br Fig Overexpression of Cx
Fig. 6. Overexpression of Cx43 enhances sensitivity of MDA-MB-231 PFK-158 to Av treatment. (A) Western blot analysis of total protein obtained from cell lysates of Av-treated MDA-Cx43D cells for NF-κB p65 and GAPDH. (B) Densitometric quantification of NF-κB p65. (C&D) Immunofluorescence staining of RelA in MDA-Cx43D cells treated with Av for 24 h followed by quantification of nuclear:cytoplasmic ratios of Rel A in a time course study of NF-κB nuclear translocation. (E) A representative flow cytometry graph where the shift in MFI is shown. Results are representative of three diﬀerent independent experiments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.
3.1.1. Av treatment induces a mesenchymal like phenotype in MDA-MB-
MDA-MB-231 cells treated with Av showed a change in morphology towards a more mesenchymal-like shape compared to a more cuboidal
shape in control cells (Fig. 1A). This morphological change was ac-companied by a significant 50% increase in proliferation of MDA-MB-231 cells treated with Av compared to control untreated cells (Fig. 1B).
3.1.2. Av treatment increases inflammation in MDA-MB-231 cells
The expression of diﬀerent pro-inflammatory mediators (IL-17, IL-6, IL-12a, IL-1β, TNF-α and RAGE) and the anti-inflammatory cytokines IL-10 and IL-13 was examined in Av-treated cells using qPCR. Results
Fig. 7. Cx43 overexpression leads to the downregulation of the mRNA and protein levels of inflammatory mediators (IL-1β and TNF-α) and VEGF in MDA-MB-231 cells. qPCR showing the eﬀect of Cx43 overexpression on IL-1β mRNA levels (A), TNF-α (B), VEGF (C). (D, E and F) Levels of secreted IL-1β, TNF-α and VEGF, respectively, as determined by ELISA. Results are representative of three diﬀerent independent experiments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.
showed that Av treatment significantly increased IL-17, IL-6, IL-12a, IL-1β, TNF-α and RAGE and significantly decreased IL-10 mRNA levels, as compared to untreated cells (Fig. 1C and D).
Evaluation of secretion levels of TNF-α and IL-1β confirmed the data obtained by qPCR where their secretion levels increased by 75% fol-lowing Av treatment (Fig. 1E).
Upregulation of pro-inflammatory cytokines mRNA and protein le-vels by Av treatment lead to the investigation of the NF-κB pathway activation and subsequent NF-κB translocation into the nucleus. Immunofluorescence revealed a significant increase in nuclear: cyto-plasmic ratio of NF-κB in Av-treated cells (Fig. 2A and B). In addition, a significant increase in NF-κB p65 protein expression and IκB-α phos-phorylation was observed accompanied by a significant decrease in IκB- α protein levels (Fig. 2C and D).
3.1.3. Av significantly down regulates transcriptional levels of VEGF while it significantly upregulates transcription of invasion and EMT markers
To examine the eﬀect of Av on angiogenic, communication and EMT genes, we studied the expression of VEGF, Cx43, MMP2, MMP9, Twist, Snail, N-Cadherin and E-Cadherin (E-Cad) by qPCR. Av significantly downregulated VEGF mRNA expression (Fig. 3A) but showed no eﬀect on Cx43 mRNA levels at 24 h of Av treatment (Fig. 3B and C). In ad-dition, Av caused a time-dependent increase in Twist, Snail, N-Cad-herin, MMP2 and MMP9 (Figs. 4 and 5A and B), and concurrently re-sulted in a time-dependent decrease in E-Cadherin mRNA levels, a mesenchymal-to-epithelial marker, reaching a maximal 50% decrease at 48 h following Av treatment (Fig. 4D).
3.1.4. Av treatment increases migration and invasion of MDA-MB-231 cells The enzymatic activities of MMP2 and MMP9 showed a time-de-pendent increase, 24 h and 48 h after Av treatment of MDA-MB-231 cells as revealed by Gelatin zymography (Fig. 5C). This increase was
correlated with a higher invasive potential of MDA-MB-231 cells fol-lowing Av treatment (Fig. 5D).
3.1.5. Overexpression of Cx43 enhances the sensitivity of MDA-MB-231 cells to Av
We next investigated the eﬀect of Cx43 overexpression on Av-in-duced inflammation. Overexpression of Cx43 in MDA-MB-231 cells lead to a significant decrease of the protein levels of NF-κB p65 and phos-phorylated IκBα and to a decrease of the levels of transcripts of in-flammatory mediators (IL-1β and TNF-α) and the angiogenic factor, VEGF. Consistently, Av treatment of MDA-Cx43D cells caused a similar decrease in NF-κB pathway components as compared to untreated cells (Figs. 6 and 7). Furthermore, Av-treated MDA-Cx43D cells showed a decrease in the protein expression levels of CD44, a target of NF-κB signaling. Fig. 6E, shows a shift of the histogram to the left indicative of a decrease in mean fluorescence intensity (MFI) and thus of CD44 protein expression levels in Av-treated cells. Immunofluorescence showed a significant decrease in nuclear: cytoplasmic ratio of NF-κB (Fig. 6C and D), further confirming that Cx43 overexpression alleviates the Av-induced inflammatory state in MDA-MB-231 cells. The decrease of NF-κB translocation in Av-treated MDA-Cx43D cells was paralleld by a decrease in the migration and invasion of Av-treated MDA-Cx43D cells (Fig. 8B and C), this was supported by a decreased MMP9 pro-teolytic activity (Fig. 8D and E).