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

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.

Andrographolide: A review of its pharmacology, pharmacokinetics, toxicity and clinical trials and pharmaceutical researches

“…Given that the toxicity and side effects has been acknowledged with the vast majority of chemotherapeutic agents, in the past few decades, researchers have paid increasing attention to the anticancer investigation of natural active ingredients, especially andrographolide (Islam et al, 2018; Malik et al, 2021; Soo et al, 2019). Published projects have validated the multiple anticancer effects, such as its ability to colon cancer (Banerjee et al, 2021; Vukmirovic, Vo, Seymour, Rollo, & Mothersill, 2021), lung cancer (Luo et al, 2021), cervical cancer (Pasha et al, 2021), breast cancer (Wanandi et al, 2020), hepatoma cancer (Shi, Zhang, Zheng, Lu, & Ji, 2017) as well as renal carcinoma (Bi et al, 2020) and so on, which the cytotoxic effects on these cancer cells above were bound up with inhibiting proliferation (Khan, Mahfooz, & Ansari, 2020), migration and invasion (Pearngam, Kumkate, Okada, & Janvilisri, 2019), blocking cell cycle (Khan, Mahfooz, Faisal, Alatar, & Ansari, 2020), inducing apoptosis (Wang et al, 2020), restraining tumor angiogenesis (Kajal et al, 2019), as well as enhancing chemosensitivity (Li et al, 2020; Mao, He, Wang, Wu, & Wei, 2019). The main anticancer mechanisms of andrographolide are depicted in Figure 4.…”

An insight into the pharmacological and analytical potential of Andrographolide

“…UV visible spectroscopic, melting temperature, and molecular fluorescent studies confirmed that annonalide interacts with CT DNA through intercalation with a stoichiometric ratio of 1:1. Docking study revealed that diterpene and DNA interact through van der Waals interaction [112]. Multiple interaction studies have been performed using different spectroscopic techniques and computational tools to extract the therapeutic value of andrographolide [113,114].…”

Advances in ameliorating inflammatory diseases and cancers by andrographolide: Pharmacokinetics, pharmacodynamics, and perspective

“…254 Andrographolide could also inhibit cervical cancer cell proliferation and promote apoptosis via decreasing inducible nitric oxide synthase (iNOS). 255 Beyond the above non-solid tumors, According to Loh et al, andrographolide inhibited cell survival and migration in human cervical cancer cells. This study showed therapeutic effects in treating cervical cancer may occur in andrographolide.…”

“…Parthenolide and andrographolide, according to Gunn et al, have anticancer stem cell action in multiple myeloma stem cells 254 . Andrographolide could also inhibit cervical cancer cell proliferation and promote apoptosis via decreasing inducible nitric oxide synthase (iNOS) 255 . Beyond the above non‐solid tumors, According to Loh et al, andrographolide inhibited cell survival and migration in human cervical cancer cells.…”