Growth inhibition of the pathogen Staphylococcus aureus with currently available antibiotics is problematic in part due to bacterial biofilm protection. Although recently characterized natural products, including 3',4',5-trihydroxy-6,7-dimethoxy-flavone [1], 3',4',5,6,7-pentahydroxy-flavone [2], and 5-hydroxy-4',7-dimethoxy-flavone [3], exhibit both antibiotic and biofilm inhibitory activities, the mode of action of such hydroxylated flavonoids with respect to S. aureus inhibition is yet to be characterized. Enzymatic digestion and high-resolution MS analysis of differentially expressed proteins from S. aureus with and without exposure to antibiotic flavonoids (1-3) allowed for the characterization of global protein alterations induced by metabolite treatment. A total of 56, 92, and 110 proteins were differentially expressed with bacterial exposure to 1, 2, or 3, respectively. The connectivity of the identified proteins was characterized using a search tool for the retrieval of interacting genes/proteins (STRING) with multitargeted S. aureus inhibition of energy metabolism and biosynthesis by the assayed flavonoids. Identifying the mode of action of natural products as antibacterial agents is expected to provide insight into the potential use of flavonoids alone or in combination with known therapeutic agents to effectively control S. aureus infection.

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 3.25 ± 0.23 (Mean ± S.E.M., n = 15, P < 0.001, t-test) and 3.13 ± 0.33 (Mean ± S.E.M., n = 6, P < 0.001, t-test), respectively. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP β and δ or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP β and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP.

The effect of statins on heme oxygenase-1 (HO-1) was compared in 2 murine cell lines, RAW 264.7 and J774A.1 cell lines, and in primary peritoneal macrophages of BALB/c or C57BL/6 mice. The role of endogenous nitric oxide and the type of transcription factors involved were explored. Simvastatin and fluvastatin induced HO-1. Pretreatment of cells with l-NMMA or 1400 W, two different nitric oxide synthase inhibitors, partially blocked statin-dependent induction of HO-1 in RAW 264.7 and J774A.1 but not in primary peritoneal macrophages. Induction of HO-1 by statins was dependent on p-38 MAP kinase activation in all types of macrophages. In RAW 264.7 cells, both statins increased the activity of reporter genes linked to the proximal 1.3 kbp promoter of HO-1 (EC50 of 1.4±0.3 µM for simvastatin and 0.6±0.03 µM for fluvastatin). This effect was significantly blocked by 1400 W (80±5.2% inhibition, p<0.02) and mevalonate, the direct metabolite of HMGCoA reductase. Gel retardation experiments implicated C/EBPβ, AP-1 but not USF, for both RAW 264.7 and primary peritoneal macrophages of C57BL/6 mice. Collectively we showed a differential role of endogenous nitric oxide between macrophage cell lines and primary macrophages and an effect of statins in the protection against inflammation by increasing HO-1 expression.

Heme‐oxygenase‐1 (HO‐1) is an antioxidant and anti‐inflammatory enzyme responsible for the degradation of heme into biliverdin, carbon monoxide and iron. Statins have pleiotropic effects in addition to lowering cholesterol. We have inverstigated the effect of statins on HO‐1 in Raw 267.4 cells and primary cells. Induction of HO‐1 was obtained with 25 μM simvastatin or 10 μM fluvastatin in both Raw 267.4 and peritoneal macrophages. Statins alone increased NO formation and iNOS expression in Raw 264.7 cells and pretreatment with inhibitors of NO synthase, l‐NMMA and 1400W blocked this effect. This induction was NO independent in primary eMPM cells. Both statins increased the promoter activity of the mouse proximal and distal promoter in Raw 264.7 cells, effect inhibited by mevalonate and 1400W. Gel retardation experiments were performed and both statins induced nuclear protein‐DNA complexes compared to untreated cells after 12 or 24‐hour incubation with probes for C/EBP, which were supershifted with antibodies specific for C/EBP β, or AP‐1 but not USF. In NIH 3T3 fibroblast cells, statins induced a significant increase in HO‐1 protein and mRNA levels and stimulated activity of mouse HO‐1 promoter (−1287 bp to +73 bp)/luciferase reporter gene by 3.25 ± 0.23 (Mean ± S.E.M., n= 15, p<0.001, t‐test) and 3.13 ± 0.33 (Mean ± S.E.M., n= 6, p<0.001, t‐test), respectively. Gel retardation experiments for C/EBP and USF on statin‐treated cells showed significant nuclear protein‐DNA complexes which were supershifted with antibodies specific for C/EBP or USF. In summary, statins induce HO‐1 and involve signaling molecules and mechanisms that vary among different cell types.