A 61603 br Av increased the expression of metalloproteinases
Av increased the expression of metalloproteinases (MMP2 and MMP9) and EMT markers (Twist and Snail). Consistent with our results, previous studies showed that NF-κB regulates expression of EMT mar-kers . Moreover, MMP2 and MMP9 are targets of many therapeutic treatments since their expression levels and activities are associated with higher invasive phenotypes . Previous studies elucidated that downregulation of microRNA-138 enhances the proliferation, migra-tion and invasion of cholangiocarcinoma A 61603 through the upregulation of MMP2 and MMP9 .
Moreover, Av elicited an enhanced migration not only in metastatic cells such as MDA-MB-231 cells, but also in non-invasive MCF-7 cells (Fig. 2, in Data in Brief). Noteworthy, there was no change in the morphology of Av-treated MCF-7 cells, unlike Av-treated MDA-MB-231 which became more mesenchymal (Fig. 1, in Data in Brief).
MDA-MB-231 cells express low levels of Cx43 and have sparse in-tercellular communication. Surprisingly, upregulation of Cx43 alle-viated the eﬀect of Av on inflammation . In fact, VEGF biosynthesis decreased upon upregulation of Cx43. This finding is in line with pre-vious studies that reported the dominance of epithelial phenotype and attenuation of angiogenesis upon Cx43 upregulation . Upregulation of Cx43 combined with Av treatment not only decreased the expression of inflammatory mediators but also decreased translocation of NF-κB. Moreover, it decreased the expression of CD44 that is involved in many cellular processes including cell adhesion and migration [49–52]. These observations are consistent with previous observations where inhibition of NF-κB lead to a decrease in CD44 expression which resulted in de-creased cell proliferation and invasiveness of MDA-MB-231 cells . In addition, our data showed that Av-treated MDA-MB-231 cells elicited a decrease in invasion and migration potential accompanied with a decrease in MMP9 activity. A previous study showed that, MMP9 down-
regulation is associated with a decrease in invasion and vascularization of skin cancer .
In the in vivo assays, tumor cells were injected sub-dermally into the lower neck region where a primary tumor forms. This model allows the development of solid tumors at the site of injection, that later metas-tasize. Expression levels of inflammatory mediators were assessed in lung tissues harvested at weeks 5 and 9 following xenograft. Results confirmed the observations of in vitro studies. However, at week 9, the protein expression level of all mediators decreased while that of Cx43 significantly increased. This stated result matches with a previous work that reported that exposure to inflammatory conditions increases Cx43 expression . Therefore, the observed decrease in inflammatory state at week 9 may be due to the induced upregulation of Cx43 which parallels the observation of in vitro study on cells upregulating Cx43.
Experiments done on human samples showed that inflammatory mediators (RAGE, TNF-α and IL-1β) are highly expressed in breast cancer specimens. Our finding comes in line with a previous study showing that TNF-α and IL-1β are highly expressed in tumor cells but not in normal breast epithelial cells . Contrary to expectation, IL-17 showed low mRNA levels in tumor tissue. This may be explained by a study done by Muraski et al. showing that the adoptive transfer of Th17 cells results in pronounced regression and enhanced overall cure [57,58]. Therefore, for an untreated tumor tissue Th17 cells may be low especially in highly metastatic ones. This is also in accordance with the finding of Yang et al. who indicated that the count of Treg but not Th17 cells is increased in breast cancer . Together, these results suggest that expression of these potential mediators are confined either in the tissues adjacent to the breast tumor or in the tumor itself. This finding may have an eﬀect on the systemic levels of these mediators and also on their biological eﬀects at various stages of cancer progression in pa-tients. Conversely, TNBC showed high levels of IL-17 and low levels of the anti-inflammatory cytokine IL-13. Given the lack of validated mo-lecular targets and the poor outcome in patients with TNBC, it remains a chief challenge in today's clinical practice . Our data highlight the fact that TNBC usually have high levels of inflammatory mediators, and the Av treatment further induces this inflammatory microenvironment. As a result, Av may increase the challenges of treating this type of cancer. Our findings come in accordance with previous work that identified only 32% of TNBC patients having low pro-inflammatory interleukin 8 had good prognosis .
Our study postulates that anti-angiogenesis therapy is more sig-nificant in tumors that express Cx43.