Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation of JNK and inhibition of mTOR, ERK1/2, and AKT plays key roles in regorafenib and sildenafil toxicity. (A) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 3 h. Cells were isolated and the phosphorylation/expression of the indicated proteins determined by western immunoblotting. (B and C) HuH7 and HT29 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point cells were fixed in place and probed for the expression and phosphorylation of MEK1, AKT, ERK1/2, and JNK1/2 and images obtained using a Hermes WiScan system. (D) Pictorial data shown in part C was analyzed and quantified using software provided with the WiScan system. The intensity of immuno-staining fluorescence using each Phospho-Antibody and in parallel total expression antibody was determined and the arbitrary “specific activity” of each kinase determine in multiple wells from multiple experiments (n = 3, ±SEM). #P < 0.05 less than vehicle control value; ##P < 0.05 less than regorafenib value; *P < 0.05 greater than regorafenib value. (E) HuH7 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point, cells were fixed in place and probed for the expression and phosphorylation of mTOR/mTOR (S2448) and images obtained using a Hermes WiScan system. *P < 0.05 expression/phosphorylation of mTOR lower than corresponding vehicle control. (F) HuH7 and HEPG2 cells were transfected with plasmids: CMV (empty vector); to express activated MEK1, caMEK1; to express activated AKT, caAKT; or to express activated mTOR, ca-mTOR. Twenty-four hours after transfection a portion of vehicle control cells were treated with the JNK inhibitory peptide (JNK-IP, 10 μM). Cells were then treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2.0 μM) and cell viability determined after 24 h using a live/dead assay using a Hermes WiScan instrument (n = 3, ±SEM). *P < 0.05 greater than vehicle control; #P < 0.05 lower than corresponding value in CMV transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835179&req=5

Figure 7: Activation of JNK and inhibition of mTOR, ERK1/2, and AKT plays key roles in regorafenib and sildenafil toxicity. (A) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 3 h. Cells were isolated and the phosphorylation/expression of the indicated proteins determined by western immunoblotting. (B and C) HuH7 and HT29 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point cells were fixed in place and probed for the expression and phosphorylation of MEK1, AKT, ERK1/2, and JNK1/2 and images obtained using a Hermes WiScan system. (D) Pictorial data shown in part C was analyzed and quantified using software provided with the WiScan system. The intensity of immuno-staining fluorescence using each Phospho-Antibody and in parallel total expression antibody was determined and the arbitrary “specific activity” of each kinase determine in multiple wells from multiple experiments (n = 3, ±SEM). #P < 0.05 less than vehicle control value; ##P < 0.05 less than regorafenib value; *P < 0.05 greater than regorafenib value. (E) HuH7 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point, cells were fixed in place and probed for the expression and phosphorylation of mTOR/mTOR (S2448) and images obtained using a Hermes WiScan system. *P < 0.05 expression/phosphorylation of mTOR lower than corresponding vehicle control. (F) HuH7 and HEPG2 cells were transfected with plasmids: CMV (empty vector); to express activated MEK1, caMEK1; to express activated AKT, caAKT; or to express activated mTOR, ca-mTOR. Twenty-four hours after transfection a portion of vehicle control cells were treated with the JNK inhibitory peptide (JNK-IP, 10 μM). Cells were then treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2.0 μM) and cell viability determined after 24 h using a live/dead assay using a Hermes WiScan instrument (n = 3, ±SEM). *P < 0.05 greater than vehicle control; #P < 0.05 lower than corresponding value in CMV transfected cells.

Mentions: Treatment of hepatoma cells in vitro increased the phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α (S51) as judged by western blotting (Fig. 7A). Drug combination treatment reduced the phosphorylation of MEK1 (S218/S222), AKT (T308), and of ERK1/2, and enhanced phosphorylation of JNK1/2. No obvious change was noted in MEK1 S298 phosphorylation. Similar data were obtained when cells were fixed in place and probed by immuno-fluorescence for phosphorylation status by immuno-fluorescence (Fig. 7B–D). The phosphorylation of the autophagy regulatory kinase mTOR was significantly reduced by drug combination treatment, which also was associated to a lesser extent with reduced expression of mTOR protein itself (Fig. 7E) i.e., both total protein and the specific activity of the mTOR protein declined. Expression of activated forms of MEK1 or AKT incompletely suppressed killing by regorafenib and sildenafil treatment (Fig. 7F). Inhibition of JNK1/2 signaling or expression of an activated form of mTOR abolished drug combination toxicity.


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)

Activation of JNK and inhibition of mTOR, ERK1/2, and AKT plays key roles in regorafenib and sildenafil toxicity. (A) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 3 h. Cells were isolated and the phosphorylation/expression of the indicated proteins determined by western immunoblotting. (B and C) HuH7 and HT29 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point cells were fixed in place and probed for the expression and phosphorylation of MEK1, AKT, ERK1/2, and JNK1/2 and images obtained using a Hermes WiScan system. (D) Pictorial data shown in part C was analyzed and quantified using software provided with the WiScan system. The intensity of immuno-staining fluorescence using each Phospho-Antibody and in parallel total expression antibody was determined and the arbitrary “specific activity” of each kinase determine in multiple wells from multiple experiments (n = 3, ±SEM). #P < 0.05 less than vehicle control value; ##P < 0.05 less than regorafenib value; *P < 0.05 greater than regorafenib value. (E) HuH7 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point, cells were fixed in place and probed for the expression and phosphorylation of mTOR/mTOR (S2448) and images obtained using a Hermes WiScan system. *P < 0.05 expression/phosphorylation of mTOR lower than corresponding vehicle control. (F) HuH7 and HEPG2 cells were transfected with plasmids: CMV (empty vector); to express activated MEK1, caMEK1; to express activated AKT, caAKT; or to express activated mTOR, ca-mTOR. Twenty-four hours after transfection a portion of vehicle control cells were treated with the JNK inhibitory peptide (JNK-IP, 10 μM). Cells were then treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2.0 μM) and cell viability determined after 24 h using a live/dead assay using a Hermes WiScan instrument (n = 3, ±SEM). *P < 0.05 greater than vehicle control; #P < 0.05 lower than corresponding value in CMV transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835179&req=5

Figure 7: Activation of JNK and inhibition of mTOR, ERK1/2, and AKT plays key roles in regorafenib and sildenafil toxicity. (A) HuH7 cells were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 3 h. Cells were isolated and the phosphorylation/expression of the indicated proteins determined by western immunoblotting. (B and C) HuH7 and HT29 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point cells were fixed in place and probed for the expression and phosphorylation of MEK1, AKT, ERK1/2, and JNK1/2 and images obtained using a Hermes WiScan system. (D) Pictorial data shown in part C was analyzed and quantified using software provided with the WiScan system. The intensity of immuno-staining fluorescence using each Phospho-Antibody and in parallel total expression antibody was determined and the arbitrary “specific activity” of each kinase determine in multiple wells from multiple experiments (n = 3, ±SEM). #P < 0.05 less than vehicle control value; ##P < 0.05 less than regorafenib value; *P < 0.05 greater than regorafenib value. (E) HuH7 cells growing in 96-well plates were treated with vehicle, regorafenib (0.5 μM), sildenafil (2 μM), or the drugs in combination for 6 h or for 12 h. At each time point, cells were fixed in place and probed for the expression and phosphorylation of mTOR/mTOR (S2448) and images obtained using a Hermes WiScan system. *P < 0.05 expression/phosphorylation of mTOR lower than corresponding vehicle control. (F) HuH7 and HEPG2 cells were transfected with plasmids: CMV (empty vector); to express activated MEK1, caMEK1; to express activated AKT, caAKT; or to express activated mTOR, ca-mTOR. Twenty-four hours after transfection a portion of vehicle control cells were treated with the JNK inhibitory peptide (JNK-IP, 10 μM). Cells were then treated with vehicle or with regorafenib (0.5 μM) and sildenafil (2.0 μM) and cell viability determined after 24 h using a live/dead assay using a Hermes WiScan instrument (n = 3, ±SEM). *P < 0.05 greater than vehicle control; #P < 0.05 lower than corresponding value in CMV transfected cells.
Mentions: Treatment of hepatoma cells in vitro increased the phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α (S51) as judged by western blotting (Fig. 7A). Drug combination treatment reduced the phosphorylation of MEK1 (S218/S222), AKT (T308), and of ERK1/2, and enhanced phosphorylation of JNK1/2. No obvious change was noted in MEK1 S298 phosphorylation. Similar data were obtained when cells were fixed in place and probed by immuno-fluorescence for phosphorylation status by immuno-fluorescence (Fig. 7B–D). The phosphorylation of the autophagy regulatory kinase mTOR was significantly reduced by drug combination treatment, which also was associated to a lesser extent with reduced expression of mTOR protein itself (Fig. 7E) i.e., both total protein and the specific activity of the mTOR protein declined. Expression of activated forms of MEK1 or AKT incompletely suppressed killing by regorafenib and sildenafil treatment (Fig. 7F). Inhibition of JNK1/2 signaling or expression of an activated form of mTOR abolished drug combination toxicity.

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