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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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Related in: MedlinePlus

Sildenafil facilitates a rapid further reduction in GRP78 protein expression levels in cells treated with OSU-03012. A: GBM cells were treated with vehicle, celecoxib (5 μM)/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 GRP78. B–E: GBM cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for the indicated amounts of time. At each time point, cells were fixed with paraformaldehyde and portions of fixed cells also being permeabilized with Triton X100. F: HuH7 (hepatoma) and HT1080 (sarcoma) were treated with vehicle, OSU-03012 (0–3 μM), sildenafil (2 μM), or the drugs in combination. Cells after 2 and 6 h drug exposure were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78; HSP90; HSP70. G: Primary mouse hepatocytes (wild type C57 black or CHOP / C57 black) were plated and 12 h after plating treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination. Six hours after drug exposure cells were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78. H,I: GBM cells were transfected with scrambled siRNA or siRNA molecules to knock down the expression of PERK, PKR, GCN2, or HRI. Twenty-four hours after transfection, cells were treated with vehicle or OSU-03012 (1 μM) + sildenafil and the cells fixed and permeabilized 6 h later. Immunofluorescence antibody staining was performed to determine the total levels of phospho-eIF2α serine 51 under each condition.
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Figure 8: Sildenafil facilitates a rapid further reduction in GRP78 protein expression levels in cells treated with OSU-03012. A: GBM cells were treated with vehicle, celecoxib (5 μM)/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 GRP78. B–E: GBM cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for the indicated amounts of time. At each time point, cells were fixed with paraformaldehyde and portions of fixed cells also being permeabilized with Triton X100. F: HuH7 (hepatoma) and HT1080 (sarcoma) were treated with vehicle, OSU-03012 (0–3 μM), sildenafil (2 μM), or the drugs in combination. Cells after 2 and 6 h drug exposure were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78; HSP90; HSP70. G: Primary mouse hepatocytes (wild type C57 black or CHOP / C57 black) were plated and 12 h after plating treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination. Six hours after drug exposure cells were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78. H,I: GBM cells were transfected with scrambled siRNA or siRNA molecules to knock down the expression of PERK, PKR, GCN2, or HRI. Twenty-four hours after transfection, cells were treated with vehicle or OSU-03012 (1 μM) + sildenafil and the cells fixed and permeabilized 6 h later. Immunofluorescence antibody staining was performed to determine the total levels of phospho-eIF2α serine 51 under each condition.

Mentions: In prior studies, we have shown that OSU-03012 reduces the expression of GRP78 through protein destabilization/ lower half-life without significantly altering GRP78 promoter activity and that celecoxib increases GRP78 expression, with both drugs causing similar amounts of PERK phosphorylation. This implies that both drugs, that are very similar in structure, are probably binding to GRP78 with OSU-0312 destabilizing the protein and celecoxib likely inhibiting the chaperone function of the protein, which both collectively result in increased PERK activity. Treatment of GBM cells with celecoxib and sildenafil increased GRP78 expression, that is, sildenafil did not alter the ability of celecoxib to increase GRP78 levels (Fig. 8A). Nevertheless, this increase in GRP78 expression was associated with increased eIF2α phosphorylation. Treatment of GBM cells with OSU-03012 and sildenafil reduced expression of GRP78 and strongly enhanced eIF2α phosphorylation. Treatment of GBM cells with OSU-03012 rapidly reduced cell surface and total expression of GRP78, an effect that was further enhanced by sildenafil (Fig. 8B–E). In dose–response studies, we discovered that OSU-03012 + sildenafil treatment more rapidly and completely reduced the expression of GRP78, HSP70, and HSP90 than OSU-03012 treatment alone (Fig. 8F). Similar data were obtained in primary mouse hepatocytes, and of note, as judged by DAPI staining no cell killing was observed (Fig. 8G). There are at least four kinases that phosphorylate eIF2α: PERK; PKR; GCN2; HRI. The nitric oxide synthase inhibitor L-NAME abolished the ability of OSU-03012 to reduce GRP78 levels (Fig. 8H). However, the cGMP-dependent kinase inhibitor reduced the ability of both OSU-03012 and [OSU-03012 + sildenafil] to lower GRP78 levels arguing that OSU-03012 may mediate some of its biologic effects through modulation of cGMP signaling. Knock down of PERK uniformly reduced the ability of OSU + SIL to increase eIF2α phosphorylation in all lines tested (Fig. 8I). However, in a cell isolate dependent fashion, we also found that PKR and HRI could contribute to the drug-induced eIF2α phosphorylation (Fig. 8I,J).


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)

Sildenafil facilitates a rapid further reduction in GRP78 protein expression levels in cells treated with OSU-03012. A: GBM cells were treated with vehicle, celecoxib (5 μM)/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 GRP78. B–E: GBM cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for the indicated amounts of time. At each time point, cells were fixed with paraformaldehyde and portions of fixed cells also being permeabilized with Triton X100. F: HuH7 (hepatoma) and HT1080 (sarcoma) were treated with vehicle, OSU-03012 (0–3 μM), sildenafil (2 μM), or the drugs in combination. Cells after 2 and 6 h drug exposure were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78; HSP90; HSP70. G: Primary mouse hepatocytes (wild type C57 black or CHOP / C57 black) were plated and 12 h after plating treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination. Six hours after drug exposure cells were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78. H,I: GBM cells were transfected with scrambled siRNA or siRNA molecules to knock down the expression of PERK, PKR, GCN2, or HRI. Twenty-four hours after transfection, cells were treated with vehicle or OSU-03012 (1 μM) + sildenafil and the cells fixed and permeabilized 6 h later. Immunofluorescence antibody staining was performed to determine the total levels of phospho-eIF2α serine 51 under each condition.
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Show All Figures
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Figure 8: Sildenafil facilitates a rapid further reduction in GRP78 protein expression levels in cells treated with OSU-03012. A: GBM cells were treated with vehicle, celecoxib (5 μM)/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 GRP78. B–E: GBM cells were treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for the indicated amounts of time. At each time point, cells were fixed with paraformaldehyde and portions of fixed cells also being permeabilized with Triton X100. F: HuH7 (hepatoma) and HT1080 (sarcoma) were treated with vehicle, OSU-03012 (0–3 μM), sildenafil (2 μM), or the drugs in combination. Cells after 2 and 6 h drug exposure were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78; HSP90; HSP70. G: Primary mouse hepatocytes (wild type C57 black or CHOP / C57 black) were plated and 12 h after plating treated with vehicle, OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination. Six hours after drug exposure cells were fixed in place and permeabilized. Immunofluorescence was performed to determine the total cellular levels of GRP78. H,I: GBM cells were transfected with scrambled siRNA or siRNA molecules to knock down the expression of PERK, PKR, GCN2, or HRI. Twenty-four hours after transfection, cells were treated with vehicle or OSU-03012 (1 μM) + sildenafil and the cells fixed and permeabilized 6 h later. Immunofluorescence antibody staining was performed to determine the total levels of phospho-eIF2α serine 51 under each condition.
Mentions: In prior studies, we have shown that OSU-03012 reduces the expression of GRP78 through protein destabilization/ lower half-life without significantly altering GRP78 promoter activity and that celecoxib increases GRP78 expression, with both drugs causing similar amounts of PERK phosphorylation. This implies that both drugs, that are very similar in structure, are probably binding to GRP78 with OSU-0312 destabilizing the protein and celecoxib likely inhibiting the chaperone function of the protein, which both collectively result in increased PERK activity. Treatment of GBM cells with celecoxib and sildenafil increased GRP78 expression, that is, sildenafil did not alter the ability of celecoxib to increase GRP78 levels (Fig. 8A). Nevertheless, this increase in GRP78 expression was associated with increased eIF2α phosphorylation. Treatment of GBM cells with OSU-03012 and sildenafil reduced expression of GRP78 and strongly enhanced eIF2α phosphorylation. Treatment of GBM cells with OSU-03012 rapidly reduced cell surface and total expression of GRP78, an effect that was further enhanced by sildenafil (Fig. 8B–E). In dose–response studies, we discovered that OSU-03012 + sildenafil treatment more rapidly and completely reduced the expression of GRP78, HSP70, and HSP90 than OSU-03012 treatment alone (Fig. 8F). Similar data were obtained in primary mouse hepatocytes, and of note, as judged by DAPI staining no cell killing was observed (Fig. 8G). There are at least four kinases that phosphorylate eIF2α: PERK; PKR; GCN2; HRI. The nitric oxide synthase inhibitor L-NAME abolished the ability of OSU-03012 to reduce GRP78 levels (Fig. 8H). However, the cGMP-dependent kinase inhibitor reduced the ability of both OSU-03012 and [OSU-03012 + sildenafil] to lower GRP78 levels arguing that OSU-03012 may mediate some of its biologic effects through modulation of cGMP signaling. Knock down of PERK uniformly reduced the ability of OSU + SIL to increase eIF2α phosphorylation in all lines tested (Fig. 8I). However, in a cell isolate dependent fashion, we also found that PKR and HRI could contribute to the drug-induced eIF2α phosphorylation (Fig. 8I,J).

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