Endotoxin Triggers Tumor Initiation Events in Nontumorigenic Breast Epithelial Cells and Enhances Invasion-Related Phenotype in Pretumorigenic and Tumorigenic Breast Epithelial Cells

Figure 4

Pretumorigenic Cx43-KO S1 human breast epithelial cells secrete higher levels of MMP-9, and their invasive ability is enhanced upon endotoxin exposure more than their nontumorigenic S1 counterparts. (a) Short-term and long-term exposures of S1 and Cx43-KO S1 cells to 10 µg/ml ET enhanced the invasion across Matrigel. Scale bar is 10 µm. (b) Fold increase in the number of invading cells subjected to short-term and long-term ET treatment at 10 µg/ml ET relative to untreated controls (0 µg/ml). (c) Gelatin zymography of untreated S1 and Cx43-KO S1 media collected from 2D cultures upon reaching 75% confluence showed upregulation of MMP-9 activity in Cx43-KO S1 compared to S1 cells. (d) Quantification of fold increase in peak area of MMP-9 bands shown in the previous zymogram, corrected for the 30% increase in cell counts observed with Cx43-KO S1 compared to S1 cells. (e) Gelatin zymography and (f) quantification of fold increase in peak area of MMP-9 bands of conditioned media collected from 2D cultures of S1 and Cx43-KO S1 cells, including untreated controls (0 µg/ml) and ET-treated (10 µg/ml) cells, upon reaching 75% confluence after treatment and after long-term exposure (one month), corrected for the 30% increase in cell counts of Cx43-KO S1 compared to S1 cells. Results showed upregulation of MMP-9 activity in S1 and Cx43-KO S1 culture media after both ET exposures, as compared to untreated controls of each cell type. Samples of each cell type were run on the same gel and under the same conditions. Each bar represents triplicate analyses of mean ± SD. ; ; compared to the untreated control.

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