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Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

Woo SM, Min KJ, Seo BR, Nam JO, Choi KS, Yoo YH, Kwon TK - Cell Death Dis (2014)

Bottom Line: Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models.We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation.Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, School of Medicine, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea.

ABSTRACT
Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

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Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. (a) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. (b) Caki cells were treated with 30 μM cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (c) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (d and e) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA (c) and Bim (d) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM cafestol (Caf) and 0.1 μM ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry (d and e, upper panel). Equal amounts of cell lysates (60 μg) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels (d and e) represent the mean±S.D. from three independent samples. *P<0.05 compared with ABT-737 plus cafestol-treated control siRNA. The data represent three independent experiments
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fig5: Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. (a) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. (b) Caki cells were treated with 30 μM cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (c) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (d and e) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA (c) and Bim (d) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM cafestol (Caf) and 0.1 μM ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry (d and e, upper panel). Equal amounts of cell lysates (60 μg) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels (d and e) represent the mean±S.D. from three independent samples. *P<0.05 compared with ABT-737 plus cafestol-treated control siRNA. The data represent three independent experiments

Mentions: Next, we investigated whether cafestol regulated other apoptosis-related proteins. Although cafestol had no effect on other proteins, it dose-dependently increased the protein levels of PUMA and Bim (Figure 5a). Protein levels of PUMA were increased from 12 h of 30 μM cafestrol treatment and those of Bim were enhanced from 6 h (Figure 5b). Furthermore, cafestol also dose-dependently increased the protein levels of PUMA and Bim in MDA-MB-231 and U251MG cells (Figure 5c). Therefore, we investigated whether these proteins were related to ABT-737 plus cafestol-induced apoptosis. Downregulation of PUMA expression by siRNA did not block apoptosis (Figure 5d). However, ABT-737 plus cafestol-induced apoptosis was markedly inhibited by downregulation of Bim expression by siRNA (Figure 5e). These data suggest that upregulation of Bim expression critically contributed to the sensitization of ABT-737-mediated apoptosis.


Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

Woo SM, Min KJ, Seo BR, Nam JO, Choi KS, Yoo YH, Kwon TK - Cell Death Dis (2014)

Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. (a) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. (b) Caki cells were treated with 30 μM cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (c) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (d and e) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA (c) and Bim (d) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM cafestol (Caf) and 0.1 μM ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry (d and e, upper panel). Equal amounts of cell lysates (60 μg) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels (d and e) represent the mean±S.D. from three independent samples. *P<0.05 compared with ABT-737 plus cafestol-treated control siRNA. The data represent three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Upregulation of Bim expression is associated with cafestol plus ABT-737-induced apoptosis. (a) Caki cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of Bcl-2, Bcl-xL, PUMA, Bim, Bax, cIAP1, cIAP2, XIAP, and c-FLIP were determined by western blotting. Actin served as a control for protein loadings. (b) Caki cells were treated with 30 μM cafestol for the indicated time periods. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (c) MDA-MB231 and U251MG cells were treated with the indicated concentrations of cafestol for 24 h. The protein expression levels of PUMA and Bim were determined by western blotting. Actin served as a control for protein loadings. (d and e) Mcl-1-overexpressed cells (Caki/Mcl-1) were transiently transfected with PUMA (c) and Bim (d) siRNA or control siRNA. Overnight after transfection, cells were treated with 30 μM cafestol (Caf) and 0.1 μM ABT-737 (ABT) for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry (d and e, upper panel). Equal amounts of cell lysates (60 μg) were separated by gel electrophoresis and analyzed by western blotting for poly ADP-ribose polymerase (PARP), PUMA, and Bim. Actin served as a control for protein loadings. The values in panels (d and e) represent the mean±S.D. from three independent samples. *P<0.05 compared with ABT-737 plus cafestol-treated control siRNA. The data represent three independent experiments
Mentions: Next, we investigated whether cafestol regulated other apoptosis-related proteins. Although cafestol had no effect on other proteins, it dose-dependently increased the protein levels of PUMA and Bim (Figure 5a). Protein levels of PUMA were increased from 12 h of 30 μM cafestrol treatment and those of Bim were enhanced from 6 h (Figure 5b). Furthermore, cafestol also dose-dependently increased the protein levels of PUMA and Bim in MDA-MB-231 and U251MG cells (Figure 5c). Therefore, we investigated whether these proteins were related to ABT-737 plus cafestol-induced apoptosis. Downregulation of PUMA expression by siRNA did not block apoptosis (Figure 5d). However, ABT-737 plus cafestol-induced apoptosis was markedly inhibited by downregulation of Bim expression by siRNA (Figure 5e). These data suggest that upregulation of Bim expression critically contributed to the sensitization of ABT-737-mediated apoptosis.

Bottom Line: Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models.We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation.Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, School of Medicine, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea.

ABSTRACT
Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

Show MeSH
Related in: MedlinePlus