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Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells.

Mahyar-Roemer M, Köhler H, Roemer K - BMC Cancer (2002)

Bottom Line: In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent.Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis.Both can limit the ability of the cells to form colonies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology, Institute of Medical Microbiology, University of Saarland Medical School, D-66421 Homburg/Saar, Germany. inmmah@uniklinik-saarland.de

ABSTRACT

Background: The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax.

Methods: The expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated.

Results: Low to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells.

Conclusion: Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies.

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Expression of Bax and Bcl-XL, and apoptosis in human HCT116 colon carcinoma cells treated with low doses of resveratrol. Panel A: Total protein extracts (15 μg) of HCT116 cultures treated with 40 μM resveratrol or only DMSO (the solvent) were prepared at different times and analyzed by immunoblotting, employing the anti-Bax N20 (1:500) and anti-Bcl-XL antibodies (1:200). Note that at this low drug concentration, neither the level of Bax nor of Bcl-XL changed significantly. Panel B: FACS analyses on exponentially growing HCT116 cultures either mock-treated or treated with low doses of resveratrol for 48 h exhibited significant apoptosis in the drug-treated cultures. Error bars depict standard deviations of the means of five experiments.
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Figure 1: Expression of Bax and Bcl-XL, and apoptosis in human HCT116 colon carcinoma cells treated with low doses of resveratrol. Panel A: Total protein extracts (15 μg) of HCT116 cultures treated with 40 μM resveratrol or only DMSO (the solvent) were prepared at different times and analyzed by immunoblotting, employing the anti-Bax N20 (1:500) and anti-Bcl-XL antibodies (1:200). Note that at this low drug concentration, neither the level of Bax nor of Bcl-XL changed significantly. Panel B: FACS analyses on exponentially growing HCT116 cultures either mock-treated or treated with low doses of resveratrol for 48 h exhibited significant apoptosis in the drug-treated cultures. Error bars depict standard deviations of the means of five experiments.

Mentions: Human colorectal adenocarcinoma HCT116 is a poorly differentiated, growth factor-insensitive cell line deficient for hMLH1, a homologue of the bacterial Mut L protein and crucial component of DNA mismatch repair. The present study has made use primarily of HCT116 and the isogenic cell lines HCT116 p53-/- and HCT116 bax-/- derived from the parental line by targeted disruption of the p53 alleles or – in the case of the bax alleles – by a combination of spontaneous frameshift mutation and targeted disruption [31,32]. HCT116 cells are responsive to a variety of stresses including DNA damage and spindle disruption, in part owing to the integrity of the wild-type p53 tumor suppressor pathway. For this reason, and since deregulated colon epithelial cells are the supposedly primary targets of natural cancer chemopreventive food constituents, this cell line has been frequently employed for the study of the molecular mechanisms of drug action. We have recently reported that the polyphenol resveratrol present in some foods can increase the steady-state levels of pro-apoptotic Bax protein and induce a mitochondria-mediated apoptosis in HCT116 cells independently of p53 [14]. However, although Bax overproduction in response to 100 μM resveratrol was weak, and at lower drug doses (< 40 μM), undetectable (Figure 1A), doses of 10 to 20 μM were able to provoke significant apoptotic cell death (typically 9 to 25 % within 48 h vs. 5 % in mock-treated cultures, Figure 1B). We therefore asked what role, if any, Bax might have in the resveratrol-induced apoptosis of HCT116 cells.


Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells.

Mahyar-Roemer M, Köhler H, Roemer K - BMC Cancer (2002)

Expression of Bax and Bcl-XL, and apoptosis in human HCT116 colon carcinoma cells treated with low doses of resveratrol. Panel A: Total protein extracts (15 μg) of HCT116 cultures treated with 40 μM resveratrol or only DMSO (the solvent) were prepared at different times and analyzed by immunoblotting, employing the anti-Bax N20 (1:500) and anti-Bcl-XL antibodies (1:200). Note that at this low drug concentration, neither the level of Bax nor of Bcl-XL changed significantly. Panel B: FACS analyses on exponentially growing HCT116 cultures either mock-treated or treated with low doses of resveratrol for 48 h exhibited significant apoptosis in the drug-treated cultures. Error bars depict standard deviations of the means of five experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC130964&req=5

Figure 1: Expression of Bax and Bcl-XL, and apoptosis in human HCT116 colon carcinoma cells treated with low doses of resveratrol. Panel A: Total protein extracts (15 μg) of HCT116 cultures treated with 40 μM resveratrol or only DMSO (the solvent) were prepared at different times and analyzed by immunoblotting, employing the anti-Bax N20 (1:500) and anti-Bcl-XL antibodies (1:200). Note that at this low drug concentration, neither the level of Bax nor of Bcl-XL changed significantly. Panel B: FACS analyses on exponentially growing HCT116 cultures either mock-treated or treated with low doses of resveratrol for 48 h exhibited significant apoptosis in the drug-treated cultures. Error bars depict standard deviations of the means of five experiments.
Mentions: Human colorectal adenocarcinoma HCT116 is a poorly differentiated, growth factor-insensitive cell line deficient for hMLH1, a homologue of the bacterial Mut L protein and crucial component of DNA mismatch repair. The present study has made use primarily of HCT116 and the isogenic cell lines HCT116 p53-/- and HCT116 bax-/- derived from the parental line by targeted disruption of the p53 alleles or – in the case of the bax alleles – by a combination of spontaneous frameshift mutation and targeted disruption [31,32]. HCT116 cells are responsive to a variety of stresses including DNA damage and spindle disruption, in part owing to the integrity of the wild-type p53 tumor suppressor pathway. For this reason, and since deregulated colon epithelial cells are the supposedly primary targets of natural cancer chemopreventive food constituents, this cell line has been frequently employed for the study of the molecular mechanisms of drug action. We have recently reported that the polyphenol resveratrol present in some foods can increase the steady-state levels of pro-apoptotic Bax protein and induce a mitochondria-mediated apoptosis in HCT116 cells independently of p53 [14]. However, although Bax overproduction in response to 100 μM resveratrol was weak, and at lower drug doses (< 40 μM), undetectable (Figure 1A), doses of 10 to 20 μM were able to provoke significant apoptotic cell death (typically 9 to 25 % within 48 h vs. 5 % in mock-treated cultures, Figure 1B). We therefore asked what role, if any, Bax might have in the resveratrol-induced apoptosis of HCT116 cells.

Bottom Line: In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent.Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis.Both can limit the ability of the cells to form colonies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology, Institute of Medical Microbiology, University of Saarland Medical School, D-66421 Homburg/Saar, Germany. inmmah@uniklinik-saarland.de

ABSTRACT

Background: The natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax.

Methods: The expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated.

Results: Low to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells.

Conclusion: Resveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies.

Show MeSH
Related in: MedlinePlus