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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

The generation of ROS/RNS following regorafenib and sildenafil treatment is a key mediator of tumor cell killing. (A) HEPG2 cells in 96-well plates were loaded for 30 min with either dihydro-DCF (10 μM) which is sensitive to oxidation by hydroxyl radicals and peroxynitrite directly and hydrogen peroxide (i.e., reactive oxygen species, ROS); or 3-amino,4-aminomethyl-2′,7′-difluorescein (DAF-FM DA, 4 μM) which is sensitive to oxidation by NO (i.e., reactive nitrogen species, RNS). Cells were treated with vehicle (DMSO), regorafenib (1 μM), sildenafil (2 μM); or the drugs in combination. Cells—ROS/RNS measurements—were made in a Vector 3 plate reader at the indicated times after drug treatment (n = 3, ±SEM). *P 0.05 < greater than vehicle control. (B) Left portion of the graph: HEPG2 cells were pre-treated with vehicle, the NOS inhibitor L-NAME (1 μM) or the ROS quenching agent N-acetyl cysteine (10 mM). Cells were then treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). Right portion of the graph: HEPG2 cells were transfected with either an empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty-four hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in VEH/CMV cells; ##P < 0.05 less than corresponding value in NAC cells. (C) HEPG2 cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of eNOS or iNOS. Thirty six hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR cells. (D) HEPG2 cells were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM) in combination. Cells were isolated at the indicated time points and western immunoblotting performed to determine the expression of iNOS.
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Figure 4: The generation of ROS/RNS following regorafenib and sildenafil treatment is a key mediator of tumor cell killing. (A) HEPG2 cells in 96-well plates were loaded for 30 min with either dihydro-DCF (10 μM) which is sensitive to oxidation by hydroxyl radicals and peroxynitrite directly and hydrogen peroxide (i.e., reactive oxygen species, ROS); or 3-amino,4-aminomethyl-2′,7′-difluorescein (DAF-FM DA, 4 μM) which is sensitive to oxidation by NO (i.e., reactive nitrogen species, RNS). Cells were treated with vehicle (DMSO), regorafenib (1 μM), sildenafil (2 μM); or the drugs in combination. Cells—ROS/RNS measurements—were made in a Vector 3 plate reader at the indicated times after drug treatment (n = 3, ±SEM). *P 0.05 < greater than vehicle control. (B) Left portion of the graph: HEPG2 cells were pre-treated with vehicle, the NOS inhibitor L-NAME (1 μM) or the ROS quenching agent N-acetyl cysteine (10 mM). Cells were then treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). Right portion of the graph: HEPG2 cells were transfected with either an empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty-four hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in VEH/CMV cells; ##P < 0.05 less than corresponding value in NAC cells. (C) HEPG2 cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of eNOS or iNOS. Thirty six hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR cells. (D) HEPG2 cells were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM) in combination. Cells were isolated at the indicated time points and western immunoblotting performed to determine the expression of iNOS.

Mentions: PDE5 inhibitors are known to enhance the levels of reactive oxygen and reactive nitrogen species in cells (e.g., Das et al., 2010; Musicki et al., 2014). Treatment of tumor cells with sildenafil and regorafenib rapidly increased the levels of ROS and RNS (Fig. 4A). Inhibition of nitric oxide synthase enzymes using L-NG-Nitroarginine Methyl Ester (L-NAME) inhibited cell killing by sildenafil and sorafenib, as did quenching of ROS production by incubation with N-acetyl cysteine (NAC) or by expression of thioredoxin (TRX) (Fig. 4B). Knock down of inducible nitric oxide synthase (iNOS) or endothelial nitric oxide synthase (eNOS) expression suppressed killing by the drug combination (Fig. 4C). Of note, knock down of iNOS abolished the drug combination interaction but did not alter sorafenib toxicity as a single agent, whereas knock down of eNOS modestly but significantly reduced sorafenib toxicity. Sorafenib and sildenafil were still capable of interacting to kill in cells lacking eNOS expression. The combination of sildenafil and sorafenib surprisingly increased iNOS expression (Fig. 4D).


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)

The generation of ROS/RNS following regorafenib and sildenafil treatment is a key mediator of tumor cell killing. (A) HEPG2 cells in 96-well plates were loaded for 30 min with either dihydro-DCF (10 μM) which is sensitive to oxidation by hydroxyl radicals and peroxynitrite directly and hydrogen peroxide (i.e., reactive oxygen species, ROS); or 3-amino,4-aminomethyl-2′,7′-difluorescein (DAF-FM DA, 4 μM) which is sensitive to oxidation by NO (i.e., reactive nitrogen species, RNS). Cells were treated with vehicle (DMSO), regorafenib (1 μM), sildenafil (2 μM); or the drugs in combination. Cells—ROS/RNS measurements—were made in a Vector 3 plate reader at the indicated times after drug treatment (n = 3, ±SEM). *P 0.05 < greater than vehicle control. (B) Left portion of the graph: HEPG2 cells were pre-treated with vehicle, the NOS inhibitor L-NAME (1 μM) or the ROS quenching agent N-acetyl cysteine (10 mM). Cells were then treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). Right portion of the graph: HEPG2 cells were transfected with either an empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty-four hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in VEH/CMV cells; ##P < 0.05 less than corresponding value in NAC cells. (C) HEPG2 cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of eNOS or iNOS. Thirty six hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR cells. (D) HEPG2 cells were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM) in combination. Cells were isolated at the indicated time points and western immunoblotting performed to determine the expression of iNOS.
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Figure 4: The generation of ROS/RNS following regorafenib and sildenafil treatment is a key mediator of tumor cell killing. (A) HEPG2 cells in 96-well plates were loaded for 30 min with either dihydro-DCF (10 μM) which is sensitive to oxidation by hydroxyl radicals and peroxynitrite directly and hydrogen peroxide (i.e., reactive oxygen species, ROS); or 3-amino,4-aminomethyl-2′,7′-difluorescein (DAF-FM DA, 4 μM) which is sensitive to oxidation by NO (i.e., reactive nitrogen species, RNS). Cells were treated with vehicle (DMSO), regorafenib (1 μM), sildenafil (2 μM); or the drugs in combination. Cells—ROS/RNS measurements—were made in a Vector 3 plate reader at the indicated times after drug treatment (n = 3, ±SEM). *P 0.05 < greater than vehicle control. (B) Left portion of the graph: HEPG2 cells were pre-treated with vehicle, the NOS inhibitor L-NAME (1 μM) or the ROS quenching agent N-acetyl cysteine (10 mM). Cells were then treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). Right portion of the graph: HEPG2 cells were transfected with either an empty vector plasmid (CMV) or a plasmid to express thioredoxin (TRX). Twenty-four hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in VEH/CMV cells; ##P < 0.05 less than corresponding value in NAC cells. (C) HEPG2 cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down expression of eNOS or iNOS. Thirty six hours after transfection cells were treated with vehicle or with sildenafil (2 μM), and sorafenib (2 μM) in combination. Twenty-four hours after treatment cells were isolated and viability determined by trypan blue (n = 3, ±SEM). #P < 0.05 less than corresponding value in siSCR cells. (D) HEPG2 cells were treated with vehicle or with sildenafil (2 μM), and/or sorafenib (2 μM) in combination. Cells were isolated at the indicated time points and western immunoblotting performed to determine the expression of iNOS.
Mentions: PDE5 inhibitors are known to enhance the levels of reactive oxygen and reactive nitrogen species in cells (e.g., Das et al., 2010; Musicki et al., 2014). Treatment of tumor cells with sildenafil and regorafenib rapidly increased the levels of ROS and RNS (Fig. 4A). Inhibition of nitric oxide synthase enzymes using L-NG-Nitroarginine Methyl Ester (L-NAME) inhibited cell killing by sildenafil and sorafenib, as did quenching of ROS production by incubation with N-acetyl cysteine (NAC) or by expression of thioredoxin (TRX) (Fig. 4B). Knock down of inducible nitric oxide synthase (iNOS) or endothelial nitric oxide synthase (eNOS) expression suppressed killing by the drug combination (Fig. 4C). Of note, knock down of iNOS abolished the drug combination interaction but did not alter sorafenib toxicity as a single agent, whereas knock down of eNOS modestly but significantly reduced sorafenib toxicity. Sorafenib and sildenafil were still capable of interacting to kill in cells lacking eNOS expression. The combination of sildenafil and sorafenib surprisingly increased iNOS expression (Fig. 4D).

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