Targeting NF-κB mediated cell signaling pathway and inflammatory mediators by 1,2-diazole in A549 cells in vitro

Abstract

Lung cancer is the leading cause of cancer deaths globally. The objective of this study was to investigate the effect of 1,2-diazole (pyrazole) as an anti-cancer drug on human non-small cell lung carcinoma A549 cells. We attempt to examine the expression level of pro-inflammatory proteins such as TNF-α, NF-κB-p65, MMP-2 and E-Cadherin which are commonly associated with an inflammatory response in epithelial cells and apoptosis in A549 cells. The LPS-induced cytokines and inflammatory mediators include TNF-α, IL-6, iNOS and COX-2 levels in A549 cells and the effect of pyrazole was studied. The present study reveals that, pyrazole inhibits A549 cells by suppressing TNF-α induced MMP-2 expression, thereby inhibiting the nuclear translocation of NF-κB-p65. Pyrazole significantly up-regulate the E-cadherin level and down-regulated MMP-2 expression that could probably preventing A549 cancer cells to invade. The study further substantiated the anti-cancer property of pyrazole by regulating the above mentioned level of LPS-induced cytokines and inflammatory mediators. The observations of the present study open a possibility for the development of an effective therapeutic agent that targets inflammatory and signaling pathway mediators to challenge human non-small cell lung carcinoma.

Keywords: 1,2-diazole; A549 cells; Cell signaling; Inflammatory mediators; Lung cancer; Pyrazole.

Graphical abstract

Fig. 1

Representation of Structure of 1,2-diazole (pyrazole).

Fig. 2

RT-PCR analysis of mRNA expression: (A) TNF-α, (B) NF-κB-p65, (C) E-Cadherin, (D) MMP-2 and (E) β-actin are presented as compared with A549 control (untreated) cells. Lane 1- DNA Ladder, Lane 2- A549 control (untreated) cells, Lane 3 & 4 - Pyrazole treated A549 cells (24 & 48 h respectively).

Fig. 3

Densitometric values of mRNA expression: The densitometric values of mRNA expression of TNF-α, NF-κB-p65, E-Cadherin, and MMP-2 normalized with β-actin are presented as compared with untreated A549 control cells. Values are expressed as Mean ± SD (n = 3). *p < 0.05, the p-value is considered as significant from untreated control.

Fig. 4

Effects of pyrazole on the expressions of TNF-α, NF-κB-p65, E-Cadherin, MMP-2 and β-actin in A549 cells. Cells were treated with pyrazole (75 μM) and the expressions of TNF-α, NF-κB-p65, E-Cadherin, MMP-2 and β-actin were determined by western blot compared with A549 control (untreated) cells. Lane 1- Untreated A549 control cells, Lane 2 & 3 - pyrazole treated A549 cells (24 & 48 h respectively).

Fig. 5

Densitometric values of the expression of TNF-α, NF-κB-p65, E-Cadherin, and MMP-2 were represented as compared with untreated control A549 cells. Values are expressed as Mean ± SD (n = 3). (*p < 0.05) the p-value is considered as significant from untreated control.

Fig. 6

Effect of pyrazole on cytokines and inflammatory mediators in lipopolysaccharide (LPS) exposed A549 cells: Densitometric values of the level of TNF-α, IL-6, iNOS and COX-2 in LPS exposed A549 cells treated with pyrazole at 75 μM concentrations for 24 h and 48 h are resented as compared with untreated control cells and LPS alone exposed A549 cells. Values are expressed as Mean ± SD (n = 3). *p < 0.05, the p-value is considered as significant from untreated control.

Fig. 7

DNA fragmentation using agarose gel electrophoresis of DNA extracted from A549 cells treated with pyrazole (75 μM) for 24 and 48 h. Lane 1- DNA marker, Lane 2- A549 cells, Lane 3 & 4 - pyrazole treated A549 cells (24 & 48 h respectively).

Fig. 8

Overall, pyrazole significantly up-regulate E-cadherin expression and regulated MMP-2 expression in A549 cells. Moreover, pyrazole inhibited the expression of TNF-α and NF-κB-p65 which specifically blocked NF-κB activation which was stimulated by TNF-α. The above anti-cancer effect was also substantiated by the role of pyrazole in regulating LPS-induced levels of cytokines and inflammatory mediators such as IL-6, TNF-α, iNOS and COX-2 in A549 cells as represented in the Fig. 8.