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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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Mitochondrial membrane potential changes and caspase activation. Panel A: Quotient of JC-1 red/green fluorescence as an indicator for the membrane potential ΔΨm in HCT116 cells and the bax-/- derivatives at different times after exposure to 100 μM resveratrol. Note that the absence of Bax delays but fails to completely inhibit membrane potential collapse. Standard deviations were derived from 4 experiments. Panel B: Detection of cleaved (active) caspases (casp) 9 and 3 in total protein extracts from parental and bax-deficient cells exposed to resveratrol. The anti-caspase antibodies were used at a dilution of 1:200. Staining with anti-β-actin is to show equal protein loading.
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Figure 3: Mitochondrial membrane potential changes and caspase activation. Panel A: Quotient of JC-1 red/green fluorescence as an indicator for the membrane potential ΔΨm in HCT116 cells and the bax-/- derivatives at different times after exposure to 100 μM resveratrol. Note that the absence of Bax delays but fails to completely inhibit membrane potential collapse. Standard deviations were derived from 4 experiments. Panel B: Detection of cleaved (active) caspases (casp) 9 and 3 in total protein extracts from parental and bax-deficient cells exposed to resveratrol. The anti-caspase antibodies were used at a dilution of 1:200. Staining with anti-β-actin is to show equal protein loading.

Mentions: Active Bax located at the mitochondria can mediate apoptosis [26], and we have recently reported that resveratrol can indeed trigger the mitochondrion form of death in HCT116 cells [14]. Mitochondria-mediated apoptosis is often, though not always, associated with the collapse of the mitochondrial transmembrane potential ΔΨm as the result of leakiness of the inner mitochondrial membrane [26]. To study whether the translocation of Bax correlates with ΔΨm changes, exponentially growing cultures of bax-positive and bax-negative cells were treated with 100 μM resveratrol or mock-treated for various times and were then incubated with the J aggregate-forming lipophilic cation JC-1, which as a monomer emits green fluorescence and in a reaction driven by ΔΨm assembles into a red fluorescence-emitting dimer. In accord with the sensitivity of HCT116 cells to resveratrol-induced apoptosis, and paralleling the co-localization of Bax to the mitochondria, ΔΨm rapidly dissipated, as indicated by the incremental increase in JC-1 green fluorescence and the lack of a corresponding increase in red fluoresence (Figure 3A). Remarkably, although stable for the first eight hours of drug treatment, the membrane potential of the bax -/- cells eventually also collapsed, indicating that a Bax-independent, mitochondria-mediated death with delayed kinetics can be triggered in HCT116 cells by resveratrol. To study this further, Western blot analyses with antibodies directed against caspases 3 and 9 were performed. Mitochondria-mediated apoptosis involves the cleavage of pro-caspase 9 into the 36 kDa active caspase 9, which then cleaves and thereby activates pro-caspase 3. Resveratrol treatment (100 μM) resulted in the increase of active caspase 9 levels, and concomitantly, in the cleavage of pro-caspase 3, regardless of Bax-status (Figure 3B), supporting the suggestion that resvertrol can provoke both Bax-dependent and -independent, mitochondria- and caspase-mediated forms of apoptosis in HCT116 cells. As reported before [14], we failed to establish a role of caspase 8 in the resveratrol-provoked cell death, as neither caspase 8 activation in Western blot analyses nor inhibitory effects of the caspase 8 inhibitor IETH-CHO could be observed.


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

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

Mitochondrial membrane potential changes and caspase activation. Panel A: Quotient of JC-1 red/green fluorescence as an indicator for the membrane potential ΔΨm in HCT116 cells and the bax-/- derivatives at different times after exposure to 100 μM resveratrol. Note that the absence of Bax delays but fails to completely inhibit membrane potential collapse. Standard deviations were derived from 4 experiments. Panel B: Detection of cleaved (active) caspases (casp) 9 and 3 in total protein extracts from parental and bax-deficient cells exposed to resveratrol. The anti-caspase antibodies were used at a dilution of 1:200. Staining with anti-β-actin is to show equal protein loading.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC130964&req=5

Figure 3: Mitochondrial membrane potential changes and caspase activation. Panel A: Quotient of JC-1 red/green fluorescence as an indicator for the membrane potential ΔΨm in HCT116 cells and the bax-/- derivatives at different times after exposure to 100 μM resveratrol. Note that the absence of Bax delays but fails to completely inhibit membrane potential collapse. Standard deviations were derived from 4 experiments. Panel B: Detection of cleaved (active) caspases (casp) 9 and 3 in total protein extracts from parental and bax-deficient cells exposed to resveratrol. The anti-caspase antibodies were used at a dilution of 1:200. Staining with anti-β-actin is to show equal protein loading.
Mentions: Active Bax located at the mitochondria can mediate apoptosis [26], and we have recently reported that resveratrol can indeed trigger the mitochondrion form of death in HCT116 cells [14]. Mitochondria-mediated apoptosis is often, though not always, associated with the collapse of the mitochondrial transmembrane potential ΔΨm as the result of leakiness of the inner mitochondrial membrane [26]. To study whether the translocation of Bax correlates with ΔΨm changes, exponentially growing cultures of bax-positive and bax-negative cells were treated with 100 μM resveratrol or mock-treated for various times and were then incubated with the J aggregate-forming lipophilic cation JC-1, which as a monomer emits green fluorescence and in a reaction driven by ΔΨm assembles into a red fluorescence-emitting dimer. In accord with the sensitivity of HCT116 cells to resveratrol-induced apoptosis, and paralleling the co-localization of Bax to the mitochondria, ΔΨm rapidly dissipated, as indicated by the incremental increase in JC-1 green fluorescence and the lack of a corresponding increase in red fluoresence (Figure 3A). Remarkably, although stable for the first eight hours of drug treatment, the membrane potential of the bax -/- cells eventually also collapsed, indicating that a Bax-independent, mitochondria-mediated death with delayed kinetics can be triggered in HCT116 cells by resveratrol. To study this further, Western blot analyses with antibodies directed against caspases 3 and 9 were performed. Mitochondria-mediated apoptosis involves the cleavage of pro-caspase 9 into the 36 kDa active caspase 9, which then cleaves and thereby activates pro-caspase 3. Resveratrol treatment (100 μM) resulted in the increase of active caspase 9 levels, and concomitantly, in the cleavage of pro-caspase 3, regardless of Bax-status (Figure 3B), supporting the suggestion that resvertrol can provoke both Bax-dependent and -independent, mitochondria- and caspase-mediated forms of apoptosis in HCT116 cells. As reported before [14], we failed to establish a role of caspase 8 in the resveratrol-provoked cell death, as neither caspase 8 activation in Western blot analyses nor inhibitory effects of the caspase 8 inhibitor IETH-CHO could be observed.

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