Diallyl disulfide inhibits TNFα-induced CCL2 release by MDA-MB-231 cells

Abstract

Monocyte chemotactic protein-1 (MCP-1/CCL2) is released by tumor tissues, serving as a potent chemokine enabling directional homing of mononuclear cells to tumor tissue, which subsequently differentiate into tumor-associated macrophages (TAMs) via TGFβ1 signaling. TAMs readily invade tumor tissue and continue to synthesize pro-oncogenic proteins including tumor growth factors, matrix proteases (metastasis), angiogenic factors (neovascularization) and CCL2. Substances, which can attenuate or block the initial release of CCL2 have been shown to prevent cancer-associated inflammative pro-oncogenic processes. In the current study, we investigated the effects of the organosulfur compound diallyl disulfide (DADS), a natural constituent of Allium sativum (garlic) on suppression of TNFα-induced release of CCL2 from triple-negative human breast tumor (MDA-MB-231) cells. Using an initial adipokine/chemokine protein panel microarray, the data show a predominant expression profile in resting/untreated MDA-MB-231 cells for sustained release of IL6, IL8, plasminogen Activator Inhibitor 1 and TIMP1/2. Treatment with TNFα (40 ng/ml) had no effect on many of these molecules, with a single major elevation in release of CCL2 (~1,300-fold up-regulation). TNFα-induced CCL2 release was reversed by a sub-lethal concentration of DADS (100 μM), evident in antibody based assays. These findings provide evidence to support another avenue of anticancer/chemopreventative properties attributable to garlic constituents through immunomodulation.

Keywords: Tumor-associated macrophages; diallyl disulfide; garlic constituents; monocyte chemotactic protein-1.

Figure 1

The effect of DADS on cell viability of MDA-MB-231 cells at 5% CO₂/Atm for 24 hr. The data are presented as mean±S.E.M. (n=4). Significance of differences from the control were determined by a one-way ANOVA, with a Tukey post hoc test. *p<0.05 compared to control.

Figure 2

The effect of TNFα on cell viability of MDA-MB-231 cells at 5% CO₂/Atm for 24 h. The data are presented as the mean±S.E.M. (n=4). Significance of differences from the control were determined by a one-way ANOVA, with a Tukey post-hoc test. *p<0.05.

Figure 3

A: Microarray layout. OSM, Oncostatin M; TPO, thrombopoietin; POS, positive control; NEG, negative control. Each protein is in duplicate. Positive controls are located in the upper left (n=4) and lower right (n=2) corners to insure equal distribution of supernatant (top). B: Microarray chemiluminescent spot intensity analysis of supernatant derived from resting MDA-MB-231 cells. POS controls are located in the upper left and lower right corners with dominant cytokines demarcated. C: Baseline cytokine release in untreated MDA-MB-231 cells corresponding to image. The data are presented as spot intensity and are the mean±S.E.M. (n=6). See Table I for cytokine abbreviations.

Figure 4

TNFα induced cytokine expression by a dominant fold change in MDA-MB-231 cells. The data show a large differential up-regulation of CCL2 protein release amongst the 62 proteins evaluated. The data are presented as fold change and are the mean±S.E.M. (n=6).

Figure 5

Spot intensity analysis (A) of antibody- coated array membranes with quantitative analysis of chemiluminescent signal (B) and ELISA (C) for controls, DADS-treated (100 μM), TNFα-treated (40 ng/ml) and co-treated cells. The data are presented as the mean±S.E.M. (n=6) and significance of differences were determined by t-test. *p<0.05.

Figure 6

Soluble TNFRI release in MDA-MB-231 cells for groups: control, DADS-treated (100 μM), TNFα-treated (40 ng/ml) and co-treated cells. The data are presented the mean±S.E.M. as percentage of control (n=6). Significance of differences from the controls in both groups were determined by t-test. *p<0.05.