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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

Regorafenib and sildenafil interact to increase ceramide levels. (A and B) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). *P < 0.05 greater than corresponding value in VEH cells; #P < 0.05 less than corresponding value in VEH treatment; % P < 0.05 greater than value in regorafenib alone treatment. Part A, upper inset part: Treatment of cells with sildenafil (2 μM) for 3 h increases nitro-tyrosine (Y) levels in ceramide synthase 6 (LASS6). (C) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or Fumonisin B1 (FB1, 25.0 μM) or FTY720 (50 nM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). (D and E) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or FTY720 (50 nM). Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (F and G) Tumor cells were treated with vehicle, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], the HDAC inhibitors vorinostat (500 nM), AR-42 (250 nM), Valproate (0.1 mM), or regorafenib + sildenafil + HDAC inhibitor. Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (H) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or ATRA (150 nM). Cells were examined 18 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (I) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) followed 30 min later by exposure to ionizing radiation. Cells were examined 12 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (J) Upper blots: Tumor cells were treated with 50 nM FTY720 for the indicated times and cell lysates western blotted to determine expression of the death receptor CD95 (n = 3); Lower IHC: Tumor cells in situ in a 96-well plate were treated with FTY720 (50 nM) for the indicated times, and cells fixed and probed for expression of FAS ligand (FAS-L) (n = 3). (K) HEPG2 cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Left: Cells were isolated after 30 min and immuno-precipitation of IκB was performed in duplicate. On separate blots assessment of IκB nitro-tyrosine and total IκB was performed. Center: Cells were isolated after 6 h and the expression of FAS-L and CD95 determined. Right: HEPG2 cells were transfected with empty vector plasmid or plasmid to express dominant negative IκB S32A S36A. Twenty-four hours after transfection cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Cells were isolated after 6 h and the expression of CD95 determined.
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Figure 8: Regorafenib and sildenafil interact to increase ceramide levels. (A and B) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). *P < 0.05 greater than corresponding value in VEH cells; #P < 0.05 less than corresponding value in VEH treatment; % P < 0.05 greater than value in regorafenib alone treatment. Part A, upper inset part: Treatment of cells with sildenafil (2 μM) for 3 h increases nitro-tyrosine (Y) levels in ceramide synthase 6 (LASS6). (C) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or Fumonisin B1 (FB1, 25.0 μM) or FTY720 (50 nM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). (D and E) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or FTY720 (50 nM). Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (F and G) Tumor cells were treated with vehicle, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], the HDAC inhibitors vorinostat (500 nM), AR-42 (250 nM), Valproate (0.1 mM), or regorafenib + sildenafil + HDAC inhibitor. Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (H) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or ATRA (150 nM). Cells were examined 18 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (I) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) followed 30 min later by exposure to ionizing radiation. Cells were examined 12 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (J) Upper blots: Tumor cells were treated with 50 nM FTY720 for the indicated times and cell lysates western blotted to determine expression of the death receptor CD95 (n = 3); Lower IHC: Tumor cells in situ in a 96-well plate were treated with FTY720 (50 nM) for the indicated times, and cells fixed and probed for expression of FAS ligand (FAS-L) (n = 3). (K) HEPG2 cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Left: Cells were isolated after 30 min and immuno-precipitation of IκB was performed in duplicate. On separate blots assessment of IκB nitro-tyrosine and total IκB was performed. Center: Cells were isolated after 6 h and the expression of FAS-L and CD95 determined. Right: HEPG2 cells were transfected with empty vector plasmid or plasmid to express dominant negative IκB S32A S36A. Twenty-four hours after transfection cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Cells were isolated after 6 h and the expression of CD95 determined.

Mentions: Prior reports from our group have shown that sorafenib can increase the levels of ceramide in hepatoma cells (Park et al., 2010a,b). Regorafenib and sildenafil treatment increased the levels of multiple ceramides and dihydro-ceramides (Fig. 8A and B). Regorafenib, and sorafenib, both increased the levels of sphingosine-1-phosphate and di-hydro sphingosine-1-phosphate (Fig. 8C, data not shown). The approved multiple sclerosis drug FTY720 (Gilenya, Fingolimod) inhibits the production of sphingosine-1-phosphate as an active site inhibitor of sphingosine kinases 1 and 2, and when phosphorylated by these kinases also down-regulates the expression of the S1P receptor 1 (EDG-1, see Fig. 2). Treatment of cells with low clinically relevant concentrations of FTY720 suppressed the ability of regorafenib to increase sphingosine-1-phosphate levels, and also increased the production of ceramide species by regorafenib (Fig. 8C). Furthermore, at early time points, prior to evident toxicity caused by (regorafenib + sildenafil), or by FTY720 treatment as a single agent, the combination of (regorafenib + sildenafil + FTY720) caused tumor cell killing (Fig. 8D and E). Similar data to that generated using FTY720 were obtained using histone deacetylase inhibitors; the well-established chemotherapy drug all-trans retinoic acid (ATRA); and ionizing radiation (Fig. 8F–I). The effect of vorinostat is in concordance with our pre-clinical and clinical findings that some hepatocellular tumors are effectively inhibited in their growth and radiosensitized by sorafenib and vorinostat treatment (see our on-going Phase I trial NCT01075113, Dent and Poklepovic, unpublished observations).


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)

Regorafenib and sildenafil interact to increase ceramide levels. (A and B) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). *P < 0.05 greater than corresponding value in VEH cells; #P < 0.05 less than corresponding value in VEH treatment; % P < 0.05 greater than value in regorafenib alone treatment. Part A, upper inset part: Treatment of cells with sildenafil (2 μM) for 3 h increases nitro-tyrosine (Y) levels in ceramide synthase 6 (LASS6). (C) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or Fumonisin B1 (FB1, 25.0 μM) or FTY720 (50 nM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). (D and E) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or FTY720 (50 nM). Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (F and G) Tumor cells were treated with vehicle, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], the HDAC inhibitors vorinostat (500 nM), AR-42 (250 nM), Valproate (0.1 mM), or regorafenib + sildenafil + HDAC inhibitor. Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (H) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or ATRA (150 nM). Cells were examined 18 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (I) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) followed 30 min later by exposure to ionizing radiation. Cells were examined 12 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (J) Upper blots: Tumor cells were treated with 50 nM FTY720 for the indicated times and cell lysates western blotted to determine expression of the death receptor CD95 (n = 3); Lower IHC: Tumor cells in situ in a 96-well plate were treated with FTY720 (50 nM) for the indicated times, and cells fixed and probed for expression of FAS ligand (FAS-L) (n = 3). (K) HEPG2 cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Left: Cells were isolated after 30 min and immuno-precipitation of IκB was performed in duplicate. On separate blots assessment of IκB nitro-tyrosine and total IκB was performed. Center: Cells were isolated after 6 h and the expression of FAS-L and CD95 determined. Right: HEPG2 cells were transfected with empty vector plasmid or plasmid to express dominant negative IκB S32A S36A. Twenty-four hours after transfection cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Cells were isolated after 6 h and the expression of CD95 determined.
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Figure 8: Regorafenib and sildenafil interact to increase ceramide levels. (A and B) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). *P < 0.05 greater than corresponding value in VEH cells; #P < 0.05 less than corresponding value in VEH treatment; % P < 0.05 greater than value in regorafenib alone treatment. Part A, upper inset part: Treatment of cells with sildenafil (2 μM) for 3 h increases nitro-tyrosine (Y) levels in ceramide synthase 6 (LASS6). (C) HuH7 cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or Fumonisin B1 (FB1, 25.0 μM) or FTY720 (50 nM). Six hours after treatment cells were isolated and bioactive lipids extracted. Multiple bioactive lipid species were analyzed using GC/MS techniques (n = 2 in triplicate ±SEM). (D and E) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or FTY720 (50 nM). Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (F and G) Tumor cells were treated with vehicle, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], the HDAC inhibitors vorinostat (500 nM), AR-42 (250 nM), Valproate (0.1 mM), or regorafenib + sildenafil + HDAC inhibitor. Cells were examined 9 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (H) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) and/or ATRA (150 nM). Cells were examined 18 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (I) Tumor cells were treated with vehicle control or with regorafenib (REGO 0.5 μM) and/or sildenafil (SIL, 2.0 μM) followed 30 min later by exposure to ionizing radiation. Cells were examined 12 h after treatment using a live/dead assay in a Hermes WiScan instrument (n = 3, ±SEM). (J) Upper blots: Tumor cells were treated with 50 nM FTY720 for the indicated times and cell lysates western blotted to determine expression of the death receptor CD95 (n = 3); Lower IHC: Tumor cells in situ in a 96-well plate were treated with FTY720 (50 nM) for the indicated times, and cells fixed and probed for expression of FAS ligand (FAS-L) (n = 3). (K) HEPG2 cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Left: Cells were isolated after 30 min and immuno-precipitation of IκB was performed in duplicate. On separate blots assessment of IκB nitro-tyrosine and total IκB was performed. Center: Cells were isolated after 6 h and the expression of FAS-L and CD95 determined. Right: HEPG2 cells were transfected with empty vector plasmid or plasmid to express dominant negative IκB S32A S36A. Twenty-four hours after transfection cells were treated with vehicle (DMSO), sorafenib (2 μM), sildenafil (2 μM); or the drugs in combination. Cells were isolated after 6 h and the expression of CD95 determined.
Mentions: Prior reports from our group have shown that sorafenib can increase the levels of ceramide in hepatoma cells (Park et al., 2010a,b). Regorafenib and sildenafil treatment increased the levels of multiple ceramides and dihydro-ceramides (Fig. 8A and B). Regorafenib, and sorafenib, both increased the levels of sphingosine-1-phosphate and di-hydro sphingosine-1-phosphate (Fig. 8C, data not shown). The approved multiple sclerosis drug FTY720 (Gilenya, Fingolimod) inhibits the production of sphingosine-1-phosphate as an active site inhibitor of sphingosine kinases 1 and 2, and when phosphorylated by these kinases also down-regulates the expression of the S1P receptor 1 (EDG-1, see Fig. 2). Treatment of cells with low clinically relevant concentrations of FTY720 suppressed the ability of regorafenib to increase sphingosine-1-phosphate levels, and also increased the production of ceramide species by regorafenib (Fig. 8C). Furthermore, at early time points, prior to evident toxicity caused by (regorafenib + sildenafil), or by FTY720 treatment as a single agent, the combination of (regorafenib + sildenafil + FTY720) caused tumor cell killing (Fig. 8D and E). Similar data to that generated using FTY720 were obtained using histone deacetylase inhibitors; the well-established chemotherapy drug all-trans retinoic acid (ATRA); and ionizing radiation (Fig. 8F–I). The effect of vorinostat is in concordance with our pre-clinical and clinical findings that some hepatocellular tumors are effectively inhibited in their growth and radiosensitized by sorafenib and vorinostat treatment (see our on-going Phase I trial NCT01075113, Dent and Poklepovic, unpublished observations).

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