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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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Subcellular localization of Bax in drug-treated HCT116 cells and the p53-/- derivatives. Panel A: HCT116 and HCT116 p53-/- cultures were treated with 100 μM resveratrol (+) or mock-treated (-) and, after 24 h, stained with the mitochondria-specific dye Mitotracker Red (MT-red) and anti-Bax N-20 antibody (1:100). Note the punctuate, cytoplasmic stain with perinuclear concentration produced by the anti-Bax antibody in cells exposed to the drug, resembling the pattern produced by MT-red. Panel B: Confocal microscopy of HCT116 cells treated with resveratrol for 24 h confirmed the co-localization of part of the Bax-produced fluorescence with the MT-red fluorescence. Panel C: Immunoblot analysis on 15 μg of total protein from the high membrane (mitochondrial) fraction of cells mock-treated (-) or drug-treated (+) for 24 h. Staining with Bax and cytochrome b (control) antibody (1:500) revealed the increase in Bax levels in the mitochondria fraction of resveratrol-treated cells. The co-localization of Bax with the mitochondria was not dependent on the presence of p53.
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Figure 2: Subcellular localization of Bax in drug-treated HCT116 cells and the p53-/- derivatives. Panel A: HCT116 and HCT116 p53-/- cultures were treated with 100 μM resveratrol (+) or mock-treated (-) and, after 24 h, stained with the mitochondria-specific dye Mitotracker Red (MT-red) and anti-Bax N-20 antibody (1:100). Note the punctuate, cytoplasmic stain with perinuclear concentration produced by the anti-Bax antibody in cells exposed to the drug, resembling the pattern produced by MT-red. Panel B: Confocal microscopy of HCT116 cells treated with resveratrol for 24 h confirmed the co-localization of part of the Bax-produced fluorescence with the MT-red fluorescence. Panel C: Immunoblot analysis on 15 μg of total protein from the high membrane (mitochondrial) fraction of cells mock-treated (-) or drug-treated (+) for 24 h. Staining with Bax and cytochrome b (control) antibody (1:500) revealed the increase in Bax levels in the mitochondria fraction of resveratrol-treated cells. The co-localization of Bax with the mitochondria was not dependent on the presence of p53.

Mentions: Previous work has documented that subtle shifts in the expression levels of Bax-interacting anti-apoptotic members of the Bcl-2 family of proteins can influence cell survival even when Bax levels are unchanged [29]. We failed to detect changes of the steady-state levels of anti-apoptotic Bcl-XL in response to resveratrol (Figure 1A), and Bcl-2 was not detectably produced in HCT116 cells, indicating that the apoptotic sensitivity to resveratrol is not regulated via alterations in the Bax/Bcl-XL ratio. Recent work has furthermore shown that Bax levels do not always increase following apoptotic stimuli but that, instead, the occluded N-terminus of Bax may become exposed and the protein may then co-localize with the mitochondria ([30] and discussion therein). When mock-treated formaldehyde-fixed HCT116 cells, p53-deficient HCT116 p53 -/- cells, or HCT116 bax -/- cells were stained with anti-Bax-antibody 6A7 (Pharmingen/Transduction Labs) or an antibody raised against the conformation-sensitive N-terminus of Bax (N20, amino acids 11 to 30, Santa Cruz), both antibodies recognized a protein in the bax-positive HCT116 cells and produced a diffuse immunofluorescence signal (see Figure 2, mock-treated cultures), whereas no fluorescence was produced in the bax -/- or isotype controls (not shown), indicating that staining was specific and that the N-terminal epitope of at least a fraction of the Bax molecules was exposed in untreated HCT116 cells after standard fixation.


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

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

Subcellular localization of Bax in drug-treated HCT116 cells and the p53-/- derivatives. Panel A: HCT116 and HCT116 p53-/- cultures were treated with 100 μM resveratrol (+) or mock-treated (-) and, after 24 h, stained with the mitochondria-specific dye Mitotracker Red (MT-red) and anti-Bax N-20 antibody (1:100). Note the punctuate, cytoplasmic stain with perinuclear concentration produced by the anti-Bax antibody in cells exposed to the drug, resembling the pattern produced by MT-red. Panel B: Confocal microscopy of HCT116 cells treated with resveratrol for 24 h confirmed the co-localization of part of the Bax-produced fluorescence with the MT-red fluorescence. Panel C: Immunoblot analysis on 15 μg of total protein from the high membrane (mitochondrial) fraction of cells mock-treated (-) or drug-treated (+) for 24 h. Staining with Bax and cytochrome b (control) antibody (1:500) revealed the increase in Bax levels in the mitochondria fraction of resveratrol-treated cells. The co-localization of Bax with the mitochondria was not dependent on the presence of p53.
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Related In: Results  -  Collection

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Figure 2: Subcellular localization of Bax in drug-treated HCT116 cells and the p53-/- derivatives. Panel A: HCT116 and HCT116 p53-/- cultures were treated with 100 μM resveratrol (+) or mock-treated (-) and, after 24 h, stained with the mitochondria-specific dye Mitotracker Red (MT-red) and anti-Bax N-20 antibody (1:100). Note the punctuate, cytoplasmic stain with perinuclear concentration produced by the anti-Bax antibody in cells exposed to the drug, resembling the pattern produced by MT-red. Panel B: Confocal microscopy of HCT116 cells treated with resveratrol for 24 h confirmed the co-localization of part of the Bax-produced fluorescence with the MT-red fluorescence. Panel C: Immunoblot analysis on 15 μg of total protein from the high membrane (mitochondrial) fraction of cells mock-treated (-) or drug-treated (+) for 24 h. Staining with Bax and cytochrome b (control) antibody (1:500) revealed the increase in Bax levels in the mitochondria fraction of resveratrol-treated cells. The co-localization of Bax with the mitochondria was not dependent on the presence of p53.
Mentions: Previous work has documented that subtle shifts in the expression levels of Bax-interacting anti-apoptotic members of the Bcl-2 family of proteins can influence cell survival even when Bax levels are unchanged [29]. We failed to detect changes of the steady-state levels of anti-apoptotic Bcl-XL in response to resveratrol (Figure 1A), and Bcl-2 was not detectably produced in HCT116 cells, indicating that the apoptotic sensitivity to resveratrol is not regulated via alterations in the Bax/Bcl-XL ratio. Recent work has furthermore shown that Bax levels do not always increase following apoptotic stimuli but that, instead, the occluded N-terminus of Bax may become exposed and the protein may then co-localize with the mitochondria ([30] and discussion therein). When mock-treated formaldehyde-fixed HCT116 cells, p53-deficient HCT116 p53 -/- cells, or HCT116 bax -/- cells were stained with anti-Bax-antibody 6A7 (Pharmingen/Transduction Labs) or an antibody raised against the conformation-sensitive N-terminus of Bax (N20, amino acids 11 to 30, Santa Cruz), both antibodies recognized a protein in the bax-positive HCT116 cells and produced a diffuse immunofluorescence signal (see Figure 2, mock-treated cultures), whereas no fluorescence was produced in the bax -/- or isotype controls (not shown), indicating that staining was specific and that the N-terminal epitope of at least a fraction of the Bax molecules was exposed in untreated HCT116 cells after standard fixation.

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