We have shown previously that celecoxib could inhibit COX-2 and p65 expression levels, which promotes apoptosis in human MDA-MB-231 breast cancer cells . Until now, the pro-apopotic effects of celecoxib have been proved to be associated with multiple intracellular signaling pathways including NF-κB, Akt and caspases, which interact to regulate programmed cell death . To better understand the NF-κB/Akt signaling interactions implicated in celecoxib-mediated apoptosis, the present study overexpressed p65, the major transactivating subunit of NF-κB, in MDA-MB-231 cells and evaluated effects on celecoxib-induced Akt activation, NF-κB transcriptional activity, apoptosis and cell cycle distribution. In this study, overexpression of p65 in MDA-MB-231 cells enhanced p65 protein expression and the basal NF-κB DNA binding activity. However, transfection of MDA-MB-231 cells with p65cDNA had no significant effect on the NF-κB p50 subunit and the inhibitory molecule IκB. Celecoxib treatment, at 80 μM, which optimally induces apoptosis in MDA-MB-231 cells , inhibited NF-κB transcriptional activity and expression of NF-κB target genes. Celecoxib suppression of NF-κB was blocked in p65 transfectants since activity remained at least 2-fold higher than in mock-transfected cells. These results suggest that the exogenous upregulation of p65 could attenuate the inactivation of NF-κB caused by celecoxib treatment.
Akt has been shown to regulate cell survival and suppress apoptosis by stimulating the transactivation potential of the NF-κB p65 subunit . In contrast, the NF-κB signaling pathway has also been reported to function upstream of Akt. Overexpression of p65 led to Akt phosphorylation in the absence of extracellular stimulatory factors and caused an increase in the expression of Akt at the mRNA and protein levels . In MDA-MB-231 cells, celecoxib could inactivate pAkt with increased activation of proapoptotic protein . In our previous study, we also found that celecoxib could inhibit cell growth, induce apoptosis and alter cell cycle distribution by blocking NF-κB signaling . However, the present results indicated that overexpression of p65 had no effect on the phosphorylation of Akt, suggesting that Akt might play a role in upstream signaling pathways or via NF-κB-independent pathway in MDA-MB-231 cells. The precise molecular mechanisms remain to be determined.
NF-κB p65 can be an activator and repressor of its target genes depending upon the manner in which it is induced . For example, Anto et al.  reported that overexpression of p65 in L929 mouse fibrosarcoma cells caused resistance to curcumin-induced apoptosis. Opposite results were reported by Collett et al. , who showed that overexpression of p65 potentiated curcumin-induced apoptosis in HCT116 human colon cancer cells. Besides, the role of p65 in the carcinogenic process is also complex and may involve the interaction of multiple signaling pathways in a context-specific manner. Yu et al.  reported that increased expression of p65 was correlated with colorectal tumorigenesis and promoted tumor progression. Conversely, Ricca et al.  reported that overexpression of p65 in MCF7/ADR cells reduced their tumorigenic ability in nude mice. The present study showed that overexpression of p65 could antagonize celecoxib-mediated apoptosis, as assessed by MTT assay and cell death fragment detection. Furthermore, transfected p65cDNA resulted in inhibition of caspase-3, -9 and PARP cleavage in MDA-MB-231 cells, which may be attributed to suppression of NF-κB-dependent transcriptional activity and expression of NF-κB-dependent anti-apoptotic genes. Therefore, we investigated two NF-κB-dependent target gene families, namely IAP family members and Bcl-2 family proteins. Results showed that p65 upregulation counteracted the celecoxib-induced inhibition of survivin and XIAP, while exerted no effects on Bcl-2 family members. The IAP family was confirmed to be regulated by NF-κB signaling and closely tied to breast cancer development . The current study indicates that survivin and XIAP could be directly regulated by NF-κB p65 subunit, thus functioning anti-apoptosis in breast cancer cells. Although Bcl-2 has NF-κB binding sites and can be regulated by p65 , the expression of Bcl-2 family members remained unaffected in both mock transfectants and p65-overexpressing cells before and after celecoxib treatment. It is speculated that a putative increase in Bcl-2 mRNA might be counteracted by micro-RNA-mediated translational arrest .
Cell proliferation is the result of a rapid shift from a quiescent state to the progression of the cell cycle . Flow cytometric results showed that celecoxib induced MDA-MB-231 cell cycle arrest at the G0/G1 phase, and this effect was suppressed by p65cDNA transfection. Progress in the eukaryotic cell cycle is driven by protein kinase complexes consisting of cyclins and CDKs. CDK activity is regulated negatively by a group of proteins called CDK inhibitors, including the protein p21 and p27 . In the present study, cyclin D1 was increased in p65-overexpressing cells even in the presence of celecoxib, and p65 overexpression caused the inhibition of p21, suggesting a possible protective role for p65 expression in celecoxib-mediated cell cycle arrest. Collectively, these results indicated that p65 may have a direct role in regulating cell cycle progression, facilitating the transition of cells from G1 to S phase.
In conclusion, the present results strongly suggest that p65 expression has a protective role against celecoxib-mediated cell death of human breast cancer. In light of such a scenario, targeting p65 subunit or transfection with anti-p65 intrabody may enhance the anti-tumor effect of celecoxib in breast cancer treatment. Given that all current clinical anti-cancer drugs have reported incidences of drug resistance, development of experimental therapeutic aimed at new proliferative targets are of increasing importance. Further investigation into the molecular mechanism of p65 action may offer a novel approach to interfere with NF-κB activity in the nuclear compartment for treating breast cancer as well as other tumors.