br Av treatment increases primary tumor volume

3.2.1. Av treatment increases primary tumor volume and does not block metastasis in the xenograft mouse model
Av treatment increased the volume of primary tumors of NSG mice xenografted with MDA-MB-231 115144-35-9 (Supplementary Fig. S1). Moreover, Av treatment failed to inhibit metastases of MDA-MB-231
Fig. 8. Upregulation of Cx43 significantly decreases migration and invasion of MDA-Cx43D cells after Av treatment. (A) Proliferation, (B) Migration and (C) Invasion quantification graphs, as detected by Real-Time Cell Analysis (RTCA) assay, after normalizing cell index values to control untreated cells. Cell im-pedance readings were taken every 15 min for a minimum of 18 h. (D) Gelatin zymography of Av-treated MDA-Cx43D cells. FBS was used as an in-ternal control. Proteins were separated on a gel containing gelatin, the substrate of MMPs, in order to assess the activation status and levels of these en-zymes. (E) Quantification analysis MMP2 and MMP9 activity levels showing the effect of Cx43 over-expression on control and Av-treated cells. The in-tensity of each band was determined by densito-metry, using Image Lab software. Quantification of each band was normalized to control. Results are representative of three different independent ex-periments. *, **, *** indicate P < .05, P < .001, P < .0001, respectively.
cells to the lungs of mice xenografted with MDA-MB-231 cells. Supplementary Fig. S2, shows that metastatic lesions have appeared in the lungs of both Av-treated or vehicle control xenografted mice.
3.2.2. Av treatment increases transcriptional expression of Cx43, N-Cadherin and inflammatory mediators
Next we assessed whether Av-induced inflammation in MDA-MB-231 cells in culture translates in vivo in the xenograft mouse model. mRNA levels of several of the markers acting during Av treatment of MDA-MB-231 in vitro were quantified by qPCR in metastatic lung tis-sues obtained from xenografted mice. Results showed a significant in-crease in the transcriptional level of IL-1β at week 5 which became more pronounced at week 9 following Av treatment. RAGE expression was also significantly increased at week 9 of Av treatment (Fig. 9A and B). Although VEGF and Cx43 expression significantly decreased at week 5, their expression increased at week 9 (Fig. 9A and B). Protein levels of NF-κB pathway components were also evaluated. At week 5 following xenograft, there was an increase in the protein levels of NF-κB pathway components including inflammatory mediators (TNF-α and IL-1β) as well as N-Cadherin (Fig. 9C and D). Immunofluorescence staining on 
lung tissues obtained from Av-treated xenografted mice further con-firmed activation of inflammatory signaling pathways. Fig. 9G shows a significant increase in the phospohorylation of IκB-α. Surprisingly, at week 9 all inflammatory mediators showed a significant decrease (Fig. 9E and F). This was accompanied by a significant increase in Cx43 expression (Fig. 9E and F).
3.3. Archived cases of different breast cancer sub-types
3.3.1. Tumor tissues show higher expression of inflammatory mediators and a lower expression of anti-inflammatory cytokine (IL-10)
We assessed the inflammatory state in a cohort of breast carcinoma patients of different sub-types by measuring their mRNA levels using qPCR. Human breast tumor tissue showed a significant mRNA expres-sion of RAGE, TNF-α and IL-1β but not IL-17 as compared to their non-cancerous counterparts (Fig. 10A, B, C and F). Whereas IL-13 expression was increased, IL-10 mRNA levels were significantly decreased (Fig. 10D and E). When comparing the three sub-types, we found that TNBC tissues showed significantly higher IL-17 mRNA levels accom-panied by significantly lower levels of IL-13 as compared to normal
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counter part breast tissue (Fig. 11). Interestingly, TNF-α mRNA levels were significantly lower levels in all groups of breast cancer patients (Fig. 11B).
4. Discussion
Tumor-promoting inflammation is an enabling characteristic of cancer [34]. Therapy-induced inflammation may lead to tumor re-emergence and resistance to treatment [35]. In this study we in-vestigated whether cancer cells become refractory to Av due to therapy-induced inflammation using MDA-MB-231 human breast cancer cells in vitro and in vivo. We have shown that Av induced inflammation, where the NF-κB pathway and its down-stream components were activated. Data showed that Av treatment increased protein expression levels of NF-κB along with its translocation into the nucleus and decreased the protein levels of its regulatory protein, IκB [36]. After activation, NF-κB binds to promoter sequences of its target genes, which play key roles in cellular growth, inflammation and apoptosis [37]. Inflammatory med-iators are prime NF-κB target genes, and in this study Av-activated NF-κB altered expression of several inflammatory mediators. Data on ex-pression levels of RAGE, IL-1β and TNF-α, emphasizes this fact and il-lustrates the effect of Av on inflammation. In addition, constitutive activation of NF-κB has been also observed in breast [38], lung [39], lymphoma [40], and leukemia [41] cell lines. Moreover, increased NF-κB levels associate with poor prognostic consequences in glioblastoma [42] and ovarian cancer [43]. This can be explained by anti-tumor responses mediated by the inhibition of NF-κB signaling or by NF-κB gene knockout [44,45].