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OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

Booth L, Roberts JL, Tavallai M, Nourbakhsh A, Chuckalovcak J, Carter J, Poklepovic A, Dent P - J. Cell. Physiol. (2015)

Bottom Line: Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells.In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response.Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.

View Article: PubMed Central - PubMed

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

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Increased expression of ATF4 after [OSU-03012 + sildenafil] treatment is eIF2α dependent and the drug-induced suppression of EDG-1 expression requires ATF4/CHOP signaling. A,B: GBM cells were transfected with empty vector CMV or to express dominant negative eIF2α. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the levels of ATF4. C–F: GBM cells were transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of ATF4 or CHOP. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the total cellular levels of S1PR1 (EDG-1).
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Figure 6: Increased expression of ATF4 after [OSU-03012 + sildenafil] treatment is eIF2α dependent and the drug-induced suppression of EDG-1 expression requires ATF4/CHOP signaling. A,B: GBM cells were transfected with empty vector CMV or to express dominant negative eIF2α. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the levels of ATF4. C–F: GBM cells were transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of ATF4 or CHOP. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the total cellular levels of S1PR1 (EDG-1).

Mentions: Treatment of GBM cells with OSU + SIL resulted in a large increase in eIF2α phosphorylation compared to individual drug treatments (Fig. 5A,B). The reduction in EDG-1 expression (total and cell surface) caused by OSU + SIL was blocked by expression of a dn eIF2α S51A protein (Fig. 5C–G). Treatment of cells with OSU + SIL increased expression of ATF4 in an eIF2α-dependent fashion (Fig. 6A,B). The reduction in EDG-1 expression caused by OSU + SIL treatment was prevented by knock down of ATF4 or of CHOP (GADD153) (Fig. 6C–F). The OSU + SIL-induced increase in CHOP expression was prevented by expression of dn eIF2α and the down-regulation of EDG-1 levels was blocked by over-expression of GRP78 (Fig. 7A–D). Expression of dn eIF2α also prevented the drug-induced reduction in MCL-1 and BCL-XL expression (Fig. 7E, data not shown). Over-expression of GRP78 prevented the drug-induced increase in eIF2α phosphorylation arguing that the kinase that phosphorylates eIF2α is probably inhibited by GRP78 (Fig. 7F). Over-expression of GRP78 also prevented the drug combination-induced rapid declines in the expression of EDG-1, ERBB1, IL6R, IGF1R, and the PDGFRα/β, as well as drug efflux/blood–brain-barrier ABC transporters, suggesting that the reduction in receptor expression and transporter expression is also a result of reduced GRP78 levels and enhanced ER stress signaling (Fig. 7G,H). Knock down of CHOP expression suppressed CEL + SIL and OSU + SIL lethality (Fig. 7I).


OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

Booth L, Roberts JL, Tavallai M, Nourbakhsh A, Chuckalovcak J, Carter J, Poklepovic A, Dent P - J. Cell. Physiol. (2015)

Increased expression of ATF4 after [OSU-03012 + sildenafil] treatment is eIF2α dependent and the drug-induced suppression of EDG-1 expression requires ATF4/CHOP signaling. A,B: GBM cells were transfected with empty vector CMV or to express dominant negative eIF2α. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the levels of ATF4. C–F: GBM cells were transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of ATF4 or CHOP. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the total cellular levels of S1PR1 (EDG-1).
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Related In: Results  -  Collection

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Figure 6: Increased expression of ATF4 after [OSU-03012 + sildenafil] treatment is eIF2α dependent and the drug-induced suppression of EDG-1 expression requires ATF4/CHOP signaling. A,B: GBM cells were transfected with empty vector CMV or to express dominant negative eIF2α. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the levels of ATF4. C–F: GBM cells were transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of ATF4 or CHOP. Twenty-four hours after transfection, cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h. Cells were fixed and permeabilized and immunofluorescence performed to determine the total cellular levels of S1PR1 (EDG-1).
Mentions: Treatment of GBM cells with OSU + SIL resulted in a large increase in eIF2α phosphorylation compared to individual drug treatments (Fig. 5A,B). The reduction in EDG-1 expression (total and cell surface) caused by OSU + SIL was blocked by expression of a dn eIF2α S51A protein (Fig. 5C–G). Treatment of cells with OSU + SIL increased expression of ATF4 in an eIF2α-dependent fashion (Fig. 6A,B). The reduction in EDG-1 expression caused by OSU + SIL treatment was prevented by knock down of ATF4 or of CHOP (GADD153) (Fig. 6C–F). The OSU + SIL-induced increase in CHOP expression was prevented by expression of dn eIF2α and the down-regulation of EDG-1 levels was blocked by over-expression of GRP78 (Fig. 7A–D). Expression of dn eIF2α also prevented the drug-induced reduction in MCL-1 and BCL-XL expression (Fig. 7E, data not shown). Over-expression of GRP78 prevented the drug-induced increase in eIF2α phosphorylation arguing that the kinase that phosphorylates eIF2α is probably inhibited by GRP78 (Fig. 7F). Over-expression of GRP78 also prevented the drug combination-induced rapid declines in the expression of EDG-1, ERBB1, IL6R, IGF1R, and the PDGFRα/β, as well as drug efflux/blood–brain-barrier ABC transporters, suggesting that the reduction in receptor expression and transporter expression is also a result of reduced GRP78 levels and enhanced ER stress signaling (Fig. 7G,H). Knock down of CHOP expression suppressed CEL + SIL and OSU + SIL lethality (Fig. 7I).

Bottom Line: Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells.In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response.Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus