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Inhibition of the vacuolar ATPase induces Bnip3-dependent death of cancer cells and a reduction in tumor burden and metastasis.

Graham RM, Thompson JW, Webster KA - Oncotarget (2014)

Bottom Line: Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts.Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression.BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects.

View Article: PubMed Central - PubMed

ABSTRACT
The pro-apoptotic protein Bnip3 is induced by hypoxia and is present in the core regions of most solid tumors. Bnip3 induces programmed necrosis by an intrinsic caspase independent mitochondrial pathway. Many tumor cells have evolved pathways to evade Bnip3-mediated death attesting to the physiological relevance of the survival threat imposed by Bnip3. We have reported that acidosis can trigger the Bnip3 death pathway in hypoxic cells therefore we hypothesized that manipulation of intracellular pH by pharmacological inhibition of the vacuolar (v)ATPase proton pump, a significant pH control pathway, may activate Bnip3 and promote death of hypoxic cells within the tumor. Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts. Combined treatment of cells with BafA1 and the ERK1/2 inhibitor U0126 further augmented cell death. Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression. BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects. These results present a novel mechanism to destroy hypoxic tumor cells that may help reverse the resistance of hypoxic tumors to radiation and chemotherapy and perhaps target tumor stem cells.

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Caspase independent cell deathMCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.
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Figure 4: Caspase independent cell deathMCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.

Mentions: We have reported that cell death caused by hypoxia-acidosis is associated with the release of cytochrome c from the mitochondria but not caspase activation [32, 33]. Instead our results suggest that calpains are activated under these conditional and may be the central mediators of cell death. To determine if a similar death pathway is activated by the combination of hypoxia and Baf1A we used subcellular fractionation to measure cytochrome c release from the mitochondria. As shown in Figure 4A cytochrome c was not significantly present in the cytoplasmic fraction of aerobic or hypoxia-neutral cultures but the levels increased markedly in extracts of hypoxic cultures treated with Baf1A. Despite the apparent increased cytoplasmic cytochrome c levels there was no increase in caspase 3 activity (Figure 4B). Similarly Baf1A treatment was not associated with the cleavage of ICAD, a target of activated caspase 3 (Figure 1C). We conclude that caspase-dependent cell death does not contribute to cell loss by exposure to hypoxia-Baf1A. These results are consistent with previous studies by Zhang et al. [36]. To determine whether hypoxia- Baf1A results in calpain activation we measured the cleavage products of the calpain substrate α-fodrin. Calpains cleave α-fodrin into 150 and 145 kDA products. Treatment of hypoxic cultures with Baf1A resulted in the generation of substantial 145 kDa cleavage products by 18 hrs of treatment (Figure 4D).


Inhibition of the vacuolar ATPase induces Bnip3-dependent death of cancer cells and a reduction in tumor burden and metastasis.

Graham RM, Thompson JW, Webster KA - Oncotarget (2014)

Caspase independent cell deathMCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4012732&req=5

Figure 4: Caspase independent cell deathMCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.
Mentions: We have reported that cell death caused by hypoxia-acidosis is associated with the release of cytochrome c from the mitochondria but not caspase activation [32, 33]. Instead our results suggest that calpains are activated under these conditional and may be the central mediators of cell death. To determine if a similar death pathway is activated by the combination of hypoxia and Baf1A we used subcellular fractionation to measure cytochrome c release from the mitochondria. As shown in Figure 4A cytochrome c was not significantly present in the cytoplasmic fraction of aerobic or hypoxia-neutral cultures but the levels increased markedly in extracts of hypoxic cultures treated with Baf1A. Despite the apparent increased cytoplasmic cytochrome c levels there was no increase in caspase 3 activity (Figure 4B). Similarly Baf1A treatment was not associated with the cleavage of ICAD, a target of activated caspase 3 (Figure 1C). We conclude that caspase-dependent cell death does not contribute to cell loss by exposure to hypoxia-Baf1A. These results are consistent with previous studies by Zhang et al. [36]. To determine whether hypoxia- Baf1A results in calpain activation we measured the cleavage products of the calpain substrate α-fodrin. Calpains cleave α-fodrin into 150 and 145 kDA products. Treatment of hypoxic cultures with Baf1A resulted in the generation of substantial 145 kDa cleavage products by 18 hrs of treatment (Figure 4D).

Bottom Line: Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts.Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression.BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects.

View Article: PubMed Central - PubMed

ABSTRACT
The pro-apoptotic protein Bnip3 is induced by hypoxia and is present in the core regions of most solid tumors. Bnip3 induces programmed necrosis by an intrinsic caspase independent mitochondrial pathway. Many tumor cells have evolved pathways to evade Bnip3-mediated death attesting to the physiological relevance of the survival threat imposed by Bnip3. We have reported that acidosis can trigger the Bnip3 death pathway in hypoxic cells therefore we hypothesized that manipulation of intracellular pH by pharmacological inhibition of the vacuolar (v)ATPase proton pump, a significant pH control pathway, may activate Bnip3 and promote death of hypoxic cells within the tumor. Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts. Combined treatment of cells with BafA1 and the ERK1/2 inhibitor U0126 further augmented cell death. Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression. BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects. These results present a novel mechanism to destroy hypoxic tumor cells that may help reverse the resistance of hypoxic tumors to radiation and chemotherapy and perhaps target tumor stem cells.

Show MeSH
Related in: MedlinePlus