This work is aimed at investigating the expression levels of inducible nitric oxide synthase (iNOS) in cervical cancer and identifying a potential iNOS inhibitor. The data mining studies performed advocated iNOS to be a promising biomarker for cancer prognosis, as it is highly overexpressed in several malignant cancers. The elevated iNOS was found to be associated with poor survival and increased tumor aggressiveness in cervical cancer. Immunohistochemical and RT-PCR investigations of iNOS showed significant upregulation of endogenous iNOS expression in the cervical tumor samples, thus making iNOS a potent target for decreasing tumor inflammation and aggressiveness. Andrographolide, a plant-derived diterpenoid lactone, is widely reported to be effective against infections and inflammation, causing no adverse side effects on humans. In the current study, we investigated the effect of andrographolide on the prognostic value of iNOS expression in cervical cancer, which has not been reported previously. The binding efficacy of andrographolide was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), torsional degree of freedom, protein-root mean square fluctuations (P-RMSF), ligand RMSF, total number of intramolecular hydrogen bonds, secondary structure elements (SSE) of the protein, and protein complex with the time-dependent functions of MDS. Ligand-protein interactions revealed binding efficacy of andrographolide with tryptophan amino acid of iNOS protein. Cancer cell proliferation, cell migration, cell cycle analysis, and apoptosis-mediated cell death were assessed in vitro, post iNOS inhibition induced by andrographolide treatment (demonstrated by Western blot). Results. Andrographolide exhibited cytotoxicity by inhibiting the in vitro proliferation of cervical cancer cells and also abrogated the cancer cell migration. A significant increase in apoptosis was observed with increasing andrographolide concentration, and it also induced cell cycle arrest at G1-S phase transition. Our results substantiate that andrographolide significantly inhibits iNOS expression and exhibits antiproliferative and proapoptotic effects on cervical cancer cells.

Cervical cancer is the second most common cause of cancer deaths in women worldwide and remains the main reason of mortality amongst women of reproductive age in developing countries. Nitric oxide is involved in several physiological functions inclusive of inflammatory and immune responses. However, the function of NO in tumor biology is debatable. The inducible NOS (iNOS/NOS2) isoform is the one responsible to maintain the levels of NO and it exhibits pleotropic effects in various cancer with concentration-dependent pro-and anti-tumor effects.NOS2 triggers angiogenesis and endothelial cell migration in tumors by regulating the levels of vascular endothelial growth factor (VEGF). In drug discovery, drug repurposing involves investigations of approved drug candidates to treat various other diseases. In this study, we used FDA-approved anti-cancer drugs and small molecules to target iNOS and identify a potential selective iNOS inhibitor. The structures of ligands were geometrically optimized, and energy minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker and ligands were selected based on MolDock score,Rerank score, and H-bonding energy. In the study showed 4 compounds, Degarelix, Goserelin, Triptorelin pamoate, and venetoclax demonstrated excellent binding affinity to NOS2 protein. These compounds exhibited the lowest MolDock score, Rerank score, with better H-bonding energy to NOS2. Based on the results theses ligands project to be promising potential NOS2 inhibitors to curtail cervical cancer progression

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