘤  三氟甲基  喹唑啉衍生物  线粒体凋亡  PI3K/Akt信号通路  转移

Key words:  osteosarcoma  trifluoromethyl  quinazoline derivatives  mitochondrial pathway apoptosis  PI3K/Akt signaling pathway  metastasis

摘要:

目的 探讨新型三氟甲基喹唑啉衍生物KZL-188对骨肉瘤143B细胞的增殖、凋亡、迁移和侵袭的影响及分子机制。方法 将143B细胞分为对照组(DMSO)和低、中、高剂量实验组(0.008、0.04、0.2 μmolL-1 KZL-188)。用MTT法检测KZL-188对各组细胞增殖的影响；流式细胞术检测细胞的凋亡情况；线粒体膜电位实验检测细胞的线粒体膜电位的变化；伤口愈合实验检测细胞迁移情况；trans-well实验检测细胞侵袭情况；western blot检测线粒体凋亡途径相关蛋白、转移相关蛋白和PI3K/Akt信号通路相关蛋白的表达。结果 与对照组相比,KZL-188呈浓度依赖性抑制143B细胞的增殖；各实验组细胞中转移相关蛋白MMP2、MMP13和Vimentin表达水平降低,KZL-18可以抑制143B细胞的迁移和侵袭,同时,线粒体膜电位降低,线粒体凋亡途径相关蛋白Bcl-2、cleaved caspase-3和cleaved caspase-9表达水平降低,Bax和Cytochrome c表达水平升高,提示细胞发生了线粒体凋亡。进一步探究发现,PI3K/Akt信号通路相关蛋白p-PI3K/PI3K、p-Akt/Akt的比值均降低,下游蛋白Bim表达水平升高。结论 KZL-188可抑制骨肉瘤143B细胞的增殖,迁移和侵袭,并通过调控线粒体凋亡相关蛋白的表达诱导143B细胞发生线粒体凋亡,其可能通过PI3K/Akt信号通路发挥其作用。

Abstract:

OBJECTIVE To investigate the effects and molecular mechanisms of KZL-188, a novel trifluoromethyl quinazoline derivative, on the proliferation, migration, invasion and apoptosis of osteosarcoma 143B cells. METHODS The 143B cells were divided into a control group (DMEM) and experimental groups with low, medium and high doses (0.008, 0.04, 0.2 μmolL-1 KZL-188). MTT assay was used to detect the effect of KZL-188 on the proliferation of 143B cells. Flow cytometry was used to detect the effect on apoptosis. JC1 assay was used to detect the effect on mitochondrial membrane potential. Wound healing assay was used to detect the effect on cell migration. Trans-well assay was used to detect the effect on cell invasion. Western blot was used to detect the effects of the expression of apoptosis-related proteins of the mitochondrial pathway, metastasis-related proteins, and proteins related to the PI3K/AKT signaling pathway. RESULTS Compared with the control group, Compared with the control group, KZL-188 inhibited the proliferation of 143B cells in a concentration-dependent manner; the expression of metastasis-related proteins MMP2, MMP13 and Vimentin decreased in all experimental groups, indicating that KZL-18 could inhibit the migration and invasion of 143B cells, meanwhile, the mitochondrial membrane potential decreased, and the expression of mitochondrial apoptosis-related proteins Bcl-2, cleavedcaspase-3 and cleavedcaspase-9 decreased. The increased expression of Bax and Cytochromec indicated that mitochondrial apoptosis occurred in the 143B cells. Further exploration showed that the ratio of p-PI3K/PI3K to p-Akt/Akt related to PI3K/Akt signaling pathway decreased, while the expression level of downstream protein Bim increased. CONCLUSION KZL-188 inhibits the proliferation, migration and invasion of 143B osteosarcoma cells and induces apoptosis of osteosarcoma cells by the mitochondrial pathway, possibly exerting its effects through the PI3K/AKT signaling pathway.