Limits...
The mitochondria-independent cytotoxic effect of nelfinavir on leukemia cells can be enhanced by sorafenib-mediated mcl-1 downregulation and mitochondrial membrane destabilization.

Brüning A, Rahmeh M, Gingelmaier A, Friese K - Mol. Cancer (2010)

Bottom Line: At the same concentration and treatment conditions, less than 10% of aspirated human bone marrow cells showed nelfinavir-induced cell damage.Despite caspase activation, the upregulation of the anti-apoptotic bcl-2 family member protein mcl-1 that resulted from nelfinavir treatment stabilized the mitochondrial membrane potential, resulting in primarily mitochondria-independent cell death.Pharmacological downregulation of mcl-1 expression by treatment with sorafenib (2 microg/ml) significantly enhanced nelfinavir-induced apoptosis even at lower nelfinavir concentrations (5 microg/ml), but did not have additional detrimental effects on non-malignant bone marrow cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ludwig-Maximilians University Munich, Department of Obstetrics/Gynaecology, Molecular Biology Laboratory, Munich, Germany. ansgar.bruening@med.uni-muenchen.de

ABSTRACT

Background: Nelfinavir is an HIV protease inhibitor that has been used for a long period of time to treat HIV-infected individuals. It has recently emerged that nelfinavir could represent a prospective new anti-cancer drug, prompting us to test the effect of nelfinavir on leukemia cells.

Methods: By combining in vitro and ex vivo studies, the effect of nelfinavir on leukemia cells and non-malignant, bone marrow-derived tissue cells was analyzed.

Results: At a concentration of 9 microg/ml, nelfinavir induced death of 90% of HL60, IM9, and Jurkat cells. At the same concentration and treatment conditions, less than 10% of aspirated human bone marrow cells showed nelfinavir-induced cell damage. Nelfinavir-induced death of leukemia cells was accompanied by activation of caspases 3, 7, and 8. Despite caspase activation, the upregulation of the anti-apoptotic bcl-2 family member protein mcl-1 that resulted from nelfinavir treatment stabilized the mitochondrial membrane potential, resulting in primarily mitochondria-independent cell death. Pharmacological downregulation of mcl-1 expression by treatment with sorafenib (2 microg/ml) significantly enhanced nelfinavir-induced apoptosis even at lower nelfinavir concentrations (5 microg/ml), but did not have additional detrimental effects on non-malignant bone marrow cells.

Conclusions: The ability of nelfinavir to induce apoptosis in leukemia cells as a single agent in a mitochondria-independent manner might suggest it could be used as a second or third line of treatment for leukemia patients for whom standard mitochondria-directed treatment strategies have failed. Combination treatment with nelfinavir and sorafenib might further enhance the efficacy of nelfinavir even on chemo-resistant leukemia cells.

Show MeSH

Related in: MedlinePlus

Nelfinavir-induced cell death is independent of mitochondrial membrane depolarization. IM9 cells were treated for 24 h with 8 μg/ml nelfinavir or 500 nM staurosporine and the outer mitochondrial membrane potential was analyzed using the MitoCapture kit (Alexis, Lörrach, Germany) by either FACScan analysis or fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2836985&req=5

Figure 5: Nelfinavir-induced cell death is independent of mitochondrial membrane depolarization. IM9 cells were treated for 24 h with 8 μg/ml nelfinavir or 500 nM staurosporine and the outer mitochondrial membrane potential was analyzed using the MitoCapture kit (Alexis, Lörrach, Germany) by either FACScan analysis or fluorescence microscopy.

Mentions: In standard apoptotic conditions, pro-apoptotic bcl-2 family members such as bak or t-bid insert into the outer mitochondrial membrane and induce pore formation, resulting in the efflux of mitochondrial proteins such as cytochrome c and smac/DIABLO. The efflux of smac into the cytosol can be monitored experimentally by cell fractionation studies. In IM9 cells, the classical apoptosis-inducer staurosporine caused an accumulation of smac in the cytosol, accompanied by downregulation of mcl-1 (Fig. 4C). In contrast, nelfinavir treatment of IM9 cells enhanced mitochondrial mcl-1 expression and had no effect on the cellular distribution of smac (Fig. 4C). These results were confirmed using a fluorescent mitochondria tracker dye that accumulates within intact mitochondria as a red-fluorescent dye or within the cytosol as a monomer that exhibits green fluorescence. Both FACScan and fluorescence analysis showed that the mitochondrial membrane potential of IM9 cells is disrupted by staurosporine but not by nelfinavir treatment (Fig. 5). Even more, the percentage of cells with intact mitochondrial membrane potential appeared to be increased after nelfinavir treatment (Fig. 5). A time-dependent analysis of the expression of pro- and anti-apoptotic proteins in nelfinavir-treated IM9 cells revealed a rather immediate upregulation of mcl-1 after nelfinavir treatment, and a continuous and obviously concomitant increase in caspase and PARP cleavage products (Fig. 4D). At later stages of apoptosis, the 36 kDa mcl-1 cleavage product appeared to be further converted into a 32 kDa cleavage product (Fig. 4D).


The mitochondria-independent cytotoxic effect of nelfinavir on leukemia cells can be enhanced by sorafenib-mediated mcl-1 downregulation and mitochondrial membrane destabilization.

Brüning A, Rahmeh M, Gingelmaier A, Friese K - Mol. Cancer (2010)

Nelfinavir-induced cell death is independent of mitochondrial membrane depolarization. IM9 cells were treated for 24 h with 8 μg/ml nelfinavir or 500 nM staurosporine and the outer mitochondrial membrane potential was analyzed using the MitoCapture kit (Alexis, Lörrach, Germany) by either FACScan analysis or fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836985&req=5

Figure 5: Nelfinavir-induced cell death is independent of mitochondrial membrane depolarization. IM9 cells were treated for 24 h with 8 μg/ml nelfinavir or 500 nM staurosporine and the outer mitochondrial membrane potential was analyzed using the MitoCapture kit (Alexis, Lörrach, Germany) by either FACScan analysis or fluorescence microscopy.
Mentions: In standard apoptotic conditions, pro-apoptotic bcl-2 family members such as bak or t-bid insert into the outer mitochondrial membrane and induce pore formation, resulting in the efflux of mitochondrial proteins such as cytochrome c and smac/DIABLO. The efflux of smac into the cytosol can be monitored experimentally by cell fractionation studies. In IM9 cells, the classical apoptosis-inducer staurosporine caused an accumulation of smac in the cytosol, accompanied by downregulation of mcl-1 (Fig. 4C). In contrast, nelfinavir treatment of IM9 cells enhanced mitochondrial mcl-1 expression and had no effect on the cellular distribution of smac (Fig. 4C). These results were confirmed using a fluorescent mitochondria tracker dye that accumulates within intact mitochondria as a red-fluorescent dye or within the cytosol as a monomer that exhibits green fluorescence. Both FACScan and fluorescence analysis showed that the mitochondrial membrane potential of IM9 cells is disrupted by staurosporine but not by nelfinavir treatment (Fig. 5). Even more, the percentage of cells with intact mitochondrial membrane potential appeared to be increased after nelfinavir treatment (Fig. 5). A time-dependent analysis of the expression of pro- and anti-apoptotic proteins in nelfinavir-treated IM9 cells revealed a rather immediate upregulation of mcl-1 after nelfinavir treatment, and a continuous and obviously concomitant increase in caspase and PARP cleavage products (Fig. 4D). At later stages of apoptosis, the 36 kDa mcl-1 cleavage product appeared to be further converted into a 32 kDa cleavage product (Fig. 4D).

Bottom Line: At the same concentration and treatment conditions, less than 10% of aspirated human bone marrow cells showed nelfinavir-induced cell damage.Despite caspase activation, the upregulation of the anti-apoptotic bcl-2 family member protein mcl-1 that resulted from nelfinavir treatment stabilized the mitochondrial membrane potential, resulting in primarily mitochondria-independent cell death.Pharmacological downregulation of mcl-1 expression by treatment with sorafenib (2 microg/ml) significantly enhanced nelfinavir-induced apoptosis even at lower nelfinavir concentrations (5 microg/ml), but did not have additional detrimental effects on non-malignant bone marrow cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ludwig-Maximilians University Munich, Department of Obstetrics/Gynaecology, Molecular Biology Laboratory, Munich, Germany. ansgar.bruening@med.uni-muenchen.de

ABSTRACT

Background: Nelfinavir is an HIV protease inhibitor that has been used for a long period of time to treat HIV-infected individuals. It has recently emerged that nelfinavir could represent a prospective new anti-cancer drug, prompting us to test the effect of nelfinavir on leukemia cells.

Methods: By combining in vitro and ex vivo studies, the effect of nelfinavir on leukemia cells and non-malignant, bone marrow-derived tissue cells was analyzed.

Results: At a concentration of 9 microg/ml, nelfinavir induced death of 90% of HL60, IM9, and Jurkat cells. At the same concentration and treatment conditions, less than 10% of aspirated human bone marrow cells showed nelfinavir-induced cell damage. Nelfinavir-induced death of leukemia cells was accompanied by activation of caspases 3, 7, and 8. Despite caspase activation, the upregulation of the anti-apoptotic bcl-2 family member protein mcl-1 that resulted from nelfinavir treatment stabilized the mitochondrial membrane potential, resulting in primarily mitochondria-independent cell death. Pharmacological downregulation of mcl-1 expression by treatment with sorafenib (2 microg/ml) significantly enhanced nelfinavir-induced apoptosis even at lower nelfinavir concentrations (5 microg/ml), but did not have additional detrimental effects on non-malignant bone marrow cells.

Conclusions: The ability of nelfinavir to induce apoptosis in leukemia cells as a single agent in a mitochondria-independent manner might suggest it could be used as a second or third line of treatment for leukemia patients for whom standard mitochondria-directed treatment strategies have failed. Combination treatment with nelfinavir and sorafenib might further enhance the efficacy of nelfinavir even on chemo-resistant leukemia cells.

Show MeSH
Related in: MedlinePlus