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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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Identification of tumor cell biomarkers that respond to regorafenib and sildenafil treatment. (A–D) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239); eIF2α (S51); and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (E and F) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239), eIF2α (S51), and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (H) Tumor cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATF4 or CHOP (siATF4, siCHOP); or tumor cells were transfected with plasmids to express dominant negative PERK or eIF2α S51A. Thirty-six hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (I) Cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 6 h. Cells were then fixed and the expression levels of HSP70 + HSC70, GRP78, GRP94, and GRP58 determined. (J) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 3 h. HSP90 was immuno-precipitated and the levels of total and nitrosylated HSP90 in the precipitates determined. (K) Cells were transfected with empty vector CMV or with a plasmid to express GRP78. Twenty-four hours later cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] for a further 24 h. Cells were isolated and viability determine by a live/dead assay (n = 3, ±SEM). #P < 0.05 lower than corresponding value in CMV transfected cells. (L) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (M and N) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (O) Cells were transfected with empty vector plasmid or a plasmid to express GRP78. Twenty-four hours after transfection cells were treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors and plasma membrane pumps. (P) Cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] and crizotinib (1.5 μM), as indicated. Cell death was assessed 12 h later using live/dead assays.
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Figure 3: Identification of tumor cell biomarkers that respond to regorafenib and sildenafil treatment. (A–D) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239); eIF2α (S51); and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (E and F) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239), eIF2α (S51), and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (H) Tumor cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATF4 or CHOP (siATF4, siCHOP); or tumor cells were transfected with plasmids to express dominant negative PERK or eIF2α S51A. Thirty-six hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (I) Cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 6 h. Cells were then fixed and the expression levels of HSP70 + HSC70, GRP78, GRP94, and GRP58 determined. (J) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 3 h. HSP90 was immuno-precipitated and the levels of total and nitrosylated HSP90 in the precipitates determined. (K) Cells were transfected with empty vector CMV or with a plasmid to express GRP78. Twenty-four hours later cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] for a further 24 h. Cells were isolated and viability determine by a live/dead assay (n = 3, ±SEM). #P < 0.05 lower than corresponding value in CMV transfected cells. (L) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (M and N) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (O) Cells were transfected with empty vector plasmid or a plasmid to express GRP78. Twenty-four hours after transfection cells were treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors and plasma membrane pumps. (P) Cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] and crizotinib (1.5 μM), as indicated. Cell death was assessed 12 h later using live/dead assays.

Mentions: We next attempted to define whether biomarkers of regorafenib and sildenafil i.e., physiologic effects, could be observed in tumor cells. Sildenafil treatment increased phosphorylation of VASP-1 (S239), which is a known cGMP dependent kinase (PKG) site (Fig. 3A–E). Identical data were obtained when cells were treated with dibutyrl-cGMP (data not shown). Prior studies using sorafenib had shown that the drug could cause a modest endoplasmic reticulum stress response, with increased phosphorylation of eIF2α (S51) (Park et al., 2008). Regorafenib treatment increased eIF2α (S51) phosphorylation (Fig. 3A–E). Studies in future figures make use of the drug FTY720 (Gilenya, Fingolimod) which is a histone deacetylase inhibitor; an inhibitor of sphingosine-1-phosphate production and also down-regulates expression of sphingosine-1-phosphate receptors, e.g., EDG-1. Treatment of tumor cells for 6 h with FTY720 abolished expression of EDG-1 (Fig. 3F). Of additional note, combined treatment of cells with regorafenib and sildenafil also abolished EDG-1 expression within 6 h. Expression of a dominant negative eIF2α (S51A) protein abolished the ability of (regorafenib + sildenafil) treatment to reduce EDG-1 expression within 6 h (Fig. 3G). Inhibition of the PERK-eIF2α-ATF4-CHOP pathway protected cells from combination treatment lethality (Fig. 3H).


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)

Identification of tumor cell biomarkers that respond to regorafenib and sildenafil treatment. (A–D) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239); eIF2α (S51); and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (E and F) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239), eIF2α (S51), and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (H) Tumor cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATF4 or CHOP (siATF4, siCHOP); or tumor cells were transfected with plasmids to express dominant negative PERK or eIF2α S51A. Thirty-six hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (I) Cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 6 h. Cells were then fixed and the expression levels of HSP70 + HSC70, GRP78, GRP94, and GRP58 determined. (J) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 3 h. HSP90 was immuno-precipitated and the levels of total and nitrosylated HSP90 in the precipitates determined. (K) Cells were transfected with empty vector CMV or with a plasmid to express GRP78. Twenty-four hours later cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] for a further 24 h. Cells were isolated and viability determine by a live/dead assay (n = 3, ±SEM). #P < 0.05 lower than corresponding value in CMV transfected cells. (L) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (M and N) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (O) Cells were transfected with empty vector plasmid or a plasmid to express GRP78. Twenty-four hours after transfection cells were treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors and plasma membrane pumps. (P) Cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] and crizotinib (1.5 μM), as indicated. Cell death was assessed 12 h later using live/dead assays.
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Figure 3: Identification of tumor cell biomarkers that respond to regorafenib and sildenafil treatment. (A–D) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239); eIF2α (S51); and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (E and F) Cells were grown in 96-well plates and treated for the indicated amounts of time with regorafenib (0.5 μM), sildenafil (2 μM), FTY720 (50 nM), and the drugs in combination as indicated in each part. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the phosphorylation of VASP-1 (S239), eIF2α (S51), and the expression of the sphingosine-1-phosphate receptor (EDG-1). (G) HuH7 and HT29 cells were transfected with empty vector plasmid (CMV) or to express dominant negative eIF2α (S51A). Twenty-four hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (H) Tumor cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATF4 or CHOP (siATF4, siCHOP); or tumor cells were transfected with plasmids to express dominant negative PERK or eIF2α S51A. Thirty-six hours after transfection cells were treated with regorafenib (0.5 μM) and sildenafil (2 μM) for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the expression of the sphingosine-1-phosphate receptor (EDG-1). (I) Cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 6 h. Cells were then fixed and the expression levels of HSP70 + HSC70, GRP78, GRP94, and GRP58 determined. (J) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), and sildenafil (2.0 μM) for 3 h. HSP90 was immuno-precipitated and the levels of total and nitrosylated HSP90 in the precipitates determined. (K) Cells were transfected with empty vector CMV or with a plasmid to express GRP78. Twenty-four hours later cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] for a further 24 h. Cells were isolated and viability determine by a live/dead assay (n = 3, ±SEM). #P < 0.05 lower than corresponding value in CMV transfected cells. (L) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (M and N) Tumor cells as indicated in each part were treated with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors. (O) Cells were transfected with empty vector plasmid or a plasmid to express GRP78. Twenty-four hours after transfection cells were treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2 μM) together for 6 h. Cells were gently fixed in situ using 2% (v/v) para-formaldehyde with Triton × 100 for permeabilization and probed for the total expression of each of the indicated growth factor receptors and plasma membrane pumps. (P) Cells were treated with vehicle or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] and crizotinib (1.5 μM), as indicated. Cell death was assessed 12 h later using live/dead assays.
Mentions: We next attempted to define whether biomarkers of regorafenib and sildenafil i.e., physiologic effects, could be observed in tumor cells. Sildenafil treatment increased phosphorylation of VASP-1 (S239), which is a known cGMP dependent kinase (PKG) site (Fig. 3A–E). Identical data were obtained when cells were treated with dibutyrl-cGMP (data not shown). Prior studies using sorafenib had shown that the drug could cause a modest endoplasmic reticulum stress response, with increased phosphorylation of eIF2α (S51) (Park et al., 2008). Regorafenib treatment increased eIF2α (S51) phosphorylation (Fig. 3A–E). Studies in future figures make use of the drug FTY720 (Gilenya, Fingolimod) which is a histone deacetylase inhibitor; an inhibitor of sphingosine-1-phosphate production and also down-regulates expression of sphingosine-1-phosphate receptors, e.g., EDG-1. Treatment of tumor cells for 6 h with FTY720 abolished expression of EDG-1 (Fig. 3F). Of additional note, combined treatment of cells with regorafenib and sildenafil also abolished EDG-1 expression within 6 h. Expression of a dominant negative eIF2α (S51A) protein abolished the ability of (regorafenib + sildenafil) treatment to reduce EDG-1 expression within 6 h (Fig. 3G). Inhibition of the PERK-eIF2α-ATF4-CHOP pathway protected cells from combination treatment lethality (Fig. 3H).

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