To contribute to the understanding of cancer genesis, the breast cancer derived cell line MCF-7 has been used as a prominent model for the study of estrogen receptor-positive breast cancer cells. In MCF-7 cells Dex is able to prevent the cytotoxic effect of TNF-α, and the anti-apoptotic proteins IAP1, IAP2 and XIAP have been postulated as effector molecules . However, and despite extensive studies, the molecular mechanisms of this protection are just beginning to be described. On this respect, the role played by anti-apoptotic routes others than the one regulated by IAP proteins in the Dex protection from TNF-α cytotoxicity has not been analyzed. Also, TNF-α stimulation does not only activate cell death pathways, but survival ones too. In consequence, it can be assumed that the balance between pro- and anti-apoptotic regulators defines the apoptotic threshold of a cell. The anti-apoptotic effect of TNF-α requires the activation of PI3K and NF-κB and, as active participants of survival routes, these proteins could participate in the Dex protection against TNF-α cytotoxicity. Thus, we analyzed their participation in Dex mediated-protection against TNF-α cytotoxicity.
It has been suggested that the scarcity of breast tumor derived cell lines has led to the apparition of several sub-lines, evidenced by different results obtained for the evaluation of related phenomena , including their susceptibility to TNF-α induced apoptosis .
This led us to corroborate the ability of TNF-α to induce cell death and to evaluate the protection mediated by Dex in our cell system. As previously reported, TNF-α treatment induced cell death in a dose and time dependent fashion and co-incubation with Dex protected MCF-7 cells against TNF-α-induced cell death.
We have found that in MCF7 and ZR-70-35 human mammary tumor cells the protective effect of Dex was compromised in the presence of 2.5 μM of Bay-117082, a pharmacological inhibitor of NF-κB activation (data not shown). This result correlates with those observed in Figure 3D, where protection is lost in cells expressing the dominant negative form of IkBα, thus providing further support to the notion that Dex protection requires NF-κB activation. Furthermore, the use of the inhibitor of NF-κB lead to a marked decrease in c-IAP1 cellular content in ZR-70-35 cells (data not shown). While c-IAP1 could not be detected in TNF-α-treated cells, in the presence of TNF-α + Dex c-IAP1 content returned to control levels. This behavior reproduced the results presented in figures 4C and 4D, and documents the correlation between Dex protection and c-IAP1 cellular content.
In our system the Akt phosphorylation level was not affected by Dex treatment in the presence or absence of TNF-α. Besides, transfection of a dominant negative mutant of PI3K (ΔP85) in MCF-7 cells did not affect Dex protection, suggesting that the PI3K/Akt pathway is not involved in Dex protection against TNF-α. NF-κB activation through PI3K/Akt has been a controversial issue due to cell type variations [30, 31]. Although in some cells Akt acts upstream of NF-κB [24, 32, 27], we found that NF-κB activation is completely independent of Akt function.
In our cell system Dex did not modify the NF-κB activation in the presence or absence of TNF-α. However, a non-degradable IκBα mutant protein (dnIκBα), which prevents NF-κB nuclear translocation; completely blocked Dex protection against TNF-α induced cell death. In the absence of Dex, dnIκBα expression increased the susceptibility to TNF-α-induced death. These results suggest that the TNF-α-dependent NF-κB activation participates in the protection conferred by Dex. Furthermore, in dnIkBα transfectant MCF-7 cells, the susceptibility to TNF-α cytotoxicity correlated with the level of expression of the IκBα mutant form, suggesting a threshold for the protective action of NF-κB activation.
NF-κB regulates the expression of a great number of genes, including several antiapoptotic gene products such as members of the Bcl-2 family  and the inhibitor of apoptosis proteins XIAP, c-IAP1 and c-IAP2 . Interestingly, NF-κB regulates XIAP  and cIAP1 promoters . Thus, we analyzed the effects of interfering with NF-κB signaling pathway during Dex protection on XIAP and c-IAP1 protein content. We detected that, as previously reported , TNF-α-induced apoptosis in MCF-7 cells correlated with downregulation of XIAP and c-IAP1 proteins, postulated as effectors of the protective effect against TNF-α-mediated cytotoxicity. However, only the expression level of c-IAP1 correlated with the protective effect of Dex: In cells expressing dnIκBα stimulated with TNF-α (i) the protein level of this antiapoptotic factor was lower than in parental cells and correlated with an increased cell death and, (ii) in parental MCF-7 cells Dex treatment correlated with a slower rate of decrease of the anti-apoptotic factors content.
While estrogen dependence in mammary tumor cells is being extensively studied due to its clinical importance, the dependency on GC has received less attention. The protection conferred by Dex against TNF-mediated cytotoxicity has been extensively analyzed in MCF-7 cells and, interestingly, this synthetic GC has also been reported to confer protection against pharmacological mediators of cell death . In contrast, GCs have been reported to interfere with proliferation in MCF7, ZR-75-1, Con-8 and MDA-MB-231 mammary tumor cells [37, 38]. At present, GC therapy is not included in patients with mammary tumors, although no comparative study has been performed to discard its efficacy. Whether or not the antiproliferative effect of natural or synthetic GCs is related to the protection against TNF-mediated cytotoxicity remains to be determined.
Also, IAPs belong to a diverse group of proteins which modulate the apoptotic pathways by binding to caspases and inhibiting their proteolitic activity . In addition to this well characterized anti-apoptotic effect, some IAP isoforms have been reported to interfere with apoptosis through caspase-inhibition independent mechanisms. Expression of IAPs in MCF-7 cells in response to Dex has been previously described and is suggestive of the anti-apoptotic protection against TNF-mediated cytotoxicity. Nevertheless, the contribution of IAP expression to this protective effect remains to be shown by specific interference with IAPs expression, possibly through iRNA technology. Without this kind of experiments, it is difficult to establish the relative contribution of IAP expression to the protective effect of Dex.