Expression of

mcl-1 mRNA did not differ between ABT-737–t

Expression of

mcl-1 mRNA did not differ between ABT-737–treated cells and vehicle-treated cells (Fig. 3B), suggesting the involvement of a posttranscriptional mechanism. Because Mcl-1 is a rapid-turnover protein, the levels of Mcl-1 may be regulated by protein degradation.21 To examine this, we treated hepatoma cells with cycloheximide, a well-established protein synthesis inhibitor, in the presence or absence of ABT-737. Cycloheximide-induced rapid decline in learn more Mcl-1 expression was substantially blocked in the presence of ABT-737, suggesting that ABT-737 significantly delays degradation and prolongs the stability of Mcl-1 (Fig. 3C). Recently, it was reported that the deubiquitinase USP9X is involved in stabilization of Mcl-1.22 In this study, western blot analysis

revealed that the levels of USP9X expression were not changed in Huh7 and Hep3B with ABT-737 (Supporting Fig. 1A). Furthermore, USP9X down-regulation by small interfering RNA (siRNA) could not block the Mcl-1 up-regulation induced by ABT-737 (Supporting Fig. 1B).These results suggest that USP9X was not involved in Mcl-1 up-regulation induced by ABT-737. Of importance is the finding that Mcl-1 expression was also up-regulated after administration of ABT-737 in our xenograft PF-01367338 research buy model (Fig. 3D). Because Mcl-1 is not a target of ABT-737, relative resistance to ABT-737 of hepatoma cells may be due, at least in part, to posttranscriptional induction of Mcl-1. To examine the impact of Mcl-1 induction in hepatoma cell resistance to ABT-737, MCE公司 we silenced Mcl-1 expression through use of three different siRNAs. Western blot analysis revealed that Mcl-1 siRNA2 and siRNA3 completely knocked down Mcl-1 expression in Hep3B cells, whereas Mcl-1 siRNA1 did so only partially (Fig. 4A). Mcl-1 knockdown or a medium dose of ABT-737 (4 μM) modestly activated caspase-3/7 in Hep3B cells, whereas both substantially activated caspase-3/7 (Fig. 4B). In addition, Mcl-1 knockdown or ABT-737 alone

failed to suppress the growth of tumor cells but caused significant suppression when used together (Fig. 4C). Caspase-3 activation was also confirmed by western blots (Fig. 4A). It should be noted that Mcl-1 siRNA1 reduced Mcl-1 expression in ABT-737-treated cells to levels similar to those of untreated cells (Fig. 4A). Even in this case, mcl-1 knockdown enhanced caspase activation and growth suppression of Hep3B cells induced by ABT-737. Similar data were obtained with another hepatoma cell line, Huh7 (Fig. 4A and Supporting Fig. 2). These results indicate that Mcl-1 up-regulation induced by ABT-737 is involved in the resistance of hepatoma cells to ABT-737 and suggest that combination therapy with ABT-737 and Mcl-1 inhibitor may be predictably effective in vivo. We previously reported that, similar to Bcl-xL, Mcl-1 plays an important role in apoptosis resistance of normal hepatocytes.

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