Inhibition of Inducible Nitric Oxide Synthase (iNOS) by Andrographolide and In Vitro Evaluation of Its Antiproliferative and Proapoptotic Effects on Cervical Cancer.

Akbar Pasha,Gangappa Dharmapuri,Annapurna S D,Pavani Soujanya,I Arnold Emeson,Divya Vishambhar Kumbhakar,Vijayalaxmi Bodiga,Smita C Pawar,Preethi Basavaraju,Ravinder Doneti,Kiran Kumar,Deepthi Pasumarthi,Pavan Kumar Poleboyina,Heena S K,Guodong Zhang

doi:10.1155/2021/6692628

Abstract

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.

Highlights

Cervical cancer (CC) ironically is still the foremost common prevalent cancer in women globally, though being the most preventable disease
Various cancers known for enhanced inducible nitric oxide synthase (iNOS) levels are related to bacterial or viral infection like cervical, gastric, prostate, and esophageal cancers and hepatocellular carcinoma [16,17,18]. iNOS expression correlates with VEGF and together is very vital for tumor growth; high levels of NO produced by the NOS2 triggers the angiogenesis process in the tumors by regulating the VEGF expression
The iNOS was darkly stained in the cervical cancer tissue section, showing strong intensity with enhanced iNOS expression both in cytoplasm and nucleus (Figure 1(d)), and the cells show dark nuclei supporting the selective immunopositivity of iNOS, whereas the normal cervical epithelial cells were stained less (Figure 1(c)) showing weak iNOS expression

Summary

Introduction

Cervical cancer (CC) ironically is still the foremost common prevalent cancer in women globally, though being the most preventable disease. Inflammation is a response of the immune defense mechanism to harmful stimuli released by various infected cells; it releases cytokines, ROS, and hormones to maintain this inflammatory response This persistent inflammation plays a significant role in carcinogenesis and contributes to cellular transformation, proliferation, invasion, and angiogenesis [5]. NOS2 knockdown in SiHa and HeLa cells has shown a decrease in cell proliferation due to the decrease in the concentration of VEGF confirming that the NOS2 regulates the growth of cervical cancer cells in a VEGF-dependent process [14]; effective inhibition of iNOS expression will thereby reduce the NO levels, triggered by inflammatory stimuli in cancer cells, and pronounces to be a valuable therapeutic strategy. The aim of this study was to elucidate the interaction of andrographolide with iNOS by employing in silico molecular docking and molecular dynamic simulations and evaluate its inhibitory activity on the iNOS in cervical cancer HeLa cells

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