OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.
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.
Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia.
- Antineoplastic Combined Chemotherapy Protocols/pharmacology*
- Blood-Brain Barrier/drug effects*
- Brain Neoplasms/pathology*
- Molecular Chaperones/metabolism*
- Blotting, Western
- Cell Line, Tumor
- Disease Models, Animal
- Drug Synergism
- Piperazines/administration & dosage
- Purines/administration & dosage
- Pyrazoles/administration & dosage
- RNA, Small Interfering
- Signal Transduction/drug effects
- Sildenafil Citrate
- Sulfonamides/administration & dosage
- Xenograft Model Antitumor Assays
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Figure 4: The regulation of chaperone function and expression by OSU-03012, sildenafil and HDAC inhibitors. A: (Left blots) GBM6 cells were treated with AR-42 (250 nM) for 90 min, after which HSP90 was immunoprecipitated and probed for total HSP90 expression and HSP90 acetylation. In parallel tumor, cell lysate was immunoblotted for the total expression of GAPDH and of c-FLIP-s. Right graph: GBM6 and GBM12 cells were infected with an empty vector virus or a virus to express HSP90, and in parallel transfected with either a scrambled siRNA (siSCR) or an siRNA to knock down IRE1 expression. Twenty-four hours after infection, cells were treated with vehicle or OSU-03012 (1 μM). Cells were isolated after 24 h and viability determined by trypan blue exclusion assay (n = 3 ± SEM). *P <0.05 greater than siSCR + CMV value; **P <0.05 greater than siIRE1 + CMV value; #P <0.05 less than siIRE1 + CMV value; ?P >0.90 between siSCR + CMV and siSCR + HSP90 values. IF images above graph: GBM6/12 cells were treated with vehicle; OSU-03012 (1 μM); sildenafil (2 μM), or the drugs in combination and after 6 h treatment were fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilized cells. Immunostaining of HSP90α/β was performed using standard techniques and IF images detected using a Hermes Wiscan machine. B: GBM cells were treated for 12 h with vehicle, [OSU-03012 (1 μM) + sildenafil (2 μM)], SNX2112 (250 nM), or the three drugs combined. Cell viability under each condition was determined by live/dead assays (n = 3 ± SEM). C: GBM6 cells were transfected with siSCR or siRNA molecules to knock down expression of IRE1, XBP1, and PERK. In parallel sets of plates, cells were transfected with empty vector plasmid (CMV) or to express dominant negative PERK. Twenty-four hours after transfection, cells were treated with vehicle or with OSU-03012 (2 μM). Twenty-four hours after drug treatment, cell viability was determined by trypan blue exclusion assay (n = 3 ± SEM). #P <0.05 lower than corresponding value in siSCR cells; *P <0.05 greater than corresponding value in siSCR cells. D: (Upper blots) GBM6 cells were treated with OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 6 h after which cells were lysed. Immunoblotting and phospho-immunoblotting was performed to detect the expression/phosphorylation of the indicated proteins. Lower Graphs: GBM6 and GBM12 cells were transfected with empty vector (CMV) or to express GRP78. Twenty-four hours after transfection, cells were treated with 2 μM OSU-03012 and 24 h later viability determined via trypan blue exclusion assay (n = 3 ± SEM). #P <0.05 less than corresponding value in CMV transfected cells. E: GBM6 cells were transfected with scrambled control (siSCR) or siRNA molecules to knock down IRE1, XBP1, or PERK. Twenty-four hours after transfection, cells were treated with OSU-03012 (1 μM), sildenafil (2 μM), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion assay (n = 3 ± SEM). *P <0.05 greater than OSU-03012 single agent treatment in siSCR cells; **P <0.05 greater than corresponding values in siSCR cells; #P <0.05 less than corresponding values in siSCR cells. F–I: GBM cells were treated with vehicle; celecoxib (5 μM), sildenafil (2 μM), OSU-03012 (1 μM) as single agents or in the indicated combinations for 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilize cells. Immunostaining of total HSP70 and HSC70 expression combined (part F) or expression of GRP94 and GRP58 (parts G and H) or expression of HSP27/HSP40/HSP60 (part I) was performed using standard techniques and IF images detected using a Hermes Wiscan machine.
HDAC inhibitors increase the acetylation of HSP90, which reduces its chaperone function. The acetylation of heat shock protein (HSP) 90 was increased and the expression of the HSP90 client protein c-FLIP-s reduced by AR-42 treatment (Fig. 4A, left blots; in agreement with Panner et al., 2007). This collectively argues that the combination of OSU + SIL + AR-42 is inhibiting the functions of both GRP78 and HSP90, which may explain the strong killing interaction between the drugs. Furthermore, over-expression of HSP90 did not significantly protect cells from OSU-03012 toxicity as a single agent, though HSP90 over-expression did modestly reduce the enhancement of cell death caused by IRE1 knock down; this effect was abolished by AR-42 (Fig. 4A, right graph). Sildenafil and OSU-03012 interacted to decrease total protein expression of HSP90α/β in multiple GBM cell isolates (Fig. 4A, upper IF images). Thus OSU-03012 inhibits HSP90 function as a single agent and combined with sildenafil also considerably reduces HSP90 protein levels. Based on our findings showing the effect of sildenafil and OSU-03012 on HSP90 function, we determined whether a clinically relevant HSP90 inhibitor (SNX2112/SNX5422, 250 nM in vitro; patient C max @ 100 mg/m2 is ~3 μM) could enhance the lethality of OSU-03012 and of [OSU + SIL] in GBM cells. SNX2112 at its IC90 concentration for HSP90 inhibition enhanced the toxicity of [OSU + SIL] (Fig. 4B).