Celecoxib inhibits NLRP1 inflammasome pathway in MDA-MB-231 Cells

Abstract

NLRP1 is predominantly overexpressed in breast cancer tissue, and the evaluated activation of NLRP1 inflammasome is associated with tumor growth, angiogenesis, and metastasis. Therefore, targeting NLRP1 activation could be a crucial strategy in anticancer therapy. In this study, we investigated the hypothesis that NLRP1 pathway may contribute to the cytotoxic effects of celecoxib and nimesulide in MDA-MB-231 cells. First of all, IC₅₀ values and inhibitory effects on the colony-forming ability of drugs were evaluated in cells. Then, the alterations in the expression levels of NLRP1 inflammasome components induced by drugs were investigated. Subsequently, the release of inflammatory cytokine IL-1β and the activity of caspase-1 in drug-treated cells were measured. According to our results, celecoxib and nimesulide selectively inhibited the viability of MDA-MB-231 cells. These drugs remarkably inhibited the colony-forming ability of cells. The expression levels of NLRP1 inflammasome components decreased in celecoxib-treated cells, accompanied by decreased caspase-1 activity and IL-1β release. In contrast, nimesulide treatment led to the upregulation of the related protein expressions with unchanged caspase-1 activity and increased IL-1β secretion. Our results indicated that the NLRP1 inflammasome pathway might contribute to the antiproliferative effects of celecoxib in MDA-MB-231 cells but is not a crucial mechanism for nimesulide.

Keywords: Anticancer drug; COX-2; Celecoxib; NLRP1 inflammasome; Triple-negative breast cancer.

Fig. 1

Cell viability analysis after incubation with celecoxib or nimesulide. MDA-MB 231 (a, c) and MCF-10A (b, d) cells were treated with increasing concentrations of each drug for 48 h, and the cell viability was measured by WST-1 assay. a Celecoxib-MDA-MB-231 cells; b celecoxib-MCF-10A cells; c nimesulide-MDA-MB-231 cells; d nimesulide-MCF-10A cells. Each value shown as mean ± S.D. was expressed as a percentage of the control (0.1% DMSO), and IC₅₀ values were calculated from the dose–response curves using GraphPad Prism® 8.4.2 software. 1% Triton-X was used as a positive control (data not shown)

Fig. 2

Celecoxib and nimesulide inhibit the colony formation ability of MDA-MB-231 cells. Cells were treated with each drug at three dose levels (IC₂₅, IC₅₀, and IC₇₅) for 14 days. Following the treatment period, colonies were stained by 1.25% crystal violet. NIM, nimesulide; CXB, celecoxib. The number of surviving colonies was expressed as a percentage of the control (0.1% DMSO). Values were expressed as mean ± S.D. ***p < 0.0001 (vs. control group)

Fig. 3

Protein expression of NLRP1, cleaved-caspase-1, ASC, and β-actin. a Semi-quantitative bands probed with anti-NLRP1, and -β-actin antibodies in several breast cell lines; b densitometric analyses of NLRP1 protein expression in several breast cell lines; c semi-quantitative bands probed with anti-NLRP1, -cleaved caspase-1, -ASC, and -β-actin antibodies in MDA-MB-231 cells; d densitometric analyses of NLRP1 protein expression in MDA-MB-231 cells; e densitometric analyses of cleaved caspase-1 protein expression in MDA-MB-231 cells; f densitometric analyses of ASC protein expression in MDA-MB-231 cells. Band density was measured with ImageJ software. The protein values were normalized to β-actin. NIM, nimesulide; CXB, celecoxib. For b; ***p < 0.0001 compared with MCF-10A, for Fig. 3d–f; *p < 0.05, ***p < 0.0001 compared with the control group (0.1% DMSO)

Fig. 4

The effects of celecoxib and nimesulide on caspase-1 activity. MDA-MB-231 cells were treated with different concentrations of each drug for 48 h. Caspase-1 activity was determined fluorometrically. NIM, nimesulide; CXB, celecoxib. 0.1% DSMO was used as a solvent control. Values were represented as mean ± S.D. ***p < 0.0001, compared to control

Fig. 5

The effects of celecoxib and nimesulide on the levels of IL-1β. MDA-MB-231 cells were treated with different concentrations of each drug for 48 h. The levels of IL-1β were measured in the cell supernatant by an ELISA assay. NIM, nimesulide; CXB, celecoxib. 0.1% DMSO served as a control. Values were represented as mean ± S.D. *p < 0.05, **p < 0.001, and compared to ***p < 0.0001, compared to control

Fig. 6

A proposed schematic model presenting the mechanism of antiproliferative effects induced by celecoxib in MDA-MB-231 cells. Exposure to celecoxib downregulates the protein expression of NLRP1, which is markedly upregulated in MDA-MB-231 cells. Moreover, celecoxib inhibits the protein levels of ASC and cleaved caspase-1. The decrease in NLRP1 inflammasome activation resulted in lower levels of caspase-1 activation and IL-1β secretion