4A, knockdown of Bcl-xL gradually inhibited cell proliferation over 72 hr as determined by MTT assay

4A, knockdown of Bcl-xL gradually inhibited cell proliferation over 72 hr as determined by MTT assay. the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma. Graphical Abstract < 0.05 compared to respective controls. Clo, clofarabine; Res, resveratrol. Open in a separate window Fig. 2 Effects of resveratrol and clofarabine on Mcl-1 and Bcl-xL protein levels. (A) Cells were co-treated with resveratrol (15 M) and clofarabine (40 nM), alone or in combination, for 24 hr. (B) Cells were co-treated with resveratrol (15 M) and clofarabine (40 nM) for 1, 2, and 3 days. (C) Cells were co-treated with resveratrol (5, 10, BMS 626529 15, and 20 M) and clofarabine (40 nM) for 24 hr. Whole cell lyastes were analyzed by Western blot analysis using antibodies against Mcl-1, Bcl-xL, and -actin. *< 0.05 compared to respective controls. Clo, clofarabine; Res, resveratrol. Mcl-1 protein level, in response to resveratrol and clofarabine, is usually regulated by proteasomal activity To elucidate the possible mechanism underlying Mcl-1 downregulation by Res plus Clo, cells were co-treated with two compounds for 24 hr. As shown in Fig. 3A, there were no detectable changes in the mRNA levels in RT-PCR analysis. These data suggest that Mcl-1 protein levels in H-2452 cells in response to Res plus Clo were regulated through transcription-independent mechanism. In contrast, the amount of Mcl-1 protein upon the combination treatment significantly increased following the pretreatment with proteasome inhibitor MG132 (Fig. 3B). Next, we analyzed the effect of protein synthesis inhibitor, cycloheximide (CHX), on Res plus Clo-induced downregulation of Mcl-1. The Mcl-1 protein level was gradually declined over 160 min of treatment with CHX alone, suggesting rapid turnover of Mcl-1. However, in the presence of MG132 and CHX, the Mcl-1 protein level was retained to a greater extent compared to cells treated with CHX alone, suggestingg that BMS 626529 Mcl-1 was stabilized by proteasomal activity (Fig. 3C). These results indicate that Mcl-1 protein level, in response to Res plus Clo, was mainly regulated at the posttranslational step in H-2452 cells. Open in a separate window Fig. 3 Regulation of Mcl-1 protein level by resveratrol and clofarabine. (A) Cells were seeded in 6-well culture plate and were co-treated with resveratrol (15 BMS 626529 M) and clofarabine (40 nM) for the indicated occasions, after which the total RNA was isolated and subjected to RT-PCR analysis. GAPDH primers were used to amplify as an internal standard. (B) Cells were treated with MG132 (10 M) for 2 hr prior to incubation with resveratrol (15 M) and clofarabine (40 nM) for additional 24 hr. (C) Cells were pretreated with 0.1 M cycloheximide (CHX) alone or CHX plus 10 M MG132, for varying intervals as indicated. Whole cell lyastes were obtained and subjected to Western blot analysis using antibodies against Mcl-1, Bcl-xL, and -actin. Normalized intensity of Mcl-1 or Bcl-xL versus -actin was presented as the mean value from two impartial experiments. Clo/Res, clofarabine plus resveratrol. Co-silencing of Mcl-1and Bcl-xL induces G2/M cell cycle arrest and caspase-dependent apoptosis Rabbit Polyclonal to Gastrin To examine whether dual inhibition of Mcl-1 and Bcl-xL sensitizes H-2452 cells to Res BMS 626529 plus Clo, cells were transfected with Bcl-xL siRNA (siBcl-xL) or in combination with Mcl-1 siRNA (siMcl-1) before exposure to two compounds. As shown in Fig. 4A, knockdown of Bcl-xL gradually inhibited cell proliferation over 72 hr as determined by MTT assay. These responses were augmented following a subsequent exposure to Res plus Clo. In flow cytometric analysis, the sub-G0 peak, indicative of apoptosis, significantly increased when cells were knocked down with siBcl-xL. A strong sub-G0 peak was observed in cells co-transfected with siMcl-1.