Figure 1.
Celecoxib inhibits intracellular bacteria.
A. Effect of NSAIDs such as Indomethacin (Ind), Ibuprofen (Ib), Flurbiprofen (Fb), gallic acid (GA) and celecoxib (Ce) and antibiotic ampicillin (Amp) on the growth of Staphylococcus aureus. DMSO and MeOH (methanol) are used as solvent controls. * p-value<0.01 compared to Control. B. Effect of celecoxib, ampicillin and their combination on the survival of intracellular bacteria as measured by the CFU. * p-value<0.01 compared to corresponding Control.
Figure 2.
Combinatorial treatment with celecoxib activated antioxidant enzymes via SIRT1.
A. RT-PCR and Western blot analysis of SIRT1 in RAW 264.7 cells (Lane 1) and S aureus phagocytosed cells (Lane 2) treated with ampicillin 100 µg/ml (Lane 3), celecoxib 10 µM (Lane 4), and combination of celecoxib and ampicillin (Lane 5), Resveratrol 50 µM (Lane 6) and Suramin 25 µM (Lane 7). GAPDH and β-actin served as controls for RT-PCR and Western blot respectively. B. Activity assay of SIRT1 immunoprecipitated from whole cell lysates of cells infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. C. Nitric Oxide levels in the culture supernatants of cells infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. D. Anti-oxidant enzymes, catalase and peroxidase, activity in the whole cell lysates of cells infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7.
Figure 3.
Celecoxib-induced SIRT1 activity inhibited TLR2-JNK-NF-κB signaling.
A. Flowcytometer analysis of TLR2 expression in RAW-264.7 cells phagocytosed with S aureus (1) and treated with ampicillin (2) or celecoxib (3) or both in combination (4). B. Western blot of levels of p-JNK and JNK in S aureus phagocytosed cells (Lane 1) and treated with ampicillin 100 µg/ml (Lane 2), celecoxib 10 µM (Lane 3), and combination of celecoxib and ampicillin (Lane 4), Resveratrol 50 µM (Lane 5) Suramin 25 µM (Lane 6) and RAW-264.7 cells (Lane 7). C. Western blot of levels of Ac- NF-κB and NF-κB in cell lysates of cells without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with ampicillin (Lane 3) or celecoxib (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7). D. NF-κB p65 levels in cytoplasmic and nuclear isolates of cells treated with/without, and ampicillin and celecoxib. infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol and Suramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7.
Figure 4.
Celecoxib-induced SIRT1 activity inhibited proinflammatory gene expression.
A. COX-2 RNA and protein expression in cell lysates of cells without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with celecoxib (Lane 3) or ampicillin (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7) B. PGE2 levels in the culture supernatants of cells infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol andSuramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. C. Levels of IL-6, IL-1β, MIP-1α and IL-2 in the culture supernatants of cells infected without or with S aureus and treated with ampicillin, celecoxib, ampicillin+celecoxib, Resveratrol andSuramin. * p-value<0.01 compared to RAW 264.7 alone, @ p-value<0.01 compared to S Aureus infected RAW 264.7. D. Total p53 and Ac-p53 levels in cells treated without (Lane 1) and with phagocytosed bacteria (Lane 2) treated with ampicillin (Lane 3) or celecoxib (Lane 4) or both (Lane 5) or Resveratrol (Lane 6) or suramin (Lane 7).
Figure 5.
Celecoxib activates SIRT1 in a biochemical, in silico and in vitro cell-based assay.
A. A representative graph of three independent experiments each with duplicates showing the enzyme activity of SIRT1 in presence and absence of different concentrations of celecoxib. Suramin, a known inhibitor of SIRT1 is taken as an assay control. * p-value<0.01 compared to Control. B. Left - Model of Celecoxib bound to SIRT1 protein (1–217 residues, proposed N-terminal activation domain of SIRT1). Right - Model showing the interactions of Celecoxib with SIRT1 protein. C. A representative Western blot showing a dose-dependent decrease of Ac-H3K9 levels in presence of celecoxib. * p-value<0.01 compared to Control. D. A representative Western blot showing the signal compensation of Ac-H3K9 levels on co-treatment of cells with celecoxib and suramin at 25 µM concentration each. * p-value<0.01 compared to Control Lane 1. 5 mM sodium butyrate treated cells. Lane 2. Suramin 25 µM. Lane 3. Celecoxib 25 µM. Lane 4. Celecoxib and Suramin 25 µM. Lane 5. Resveratrol 50 µM.
Figure 6.
Schematic representation of mechanism of action of combinatorial treatment in host cells.