Rincón-Sánchez AR. Cu/Zn superoxide dismutase (SOD1) induction is implicated in the antioxidative and antiviral activity of acetylsalicylic acid in HCV-expressing cells. Am J Physiol Gastrointest Liver Physiol 302: G1264 -G1273, 2012. First published March 22, 2012 doi:10.1152/ajpgi.00237.2011We evaluated the participation of oxidative stress in the negative regulation of hepatitis C virus (HCV)-RNA induced by acetylsalicylic acid (ASA). We used the HCV subgenomic replicon cell system that stably expresses HCV-nonstructural proteins (Huh7 HCV replicon cells) and the parental cell line. Cells were exposed to 4 mM ASA at different times (12-72 h), and pyrrolidine dithiocarbamate (PDTC) was used as an antioxidant control. Reactive oxygen species (ROS) production, oxidized protein levels, cytosolic superoxide dismutase (Cu/Zn-SOD), and glutathione peroxidase (GPx) activity were measured to evaluate oxidative stress. In addition, viral RNA and prostaglandin (PGE 2) levels were determined. We observed that ASA treatment decreased ROS production and oxidized protein levels in a time-dependent fashion in both parental and HCV replicon cells with a greater extent in the latter. Similar results were found with PDTC exposure. Average GPx activity was decreased, whereas a striking increase was observed in average cytosolic SOD activity at 48 and 72 h in both cells exposed to ASA, compared with untreated cells. HCV replicon cells showed higher levels of Cu/Zn-SOD expression (mRNA and protein) with ASA treatment (48 and 72 h), whereas NS5A protein levels showed decreased expression. In addition, we found that inhibition of SOD1 expression reversed the effect of ASA. Interestingly, PDTC downregulated HCV-RNA expression (55%) and PGE2 (60%) levels, imitating ASA exposure. These results suggest that ASA treatment could reduce cellular oxidative stress markers and modify Cu/Zn-

Commonly called the Mexican prickly poppy, Argemone mexicana is a stress-resistant member of the Papaveraceae family of plants that has been used in traditional medicine for centuries by indigenous communities in Mexico and Western parts of the United States. This plant has been exploited to treat a wide variety of ailments, with reported antimicrobial and antioxidant properties, as well as cytotoxic effects against some human cancer cell lines. Due to its various therapeutic uses and its abundance of secondary metabolites, A. mexicana has great potential as a drug discovery candidate. Herein, the germination conditions of A. mexicana are described and the cytotoxic activities of different parts (seeds, leaves, inner vs. outer roots) of the plant from methanol or hexane extracts are preliminarily characterized against cells of seven unique organisms. When comparing 1 mg of each sample normalized to background solvent alone, A. mexicana methanol outer root and leaf extracts possessed the strongest antimicrobial activity, with greatest effects against the Gram-positive bacteria tested, and less activity against the Gram-negative bacteria and fungi tested. Additionally, using the MTT colorimetric assay, the outer root methanol and seed hexane extracts displayed pronounced inhibitory effects against human colon cancer cells. Quantification of c-MYC (oncogene) and APC (tumor suppressor) mRNA levels help elucidate how the A. mexicana root methanol extract may be affecting colon cancer cells. After ultra-performance liquid chromatography coupled with mass spectrometry and subsequent nuclear magnetic resonance analysis of the root and leaf methanol fractions, two main antibacterial compounds, chelerythrine and berberine, have been identified. The roots were found to possess both phytocompounds, while the leaf lacked chelerythrine. These data highlight the importance of plants as an invaluable pharmaceutical resource at a time when antimicrobial and anticancer drug discovery has plateaued.

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Previously, we described that acetylsalicylic acid (ASA) decreases HCV expression, but the mechanisms involved have not been clearly established. We evaluated the participation of inducible nitric oxide synthase (iNOS) in the regulation of HCV-RNA induced by ASA. Huh7 cells expressing non-structural HCV proteins were exposed to 4 mM ASA and incubated at the same times we reported HCV downregulation (24-72 h), and iNOS mRNA and protein levels were then measured by real-time PCR and Western blot, respectively. Nitric oxide levels were measured at the same time. Inhibition of iNOS mRNA by small interfering RNAs (siRNA) and activation of the iNOS gene promoter by ASA treatment were evaluated. In Huh7 replicon cells treated with ASA, we found decreased levels of iNOS mRNA, iNOS protein and nitrosylated protein levels at 48-72 h. ASA exposure also reduced the transactivation of the iNOS promoter in HCV replicon cells at 48 h, and this was partly due to the decrease in the affinity of transcription factor C/EBP-β for its binding site in the iNOS promoter. siRNA silencing of iNOS decreased HCV-RNA expression (65 %) and potentiated the antiviral effect (80 %) of ASA compared with control cells. ASA reduces iNOS expression by downregulating promoter activity, mRNA and protein levels at the same time that it decreases HCV expression. These findings suggest that the antiviral activity of ASA is mediated partially through the modulation of iNOS.