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.

Figure 1

(a, b) H&E (hematoxylin and eosin) staining of (a) control tissue and (b) cervical tumor tissues; (c, d) iNOS antibody-specific tissue staining: (c) control cervix section showing weak iNOS expression and (d) cervical carcinoma section with an elevated level of iNOS expression showing strong intensity. (e) Significantly elevated iNOS expression in cervical cancer tissue in contrast to normal control assessed by RT-PCR.

Figure 2

Differential expression of NOS2 gene: elevated NOS2 gene expression in different cancers. ESCA: esophageal carcinoma; CESC: cervical squamous cell carcinoma; STAD: stomach adenocarcinoma; COAD: colon adenocarcinoma; KIRC: kidney renal clear cell carcinoma; LUSC: lung squamous cell carcinoma.

Figure 3

(a) 3D docking image of andrographolide with iNOS (nitric oxide acid), (b) two-dimensional representation of the interaction formed by andrographolide at the catalytical active site of iNOS, and (c) RMSD of native iNOS (4NOS) and protein-ligand complex (4NOS-andrographolide).

Figure 4

(a) Total contacts, (b) protein-ligand H bond, and (c) MD simulation hydrogen bond percentage.

Figure 5

(a) Analysis of the torsional degree of freedom during MD simulation trajectory for the rotational bonds present in the andrographolide, (b) analysis of RMS fluctuation (RMSF) trajectories generated by Schrodinger (Desmond).

Figure 6

(a) 2D structure of andrographolide; (b) analysis of Ligand Root Mean Fluctuation (L-RMSF) of the andrographolide ligand. (c) SSE distributions by residue index throughout the protein structure iNOS. (d) SSE distribution by residue index of iNOS-andrographolide complex, summary of SSE composition for each trajectory frame throughout the simulation, (e) SSE composition for only protein, and (f) SSE composition for (protein complex).

Figure 7

Cell viability due to andrographolide treatment was analyzed using MTT assay on (a) HeLa cells and (b) HEK cells; (c) Western blot analysis showing decrease in NOS2 expression in HeLa cells incubated with andrographolide, and GAPDH was used as control; (d) dose-dependent reduction of iNOS was observed in response to andrographolide treatments. ns: not significant. ∗∗ and ∗∗∗ indicate significance at p < 0.05 level.

Figure 8

(a–c) DAPI-stained HeLa cell images of both (a) untreated control and treated andrographolide-induced apoptosis showing blue florescent nuclear fragmentation in HeLa cells at (b) 5 μM and (c) 10 μM, observed under an inverted fluorescence microscope (at magnification 20x). (d–g) Antimigration potency measured with andrographolide at (e) 5 μM and (f) 10 μM showing less migration than (d) control at 24 h. (g) Relative size of open scratch area (in percentage) in all the treatments and controls.

Figure 9

Flow cytometric analysis of andrographolide-treated HeLa cells showed dose-dependent (a–d) cell cycle arrest at G1/S transition phase, (e–h) Annexin V-FITC/PI-stained apoptotic cells. (a, e) Represent untreated control, (b, f) represent 5 μM of andrographolide, and (c, g) represent 10 μM of andrographolide. (d) Relative distribution of cell cycle percentage and (h) comparative percentage of the cell population in early and late apoptosis in control and andrographolide treatments.