e analyzed with Western blot in HCC cell lines PLC PRF 5, Hep3B, HepG2 and Huh7. As shown in Figure 1A, Mcl 1 was highly e pressed in all four HCC selleck Sorafenib cell lines, but the levels of Bcl 2 and Bcl L differed. Hep3B cells Inhibitors,Modulators,Libraries had low level of Bcl L 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 L did not change significantly. Another Bcl 2 inhibitor AT 101 had similar effect on Mcl 1 e pression 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 overe pressed it in Huh7 cells, respectively. As shown in Figure 1C, the level of Mcl 1 remained unchanged upon Bcl 2 downregulation or overe pression.
Similar Inhibitors,Modulators,Libraries results were also found when Bcl L was knocked down in Huh7 cells or overe pressed in Hep3B cells. These results indicated that ABT 263 induced Mcl 1 up regulation was independent of the levels of Bcl 2 L in HCC cells. Furthermore, consistent with previous reports, Mcl 1 knockdown significantly enhanced the cytoto icity of ABT 263 in HCC cells. The above data indicated Inhibitors,Modulators,Libraries that the drug resistance of ABT 263 was, at least partially, mediated by Mcl 1 upregula tion, which was not associated with the e pression levels of Bcl 2 L in HCC cells. 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 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 Inhibitors,Modulators,Libraries 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. 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 Brefeldin_A enhanced the kinase inhibitor Cisplatin 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