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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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Representative colony formation assay with HCT116 and HCT116 bax -/- cells. When 1.5 × 103 live cells, counted in a CASY cell counter, were seeded into 25 cm2 tissue culture flasks and were incubated for 10 days in the absence of drug, bax -/- cells produced more colonies (left figure), indicating the survival advantage per se of bax-deficiency. Right figure: 7.5 × 103 live HCT116 cells and 1.5 × 103 bax -/- cells were seeded into flasks and exposed to the solvent DMSO (mock) or resveratrol dissolved in DMSO, 24 h later. Colonies were stained with crystal violet.
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Figure 5: Representative colony formation assay with HCT116 and HCT116 bax -/- cells. When 1.5 × 103 live cells, counted in a CASY cell counter, were seeded into 25 cm2 tissue culture flasks and were incubated for 10 days in the absence of drug, bax -/- cells produced more colonies (left figure), indicating the survival advantage per se of bax-deficiency. Right figure: 7.5 × 103 live HCT116 cells and 1.5 × 103 bax -/- cells were seeded into flasks and exposed to the solvent DMSO (mock) or resveratrol dissolved in DMSO, 24 h later. Colonies were stained with crystal violet.

Mentions: To assess whether the difference in FACS analysis in the apoptosis sensitivity between bax +/+ and bax -/- cells is meaningful, standard colony formation assays were performed. In a first set of tests, it was revealed that a short initial exposure of the cultures to 100 μM resveratrol for up to 6 h and removal of the drug thereafter, had no deleterious effect on colony formation (data not shown). This contrasted with the effects of damaging drugs such as adriamycin (0.34 μM) or 5-fluorouracil (375 μM), which inhibited colony formation under these conditions, and suggested that resveratrol acts primarily through the reversible inhibition of cellular factors. In the further study, cultures were therefore exposed to resveratrol for the duration of the assay. When equal numbers of live bax +/+ and bax -/- cells were seeded and mock-treated, bax -/- cells produced more colonies (Figure 5, left figure), consistent with the much lower basal level of cell death in these cultures (see Figure 4). To be able to compare the effect of resveratrol on colony formation in bax +/+ and -/- cultures, we therefore seeded numbers of live cells that gave rise to similar colony numbers and incubated all cultures for ten days in the presence of different concentrations of resveratrol. The drug decreased the numbers of colonies, and at the highest resveratrol concentration (100 μM), no colonies formed, regardless of Bax-status (Figure 5). At lower drug concentration (20 μM) however, more colonies formed consistently in the bax -/- cultures when compared with the bax +/+ cultures (62 +15 vs. 24 +11; average + SD from 10 flasks each), and the colonies were generally larger. We interpret this to reflect the lack of inhibition of colony formation by the Bax-dependent form of apoptosis in the bax -/- cultures. In summary, our data thus show that resveratrol at concentrations present in some foods can induce in HCT116 cells i) Bax-dependent, mitochondria-mediated apoptosis, regardless of p53-status and involving Bax co-localization with mitochondria, and ii) Bax-independent, mitochondria-mediated death. Both forms of apoptosis may independently limit the ability of the cells to produce colonies.


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

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

Representative colony formation assay with HCT116 and HCT116 bax -/- cells. When 1.5 × 103 live cells, counted in a CASY cell counter, were seeded into 25 cm2 tissue culture flasks and were incubated for 10 days in the absence of drug, bax -/- cells produced more colonies (left figure), indicating the survival advantage per se of bax-deficiency. Right figure: 7.5 × 103 live HCT116 cells and 1.5 × 103 bax -/- cells were seeded into flasks and exposed to the solvent DMSO (mock) or resveratrol dissolved in DMSO, 24 h later. Colonies were stained with crystal violet.
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Related In: Results  -  Collection

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Figure 5: Representative colony formation assay with HCT116 and HCT116 bax -/- cells. When 1.5 × 103 live cells, counted in a CASY cell counter, were seeded into 25 cm2 tissue culture flasks and were incubated for 10 days in the absence of drug, bax -/- cells produced more colonies (left figure), indicating the survival advantage per se of bax-deficiency. Right figure: 7.5 × 103 live HCT116 cells and 1.5 × 103 bax -/- cells were seeded into flasks and exposed to the solvent DMSO (mock) or resveratrol dissolved in DMSO, 24 h later. Colonies were stained with crystal violet.
Mentions: To assess whether the difference in FACS analysis in the apoptosis sensitivity between bax +/+ and bax -/- cells is meaningful, standard colony formation assays were performed. In a first set of tests, it was revealed that a short initial exposure of the cultures to 100 μM resveratrol for up to 6 h and removal of the drug thereafter, had no deleterious effect on colony formation (data not shown). This contrasted with the effects of damaging drugs such as adriamycin (0.34 μM) or 5-fluorouracil (375 μM), which inhibited colony formation under these conditions, and suggested that resveratrol acts primarily through the reversible inhibition of cellular factors. In the further study, cultures were therefore exposed to resveratrol for the duration of the assay. When equal numbers of live bax +/+ and bax -/- cells were seeded and mock-treated, bax -/- cells produced more colonies (Figure 5, left figure), consistent with the much lower basal level of cell death in these cultures (see Figure 4). To be able to compare the effect of resveratrol on colony formation in bax +/+ and -/- cultures, we therefore seeded numbers of live cells that gave rise to similar colony numbers and incubated all cultures for ten days in the presence of different concentrations of resveratrol. The drug decreased the numbers of colonies, and at the highest resveratrol concentration (100 μM), no colonies formed, regardless of Bax-status (Figure 5). At lower drug concentration (20 μM) however, more colonies formed consistently in the bax -/- cultures when compared with the bax +/+ cultures (62 +15 vs. 24 +11; average + SD from 10 flasks each), and the colonies were generally larger. We interpret this to reflect the lack of inhibition of colony formation by the Bax-dependent form of apoptosis in the bax -/- cultures. In summary, our data thus show that resveratrol at concentrations present in some foods can induce in HCT116 cells i) Bax-dependent, mitochondria-mediated apoptosis, regardless of p53-status and involving Bax co-localization with mitochondria, and ii) Bax-independent, mitochondria-mediated death. Both forms of apoptosis may independently limit the ability of the cells to produce colonies.

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