These results indicate that agonists for Gs coupled receptors can activate PP2A

Results Upregulation of Mcl 1 is correlated with ABT 263 resistance in HCC cells Firstly, the AP24534 溶解度 expression levels of anti apoptotic Bcl 2 fam ily members Bcl 2, Bcl xL and Mcl 1 were analyzed with Western blot in HCC cell lines PLCPRF5, Hep3B, HepG2 and Huh7. As shown in Figure 1A, Mcl 1 was highly expressed in all four HCC cell lines, but the levels of Bcl 2 and Bcl xL differed. Hep3B cells had low level of Bcl xL and Huh7 cells had almost no detectable Bcl 2. Upon treatment with ABT 263, the level of Mcl 1 in creased dramatically in all HCC cell lines, but the levels of Bcl 2 and Bcl xL did not change significantly. Another Bcl 2 inhibitor AT 101 had similar effect on Mcl 1 expression in HCC cells. To test whether the upregulation of Mcl 1 is affected by Bcl 2 level, we knocked down Bcl 2 in Hep3B cells and overexpressed it in Huh7 cells, respectively.<br><br> As shown in Figure 1C, the level of Mcl 1 remained unchanged upon Bcl 2 downregulation or overexpression. Similar results were also found when Bcl xL was knocked down in Huh7 cells or overexpressed in Hep3B cells. These results indicated that ABT AT7519 臨床試験 263 induced Mcl 1 up regulation was independent of the levels of Bcl 2xL in HCC cells. Furthermore, consistent with previous reports, Mcl 1 knockdown significantly enhanced the cytotoxicity of ABT 263 in HCC cells. The above data indicated that the drug resistance of ABT 263 was, at least partially, mediated by Mcl 1 upregula tion, which was not associated with the expression levels of Bcl 2xL in HCC cells.<br><br> ABT 263 upregulates Mcl 1 at both mRNA and protein levels To investigate the underlying mechanism of ABT 263 induced Mcl 1 upregulation in HCC cells, both mRNA and protein levels of Mcl 1 were analyzed after treat ment with ABT 263. Since PLC and Huh7 cell lines had a higher sensitivity to ABT 263 after Mcl Alisertib Aurora キナーゼ 阻害剤 1 knockdown, they were chosen as target cells. As shown in Figure 2, ABT 263 upregulated Mcl 1 at both mRNA and protein levels in PLC and Huh7 cells revealed by RT PCR, real time PCR and Western blot. ABT 263 increases the mRNA stability of Mcl 1 To figure out the mechanisms of ABT 263 mediated Mcl 1 mRNA upregulation, the promoter region of Mcl 1 gene was cloned into re porter vector pGL3 basic, and the resulting plasmid was named as pLucM1. Meanwhile, the pro moter region containing the binding sites for several predicted transcriptional factors was also cloned into pGL3 basic, and the resulting plas mid was named as pLucM2.<br><br> Then PLC and Huh7 cells were separately transfected with pLucM1 and pLucM2 and followed by the treatment with ABT 263. As shown in Figure 3B, ABT 263 didnt affect the activ ity of Mcl 1 promoter in HCC cells, neither in pLucM1 nor in pLucM2. Subsequently, PLC and Huh7 cells were treated with transcription inhibitor actinomycin D in the presence or absence of ABT 263. As shown in Figure 3C, ABT 263 co treatment significantly enhanced the mRNA stability of Mcl 1 compared to Act D treat ment alone. These results indicated that ABT 263 upregulated Mcl 1 mRNA level via increasing the mRNA stability instead of activating its transcription in HCC cells. ABT 263 increases the protein stability of Mcl 1 To assess whether the increase of Mcl 1 protein solely results from the upregulation of Mcl 1 mRNA, the HCC cells were treated with translation inhibitor cyclohexi mide.