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GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease.

Booth L, Roberts JL, Cash DR, Tavallai S, Jean S, Fidanza A, Cruz-Luna T, Siembiba P, Cycon KA, Cornelissen CN, Dent P - J. Cell. Physiol. (2015)

Bottom Line: The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes.Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce.OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298.

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Related in: MedlinePlus

The regulation of virus receptor expression and the virus reproductive cycle by OSU-03012 and Sildenafil. (A) HT1080 sarcoma and HuH7 hepatoma cells were treated with vehicle, sildenafil (2 μM), OSU-03012 (0.5–3.0 μM) as single agents or in the indicated combinations for 2 h or 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilize cells. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (B) A549 (NSCLC), HEK293 (kidney), HuH7 and HT1080 cells were transfected with empty vector plasmid or a plasmid to express GRP78. Primary mouse hepatocytes were not transfected. Twenty four h later cells were treated with vehicle control or with OSU-03012 (1 μM) and/or sildenafil (2 μM) for 6 h. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (C) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant serotype 5 adenovirus to express green fluorescent protein (GFP). The media was changed to media still containing drugs, as indicated, and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (D) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). The media was changed to drug free media and the percentage of GFP+ cells determined after 24 h; and the percentage cell death was determined by live/dead assay determined after 24 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (E) HEK293 cells (∼10,000) were infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). One h after infection the media was changed to contain vehicle or [OSU-03012 + Sildenafil] and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (F) HEK293 cells were continuously treated/pre-treated post-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (G) and (H) HEK293 cells were transfected with empty vector plasmid or a plasmid to express GRP78. After 24 h cells were infected with Ad5.GFP or with coxsakie virus B4 and treated before/during/after/always with vehicle or OSU-03012 (1 μM) and sildenafil (2 μM). The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM). # P < 0.05 less than corresponding value in vehicle treated cells; * P < 0.05 greater than corresponding value in CMV transfected cells. (I) and (J) HEK293 cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down GRP78 expression (siGRP78). Twenty four h after transfection cells are infected with increasing amounts of Ad5.GFP or coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM) # P < 0.05 less than corresponding value in siSCR + vehicle treated cells.
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fig04: The regulation of virus receptor expression and the virus reproductive cycle by OSU-03012 and Sildenafil. (A) HT1080 sarcoma and HuH7 hepatoma cells were treated with vehicle, sildenafil (2 μM), OSU-03012 (0.5–3.0 μM) as single agents or in the indicated combinations for 2 h or 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilize cells. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (B) A549 (NSCLC), HEK293 (kidney), HuH7 and HT1080 cells were transfected with empty vector plasmid or a plasmid to express GRP78. Primary mouse hepatocytes were not transfected. Twenty four h later cells were treated with vehicle control or with OSU-03012 (1 μM) and/or sildenafil (2 μM) for 6 h. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (C) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant serotype 5 adenovirus to express green fluorescent protein (GFP). The media was changed to media still containing drugs, as indicated, and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (D) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). The media was changed to drug free media and the percentage of GFP+ cells determined after 24 h; and the percentage cell death was determined by live/dead assay determined after 24 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (E) HEK293 cells (∼10,000) were infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). One h after infection the media was changed to contain vehicle or [OSU-03012 + Sildenafil] and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (F) HEK293 cells were continuously treated/pre-treated post-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (G) and (H) HEK293 cells were transfected with empty vector plasmid or a plasmid to express GRP78. After 24 h cells were infected with Ad5.GFP or with coxsakie virus B4 and treated before/during/after/always with vehicle or OSU-03012 (1 μM) and sildenafil (2 μM). The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM). # P < 0.05 less than corresponding value in vehicle treated cells; * P < 0.05 greater than corresponding value in CMV transfected cells. (I) and (J) HEK293 cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down GRP78 expression (siGRP78). Twenty four h after transfection cells are infected with increasing amounts of Ad5.GFP or coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM) # P < 0.05 less than corresponding value in siSCR + vehicle treated cells.

Mentions: OSU-03012 + sildenafil treatment rapidly decreased expression of the coxsakie and adenovirus receptor (CAR) in a dose- and time-dependent fashion that was modestly reduced by in a cell type dependent fashion by over-expression of GRP78 (Fig. 4A and B). Pre-treatment of cells with OSU-03012 + sildenafil significantly reduced the ability of a serotype 5 virus expressing GFP to infect cells and reduced the production of GFP when drug treatment occurred after virus infection (Fig. 4C–E). OSU-03012 + sildenafil treatment following virus infection also reduced the ability of a serotype 5 adenovirus to replicate as judged by infected cell killing. Pre-treatment of cells with OSU-03012 + sildenafil or OSU-03012 + sildenafil treatment following virus infection suppressed the ability of coxsakie B4 virus to kill infected cells 18 h after infection (Figure 4F). Over-expression of GRP78 enhanced the abilities of adenovirus and coxsakie virus to kill and abolished the protective effect of OSU-03012 + sildenafil treatment (Fig. 4G and H). Knock down of GRP78 also suppressed virus –induced killing (Fig. 4I and J).


GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease.

Booth L, Roberts JL, Cash DR, Tavallai S, Jean S, Fidanza A, Cruz-Luna T, Siembiba P, Cycon KA, Cornelissen CN, Dent P - J. Cell. Physiol. (2015)

The regulation of virus receptor expression and the virus reproductive cycle by OSU-03012 and Sildenafil. (A) HT1080 sarcoma and HuH7 hepatoma cells were treated with vehicle, sildenafil (2 μM), OSU-03012 (0.5–3.0 μM) as single agents or in the indicated combinations for 2 h or 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilize cells. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (B) A549 (NSCLC), HEK293 (kidney), HuH7 and HT1080 cells were transfected with empty vector plasmid or a plasmid to express GRP78. Primary mouse hepatocytes were not transfected. Twenty four h later cells were treated with vehicle control or with OSU-03012 (1 μM) and/or sildenafil (2 μM) for 6 h. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (C) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant serotype 5 adenovirus to express green fluorescent protein (GFP). The media was changed to media still containing drugs, as indicated, and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (D) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). The media was changed to drug free media and the percentage of GFP+ cells determined after 24 h; and the percentage cell death was determined by live/dead assay determined after 24 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (E) HEK293 cells (∼10,000) were infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). One h after infection the media was changed to contain vehicle or [OSU-03012 + Sildenafil] and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (F) HEK293 cells were continuously treated/pre-treated post-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (G) and (H) HEK293 cells were transfected with empty vector plasmid or a plasmid to express GRP78. After 24 h cells were infected with Ad5.GFP or with coxsakie virus B4 and treated before/during/after/always with vehicle or OSU-03012 (1 μM) and sildenafil (2 μM). The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM). # P < 0.05 less than corresponding value in vehicle treated cells; * P < 0.05 greater than corresponding value in CMV transfected cells. (I) and (J) HEK293 cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down GRP78 expression (siGRP78). Twenty four h after transfection cells are infected with increasing amounts of Ad5.GFP or coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM) # P < 0.05 less than corresponding value in siSCR + vehicle treated cells.
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fig04: The regulation of virus receptor expression and the virus reproductive cycle by OSU-03012 and Sildenafil. (A) HT1080 sarcoma and HuH7 hepatoma cells were treated with vehicle, sildenafil (2 μM), OSU-03012 (0.5–3.0 μM) as single agents or in the indicated combinations for 2 h or 6 h. Cells were then fixed with 2% (v/v) paraformaldehyde containing Triton X100 to permeabilize cells. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (B) A549 (NSCLC), HEK293 (kidney), HuH7 and HT1080 cells were transfected with empty vector plasmid or a plasmid to express GRP78. Primary mouse hepatocytes were not transfected. Twenty four h later cells were treated with vehicle control or with OSU-03012 (1 μM) and/or sildenafil (2 μM) for 6 h. Immuno-staining of the coxsakie and adenovirus virus receptor (CAR) expression was performed using standard techniques and IF images detected using a Hermes Wiscan machine. (C) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant serotype 5 adenovirus to express green fluorescent protein (GFP). The media was changed to media still containing drugs, as indicated, and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (D) HEK293 cells were pre-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). The media was changed to drug free media and the percentage of GFP+ cells determined after 24 h; and the percentage cell death was determined by live/dead assay determined after 24 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (E) HEK293 cells (∼10,000) were infected at the indicated numbers of virus particles (total) with a recombinant adenovirus to express green fluorescent protein (GFP). One h after infection the media was changed to contain vehicle or [OSU-03012 + Sildenafil] and the percentage of GFP+ cells determined after 14 h; and the percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (F) HEK293 cells were continuously treated/pre-treated post-treated with vehicle or [OSU-03012 + Sildenafil] as above for 3 h. Cells (∼10,000) were then infected at the indicated numbers of virus particles (total) with a coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h (n = 3 +/− SEM) # P < 0.05 less than corresponding value in vehicle treated cells. (G) and (H) HEK293 cells were transfected with empty vector plasmid or a plasmid to express GRP78. After 24 h cells were infected with Ad5.GFP or with coxsakie virus B4 and treated before/during/after/always with vehicle or OSU-03012 (1 μM) and sildenafil (2 μM). The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM). # P < 0.05 less than corresponding value in vehicle treated cells; * P < 0.05 greater than corresponding value in CMV transfected cells. (I) and (J) HEK293 cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down GRP78 expression (siGRP78). Twenty four h after transfection cells are infected with increasing amounts of Ad5.GFP or coxsakie virus B4. The percentage cell death was determined by live/dead assay determined after 18 h where green cells = alive and red/yellow cells = dead (n = 3 +/− SEM) # P < 0.05 less than corresponding value in siSCR + vehicle treated cells.
Mentions: OSU-03012 + sildenafil treatment rapidly decreased expression of the coxsakie and adenovirus receptor (CAR) in a dose- and time-dependent fashion that was modestly reduced by in a cell type dependent fashion by over-expression of GRP78 (Fig. 4A and B). Pre-treatment of cells with OSU-03012 + sildenafil significantly reduced the ability of a serotype 5 virus expressing GFP to infect cells and reduced the production of GFP when drug treatment occurred after virus infection (Fig. 4C–E). OSU-03012 + sildenafil treatment following virus infection also reduced the ability of a serotype 5 adenovirus to replicate as judged by infected cell killing. Pre-treatment of cells with OSU-03012 + sildenafil or OSU-03012 + sildenafil treatment following virus infection suppressed the ability of coxsakie B4 virus to kill infected cells 18 h after infection (Figure 4F). Over-expression of GRP78 enhanced the abilities of adenovirus and coxsakie virus to kill and abolished the protective effect of OSU-03012 + sildenafil treatment (Fig. 4G and H). Knock down of GRP78 also suppressed virus –induced killing (Fig. 4I and J).

Bottom Line: The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes.Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce.OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298.

Show MeSH
Related in: MedlinePlus