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Nexavar/Stivarga and viagra interact to kill tumor cells.

Tavallai M, Hamed HA, Roberts JL, Cruickshanks N, Chuckalovcak J, Poklepovic A, Booth L, Dent P - J. Cell. Physiol. (2015)

Bottom Line: PDE5 and PDGFRα/β were over-expressed in liver tumors compared to normal liver tissue.Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs.The drug combination also reduced mTOR protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia.

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Related in: MedlinePlus

Death receptor-dependent and independent induction of cell death by sorafenib and sildenafil treatment. (A) HuH7 and HEPG2 cells were infected with recombinant adenoviruses to express empty vector (CMV); dominant negative caspase 9, BCL-XL, or c-FLIP-s. Twenty-four hours after infection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3, ±SEM). #P < 0.05 less than corresponding value in CMV infected cells. (B) HEPG2 cells were treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were lysed and prepared for immunoprecipitation of CD95. After immunoprecipitation, immunoblotting was performed to determine the levels of caspase 8 and FADD in the immunoprecipitate. Total levels of CD95 and GAPDH in the lysate are also presented. (C) HEPG2 cells grown on microscope slides were treated with vehicle (DMSO), regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 and 24 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95 and death by DAPI staining. (D) HuH7 cells were transfected with either an empty vector plasmid (CMV); a plasmid to express CD95-GFP or a plasmid to express CD95-GFP-Y232F Y291F. Twenty-four hours after transfection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by using a live/dead viability assay (n = 3, ±SEM). *P < 0.05 greater than corresponding value in CMV transfected cells. (E) HEP3B cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 or FADD. Thirty-six hours after transfection were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM). Twenty-four hours after treatment cells were isolated and viability determine by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR transfected cells. (F) HEP3B cells grown on microscope slides were pre-treated with L-NAME (1 μM) or NAC (10 mM), then treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95.
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Figure 6: Death receptor-dependent and independent induction of cell death by sorafenib and sildenafil treatment. (A) HuH7 and HEPG2 cells were infected with recombinant adenoviruses to express empty vector (CMV); dominant negative caspase 9, BCL-XL, or c-FLIP-s. Twenty-four hours after infection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3, ±SEM). #P < 0.05 less than corresponding value in CMV infected cells. (B) HEPG2 cells were treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were lysed and prepared for immunoprecipitation of CD95. After immunoprecipitation, immunoblotting was performed to determine the levels of caspase 8 and FADD in the immunoprecipitate. Total levels of CD95 and GAPDH in the lysate are also presented. (C) HEPG2 cells grown on microscope slides were treated with vehicle (DMSO), regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 and 24 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95 and death by DAPI staining. (D) HuH7 cells were transfected with either an empty vector plasmid (CMV); a plasmid to express CD95-GFP or a plasmid to express CD95-GFP-Y232F Y291F. Twenty-four hours after transfection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by using a live/dead viability assay (n = 3, ±SEM). *P < 0.05 greater than corresponding value in CMV transfected cells. (E) HEP3B cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 or FADD. Thirty-six hours after transfection were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM). Twenty-four hours after treatment cells were isolated and viability determine by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR transfected cells. (F) HEP3B cells grown on microscope slides were pre-treated with L-NAME (1 μM) or NAC (10 mM), then treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95.

Mentions: In addition to our prior studies linking sorafenib and autophagy, we have also described how sorafenib can interact with agents that generate ROS to facilitate activation of the death receptor CD95 (Park et al., 2008, 2010a). In these studies HuH7 cells, that lack endogenous CD95 expression, were particularly resistant to the tested sorafenib drug combinations. In the present studies i.e., Figure 1, we have found that sorafenib and sildenafil interact to kill both CD95 HuH7 cells and hepatoma cells that express CD95 (HEPG2, HEP3B), though drug-combination killing appears to be more efficient in HEPG2 and HEP3B cells. Thus, we next determined whether inhibition of the extrinsic and/or intrinsic apoptosis pathways could reduce cell killing by sorafenib and sildenafil. In HEPG2 cells that express CD95, expression of BCL-XL and dominant negative caspase 9 that suppress activation of the intrinsic apoptosis pathway protected cells from drug combination toxicity (Fig. 6A). Expression of the caspase 8 inhibitor c-FLIP-s in HEPG2 cells also was protective demonstrating that the extrinsic pathway was playing a role in cell killing. In HuH7 cells that are for CD95, expression of BCL-XL and dominant negative caspase 9 was protective, whereas expression of c-FLIP-s did not alter the cell death response (Fig. 6A).


Nexavar/Stivarga and viagra interact to kill tumor cells.

Tavallai M, Hamed HA, Roberts JL, Cruickshanks N, Chuckalovcak J, Poklepovic A, Booth L, Dent P - J. Cell. Physiol. (2015)

Death receptor-dependent and independent induction of cell death by sorafenib and sildenafil treatment. (A) HuH7 and HEPG2 cells were infected with recombinant adenoviruses to express empty vector (CMV); dominant negative caspase 9, BCL-XL, or c-FLIP-s. Twenty-four hours after infection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3, ±SEM). #P < 0.05 less than corresponding value in CMV infected cells. (B) HEPG2 cells were treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were lysed and prepared for immunoprecipitation of CD95. After immunoprecipitation, immunoblotting was performed to determine the levels of caspase 8 and FADD in the immunoprecipitate. Total levels of CD95 and GAPDH in the lysate are also presented. (C) HEPG2 cells grown on microscope slides were treated with vehicle (DMSO), regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 and 24 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95 and death by DAPI staining. (D) HuH7 cells were transfected with either an empty vector plasmid (CMV); a plasmid to express CD95-GFP or a plasmid to express CD95-GFP-Y232F Y291F. Twenty-four hours after transfection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by using a live/dead viability assay (n = 3, ±SEM). *P < 0.05 greater than corresponding value in CMV transfected cells. (E) HEP3B cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 or FADD. Thirty-six hours after transfection were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM). Twenty-four hours after treatment cells were isolated and viability determine by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR transfected cells. (F) HEP3B cells grown on microscope slides were pre-treated with L-NAME (1 μM) or NAC (10 mM), then treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95.
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Figure 6: Death receptor-dependent and independent induction of cell death by sorafenib and sildenafil treatment. (A) HuH7 and HEPG2 cells were infected with recombinant adenoviruses to express empty vector (CMV); dominant negative caspase 9, BCL-XL, or c-FLIP-s. Twenty-four hours after infection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by trypan blue exclusion assay (n = 3, ±SEM). #P < 0.05 less than corresponding value in CMV infected cells. (B) HEPG2 cells were treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were lysed and prepared for immunoprecipitation of CD95. After immunoprecipitation, immunoblotting was performed to determine the levels of caspase 8 and FADD in the immunoprecipitate. Total levels of CD95 and GAPDH in the lysate are also presented. (C) HEPG2 cells grown on microscope slides were treated with vehicle (DMSO), regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 and 24 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95 and death by DAPI staining. (D) HuH7 cells were transfected with either an empty vector plasmid (CMV); a plasmid to express CD95-GFP or a plasmid to express CD95-GFP-Y232F Y291F. Twenty-four hours after transfection cells were then treated with sorafenib (SOR 2.0 μM) and/or sildenafil (SIL, 2.0 μM). Twenty-four hours after drug treatment cells were isolated and viability determined by using a live/dead viability assay (n = 3, ±SEM). *P < 0.05 greater than corresponding value in CMV transfected cells. (E) HEP3B cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 or FADD. Thirty-six hours after transfection were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM). Twenty-four hours after treatment cells were isolated and viability determine by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR transfected cells. (F) HEP3B cells grown on microscope slides were pre-treated with L-NAME (1 μM) or NAC (10 mM), then treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM), or the drugs in combination. Cells were fixed (but not permeabilized) 6 h after drug exposure. IHC was performed to determine the plasma membrane levels of CD95.
Mentions: In addition to our prior studies linking sorafenib and autophagy, we have also described how sorafenib can interact with agents that generate ROS to facilitate activation of the death receptor CD95 (Park et al., 2008, 2010a). In these studies HuH7 cells, that lack endogenous CD95 expression, were particularly resistant to the tested sorafenib drug combinations. In the present studies i.e., Figure 1, we have found that sorafenib and sildenafil interact to kill both CD95 HuH7 cells and hepatoma cells that express CD95 (HEPG2, HEP3B), though drug-combination killing appears to be more efficient in HEPG2 and HEP3B cells. Thus, we next determined whether inhibition of the extrinsic and/or intrinsic apoptosis pathways could reduce cell killing by sorafenib and sildenafil. In HEPG2 cells that express CD95, expression of BCL-XL and dominant negative caspase 9 that suppress activation of the intrinsic apoptosis pathway protected cells from drug combination toxicity (Fig. 6A). Expression of the caspase 8 inhibitor c-FLIP-s in HEPG2 cells also was protective demonstrating that the extrinsic pathway was playing a role in cell killing. In HuH7 cells that are for CD95, expression of BCL-XL and dominant negative caspase 9 was protective, whereas expression of c-FLIP-s did not alter the cell death response (Fig. 6A).

Bottom Line: PDE5 and PDGFRα/β were over-expressed in liver tumors compared to normal liver tissue.Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs.The drug combination also reduced mTOR protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia.

Show MeSH
Related in: MedlinePlus