Archives

  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br Av treatment increases primary tumor volume

    2020-08-18


    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
    (caption on next page)
    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 115144-35-9 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].