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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

OSU-03012 and sildenafil interact to kill tumor cells regardless of stem-like or anoikis-like properties, and that the tumor response to this combination provides biomarkers for the addition of a third agent. A: GBM5/6/12/14 cells or stem-like selected variants of these cells were plated in 96-well plates and 24 h after plating cells were cyto-centrifuged to sediment cells to the plate, and cells were fixed with 2% paraformaldehyde containing Triton X100 for permeabilization. Immunofluorescence was performed to determine the expression of GRP78/MCL-1/P-eIF2α, EDG-1, and counter-staining was with DAPI and detected using a Hermes Wiscan machine. The brightness/contrast for each IF image was set to show the most intense staining and all other images were presented with the identical same brightness/contrast setting. B: GBM cells or stem-like variants of these cells were treated with vehicle; celecoxib (5 μM) + sildenafil (2 μM); or OSU-03012 (1 μM) + sildenafil and the viability of cells determined by trypan blue exclusion assay after 24 h. (n = 3, ±SEM) and P <0.05 greater than corresponding value in celecoxib treated cells; $P <0.05 greater than corresponding value in wild type cells; @P <0.05 less than corresponding value in wild type cells. C: Athymic mice were treated with vehicle (Cremophore); OSU-03012 (50 mg/kg) + sildenafil (10 mg/kg); celecoxib (50 mg/kg + sildenafil (10 mg/kg) for 14 days (nota bene: below in section (D) that anti-tumor effects were observed using half the doses of these drugs and for half as long). Animals were sacrificed, the normal tissue organs collected and fixed. Sections (10 μm) were taken and H&E stained by The Department of Pathology, VCU). D: Athymic nude mice were injected with 1 × 107 BT474 cells into their fourth mammary fat pad. Seven days after injection, animals with ~50 mm3 tumors were treated by oral gavage with vehicle (cremophore); celecoxib (10 mg/ kg) + sildenafil (5 mg/kg); OSU-03012 (10 mg/kg) + sildenafil (5 mg/kg) for 5 days after which tumors were isolated, and plated as single cells to determine the ex vivo colony formation ability of in vivo treated tumors (n = 3 ± SEM). #P <0.05 less than vehicle control; ##P <0.05 less than [CEL + SIL] value. E–J: Tumors treated for 5 days in section (D) were isolated as was mouse blood and plasma collected. Multiplex assays were performed on tumor lysates and on clarified mouse plasma to detect changes in signal transduction parameters and on plasma cytokine levels (n = 4 tumors ± SEM). The treatments are presented in groups of three bars, with bars on the left being vehicle control; bars in the center being [OSU + SIL]; and bars on the right being [CEL + SIL]. ~P <0.05 greater than vehicle control value; #P <0.05 less than vehicle control value. K: BT474 cells were treated with vehicle control; OSU-03012 (1 μM) and and sildenafil (2 μM); the FGFR inhibitor BGJ398 (1 μM); the JAK1/2 inhibitor ruxolitinib (1 μM) as indicated for 12 h. Cells viability was determined using a live/dead assay in a Hermes WiScan machine where red/yellow cells = dead; green cells = alive (n = 3 ± SEM). *P <0.05 greater than corresponding value in vehicle control treated cells.
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Figure 1: OSU-03012 and sildenafil interact to kill tumor cells regardless of stem-like or anoikis-like properties, and that the tumor response to this combination provides biomarkers for the addition of a third agent. A: GBM5/6/12/14 cells or stem-like selected variants of these cells were plated in 96-well plates and 24 h after plating cells were cyto-centrifuged to sediment cells to the plate, and cells were fixed with 2% paraformaldehyde containing Triton X100 for permeabilization. Immunofluorescence was performed to determine the expression of GRP78/MCL-1/P-eIF2α, EDG-1, and counter-staining was with DAPI and detected using a Hermes Wiscan machine. The brightness/contrast for each IF image was set to show the most intense staining and all other images were presented with the identical same brightness/contrast setting. B: GBM cells or stem-like variants of these cells were treated with vehicle; celecoxib (5 μM) + sildenafil (2 μM); or OSU-03012 (1 μM) + sildenafil and the viability of cells determined by trypan blue exclusion assay after 24 h. (n = 3, ±SEM) and P <0.05 greater than corresponding value in celecoxib treated cells; $P <0.05 greater than corresponding value in wild type cells; @P <0.05 less than corresponding value in wild type cells. C: Athymic mice were treated with vehicle (Cremophore); OSU-03012 (50 mg/kg) + sildenafil (10 mg/kg); celecoxib (50 mg/kg + sildenafil (10 mg/kg) for 14 days (nota bene: below in section (D) that anti-tumor effects were observed using half the doses of these drugs and for half as long). Animals were sacrificed, the normal tissue organs collected and fixed. Sections (10 μm) were taken and H&E stained by The Department of Pathology, VCU). D: Athymic nude mice were injected with 1 × 107 BT474 cells into their fourth mammary fat pad. Seven days after injection, animals with ~50 mm3 tumors were treated by oral gavage with vehicle (cremophore); celecoxib (10 mg/ kg) + sildenafil (5 mg/kg); OSU-03012 (10 mg/kg) + sildenafil (5 mg/kg) for 5 days after which tumors were isolated, and plated as single cells to determine the ex vivo colony formation ability of in vivo treated tumors (n = 3 ± SEM). #P <0.05 less than vehicle control; ##P <0.05 less than [CEL + SIL] value. E–J: Tumors treated for 5 days in section (D) were isolated as was mouse blood and plasma collected. Multiplex assays were performed on tumor lysates and on clarified mouse plasma to detect changes in signal transduction parameters and on plasma cytokine levels (n = 4 tumors ± SEM). The treatments are presented in groups of three bars, with bars on the left being vehicle control; bars in the center being [OSU + SIL]; and bars on the right being [CEL + SIL]. ~P <0.05 greater than vehicle control value; #P <0.05 less than vehicle control value. K: BT474 cells were treated with vehicle control; OSU-03012 (1 μM) and and sildenafil (2 μM); the FGFR inhibitor BGJ398 (1 μM); the JAK1/2 inhibitor ruxolitinib (1 μM) as indicated for 12 h. Cells viability was determined using a live/dead assay in a Hermes WiScan machine where red/yellow cells = dead; green cells = alive (n = 3 ± SEM). *P <0.05 greater than corresponding value in vehicle control treated cells.

Mentions: GBM stem cells compared to parental non-selected GBM cells exhibited higher basal levels of phosphorylated eIF2α, but stem cells nevertheless also expressed more GRP78/BiP/HSPA5 protein (Fig. 1A). Stem cell-like GBM cells expressed considerably more MCL-1 and sphingosine-1-phosphate receptor 1 (EDG-1) than parental WT cells. Of note, the intensity of each image was normalized to the most intense staining image for each antibody probe; WT cells did express MCL-1, EDG-1, and GRP78 albeit at lower levels than stem cells (Fig. 1A, lower images). In GBM cells, it was noted that stem cell selected cells were modestly but significantly more sensitive to OSU + sildenafil also called Viagra (SIL) treatment than parental WT cells. In the majority of GBM cell lines, treatment using 1 μM OSU-03012 caused a greater toxic response when combined with sildenafil than did treatment with the parental compound 5 μM celecoxib and sildenafil. Regardless of whether breast cancer cells were parental or generated to be anoikis resistant, the relative ability of celecoxib also called Celebrex (CEL) + SIL treatment to kill parental or anoikis cells preferentially appeared to be more unpredictable than in GBM cells and stochastic in nature (data not shown). Prolonged high concentration dosing of mice with [OSU-03012 + sildenafil] or [celecoxib + sildenafil] did not cause obvious frank damage to normal tissues from an athymic mouse (Fig. 1C). In contrast, treatment of animals with lower doses of [OSU-03012 + sildenafil] or [celecoxib + sildenafil] significantly reduced tumor volumes after 5 days of treatment and reduced the long-term viability of in vivo treated cells, as judged using ex vivo colony formation assays (Fig. 1D, data not shown).


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)

OSU-03012 and sildenafil interact to kill tumor cells regardless of stem-like or anoikis-like properties, and that the tumor response to this combination provides biomarkers for the addition of a third agent. A: GBM5/6/12/14 cells or stem-like selected variants of these cells were plated in 96-well plates and 24 h after plating cells were cyto-centrifuged to sediment cells to the plate, and cells were fixed with 2% paraformaldehyde containing Triton X100 for permeabilization. Immunofluorescence was performed to determine the expression of GRP78/MCL-1/P-eIF2α, EDG-1, and counter-staining was with DAPI and detected using a Hermes Wiscan machine. The brightness/contrast for each IF image was set to show the most intense staining and all other images were presented with the identical same brightness/contrast setting. B: GBM cells or stem-like variants of these cells were treated with vehicle; celecoxib (5 μM) + sildenafil (2 μM); or OSU-03012 (1 μM) + sildenafil and the viability of cells determined by trypan blue exclusion assay after 24 h. (n = 3, ±SEM) and P <0.05 greater than corresponding value in celecoxib treated cells; $P <0.05 greater than corresponding value in wild type cells; @P <0.05 less than corresponding value in wild type cells. C: Athymic mice were treated with vehicle (Cremophore); OSU-03012 (50 mg/kg) + sildenafil (10 mg/kg); celecoxib (50 mg/kg + sildenafil (10 mg/kg) for 14 days (nota bene: below in section (D) that anti-tumor effects were observed using half the doses of these drugs and for half as long). Animals were sacrificed, the normal tissue organs collected and fixed. Sections (10 μm) were taken and H&E stained by The Department of Pathology, VCU). D: Athymic nude mice were injected with 1 × 107 BT474 cells into their fourth mammary fat pad. Seven days after injection, animals with ~50 mm3 tumors were treated by oral gavage with vehicle (cremophore); celecoxib (10 mg/ kg) + sildenafil (5 mg/kg); OSU-03012 (10 mg/kg) + sildenafil (5 mg/kg) for 5 days after which tumors were isolated, and plated as single cells to determine the ex vivo colony formation ability of in vivo treated tumors (n = 3 ± SEM). #P <0.05 less than vehicle control; ##P <0.05 less than [CEL + SIL] value. E–J: Tumors treated for 5 days in section (D) were isolated as was mouse blood and plasma collected. Multiplex assays were performed on tumor lysates and on clarified mouse plasma to detect changes in signal transduction parameters and on plasma cytokine levels (n = 4 tumors ± SEM). The treatments are presented in groups of three bars, with bars on the left being vehicle control; bars in the center being [OSU + SIL]; and bars on the right being [CEL + SIL]. ~P <0.05 greater than vehicle control value; #P <0.05 less than vehicle control value. K: BT474 cells were treated with vehicle control; OSU-03012 (1 μM) and and sildenafil (2 μM); the FGFR inhibitor BGJ398 (1 μM); the JAK1/2 inhibitor ruxolitinib (1 μM) as indicated for 12 h. Cells viability was determined using a live/dead assay in a Hermes WiScan machine where red/yellow cells = dead; green cells = alive (n = 3 ± SEM). *P <0.05 greater than corresponding value in vehicle control treated cells.
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Figure 1: OSU-03012 and sildenafil interact to kill tumor cells regardless of stem-like or anoikis-like properties, and that the tumor response to this combination provides biomarkers for the addition of a third agent. A: GBM5/6/12/14 cells or stem-like selected variants of these cells were plated in 96-well plates and 24 h after plating cells were cyto-centrifuged to sediment cells to the plate, and cells were fixed with 2% paraformaldehyde containing Triton X100 for permeabilization. Immunofluorescence was performed to determine the expression of GRP78/MCL-1/P-eIF2α, EDG-1, and counter-staining was with DAPI and detected using a Hermes Wiscan machine. The brightness/contrast for each IF image was set to show the most intense staining and all other images were presented with the identical same brightness/contrast setting. B: GBM cells or stem-like variants of these cells were treated with vehicle; celecoxib (5 μM) + sildenafil (2 μM); or OSU-03012 (1 μM) + sildenafil and the viability of cells determined by trypan blue exclusion assay after 24 h. (n = 3, ±SEM) and P <0.05 greater than corresponding value in celecoxib treated cells; $P <0.05 greater than corresponding value in wild type cells; @P <0.05 less than corresponding value in wild type cells. C: Athymic mice were treated with vehicle (Cremophore); OSU-03012 (50 mg/kg) + sildenafil (10 mg/kg); celecoxib (50 mg/kg + sildenafil (10 mg/kg) for 14 days (nota bene: below in section (D) that anti-tumor effects were observed using half the doses of these drugs and for half as long). Animals were sacrificed, the normal tissue organs collected and fixed. Sections (10 μm) were taken and H&E stained by The Department of Pathology, VCU). D: Athymic nude mice were injected with 1 × 107 BT474 cells into their fourth mammary fat pad. Seven days after injection, animals with ~50 mm3 tumors were treated by oral gavage with vehicle (cremophore); celecoxib (10 mg/ kg) + sildenafil (5 mg/kg); OSU-03012 (10 mg/kg) + sildenafil (5 mg/kg) for 5 days after which tumors were isolated, and plated as single cells to determine the ex vivo colony formation ability of in vivo treated tumors (n = 3 ± SEM). #P <0.05 less than vehicle control; ##P <0.05 less than [CEL + SIL] value. E–J: Tumors treated for 5 days in section (D) were isolated as was mouse blood and plasma collected. Multiplex assays were performed on tumor lysates and on clarified mouse plasma to detect changes in signal transduction parameters and on plasma cytokine levels (n = 4 tumors ± SEM). The treatments are presented in groups of three bars, with bars on the left being vehicle control; bars in the center being [OSU + SIL]; and bars on the right being [CEL + SIL]. ~P <0.05 greater than vehicle control value; #P <0.05 less than vehicle control value. K: BT474 cells were treated with vehicle control; OSU-03012 (1 μM) and and sildenafil (2 μM); the FGFR inhibitor BGJ398 (1 μM); the JAK1/2 inhibitor ruxolitinib (1 μM) as indicated for 12 h. Cells viability was determined using a live/dead assay in a Hermes WiScan machine where red/yellow cells = dead; green cells = alive (n = 3 ± SEM). *P <0.05 greater than corresponding value in vehicle control treated cells.
Mentions: GBM stem cells compared to parental non-selected GBM cells exhibited higher basal levels of phosphorylated eIF2α, but stem cells nevertheless also expressed more GRP78/BiP/HSPA5 protein (Fig. 1A). Stem cell-like GBM cells expressed considerably more MCL-1 and sphingosine-1-phosphate receptor 1 (EDG-1) than parental WT cells. Of note, the intensity of each image was normalized to the most intense staining image for each antibody probe; WT cells did express MCL-1, EDG-1, and GRP78 albeit at lower levels than stem cells (Fig. 1A, lower images). In GBM cells, it was noted that stem cell selected cells were modestly but significantly more sensitive to OSU + sildenafil also called Viagra (SIL) treatment than parental WT cells. In the majority of GBM cell lines, treatment using 1 μM OSU-03012 caused a greater toxic response when combined with sildenafil than did treatment with the parental compound 5 μM celecoxib and sildenafil. Regardless of whether breast cancer cells were parental or generated to be anoikis resistant, the relative ability of celecoxib also called Celebrex (CEL) + SIL treatment to kill parental or anoikis cells preferentially appeared to be more unpredictable than in GBM cells and stochastic in nature (data not shown). Prolonged high concentration dosing of mice with [OSU-03012 + sildenafil] or [celecoxib + sildenafil] did not cause obvious frank damage to normal tissues from an athymic mouse (Fig. 1C). In contrast, treatment of animals with lower doses of [OSU-03012 + sildenafil] or [celecoxib + sildenafil] significantly reduced tumor volumes after 5 days of treatment and reduced the long-term viability of in vivo treated cells, as judged using ex vivo colony formation assays (Fig. 1D, data not shown).

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