Wang Bin, Ni Zhenhong, Dai Xufang, Qin Liyan, Li Xinzhe, Xu Liang, Lian Jiqin, He Fengtian
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, 30 Gaotanyan, Chongqing 400038, China.
Mol Cancer. 2014 Apr 30;13:98. doi: 10.1186/1476-4598-13-98.
Hepatocellular carcinoma (HCC) is one of the major causes of mortality. ABT-263 is a newly synthesized, orally available Bcl-2/xL inhibitor that shows promising efficacy in HCC therapy. ABT-263 inhibits the anti-apoptotic activity of Bcl-2 and Bcl-xL, but not Mcl-1. Previous reports have shown that ABT-263 upregulates Mcl-1 in various cancer cells, which contributes to ABT-263 resistance in cancer therapy. However, the associated mechanisms are not well known.
Western blot, RNAi and CCK-8 assays were used to investigate the relationship between Mcl-1 upregulation and ABT-263 sensitivity in HCC cells. Real-time PCR and Western blot were used to detect Mcl-1 mRNA and protein levels. Luciferase reporter assay and RNA synthesis inhibition assay were adopted to analyze the mechanism of Mcl-1 mRNA upregulation. Western blot and the inhibition assays for protein synthesis and proteasome were used to explore the mechanisms of ABT-263-enhanced Mcl-1 protein stability. Trypan blue exclusion assay and flow cytometry were used to examine cell death and apoptosis.
ABT-263 upregulated Mcl-1 mRNA and protein levels in HCC cells, which contributes to ABT-263 resistance. ABT-263 increased the mRNA level of Mcl-1 in HCC cells by enhancing the mRNA stability without influencing its transcription. Furthermore, ABT-263 increased the protein stability of Mcl-1 through promoting ERK- and JNK-induced phosphorylation of Mcl-1Thr163 and increasing the Akt-mediated inactivation of GSK-3β. Additionally, the inhibitors of ERK, JNK or Akt sensitized ABT-263-induced apoptosis in HCC cells.
ABT-263 increases Mcl-1 stability at both mRNA and protein levels in HCC cells. Inhibition of ERK, JNK or Akt activity sensitizes ABT-263-induced apoptosis. This study may provide novel insights into the Bcl-2-targeted cancer therapeutics.
肝细胞癌（HCC）是主要的致死原因之一。ABT-263是一种新合成的、口服可用的Bcl-2/xL抑制剂，在HCC治疗中显示出有前景的疗效。ABT-263抑制Bcl-2和Bcl-xL的抗凋亡活性，但不抑制Mcl-1。先前的报道表明，ABT-263在各种癌细胞中上调Mcl-1，这导致癌症治疗中对ABT-263产生抗性。然而，相关机制尚不清楚。
采用蛋白质免疫印迹法、RNA干扰和CCK-8检测法研究HCC细胞中Mcl-1上调与ABT-263敏感性之间的关系。采用实时定量PCR和蛋白质免疫印迹法检测Mcl-1 mRNA和蛋白水平。采用荧光素酶报告基因检测法和RNA合成抑制试验分析Mcl-1 mRNA上调的机制。采用蛋白质免疫印迹法以及蛋白质合成和蛋白酶体抑制试验探讨ABT-263增强Mcl-1蛋白稳定性的机制。采用台盼蓝排斥试验和流式细胞术检测细胞死亡和凋亡情况。
ABT-263上调HCC细胞中Mcl-1 mRNA和蛋白水平，这导致对ABT-263产生抗性。ABT-263通过增强mRNA稳定性而不影响其转录来提高HCC细胞中Mcl-1的mRNA水平。此外，ABT-263通过促进ERK和JNK诱导的Mcl-1苏氨酸163磷酸化以及增加Akt介导的GSK-3β失活来提高Mcl-1的蛋白稳定性。此外，ERK、JNK或Akt的抑制剂使ABT-263诱导的HCC细胞凋亡敏感化。
ABT-263在HCC细胞中在mRNA和蛋白水平上均增加Mcl-1的稳定性。抑制ERK、JNK或Akt活性可使ABT-263诱导的凋亡敏感化。本研究可能为以Bcl-2为靶点的癌症治疗提供新的见解。
