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  • The p controls transcription of several genes that


    The p53 controls transcription of several genes that affect the release of cytochrome c in the mitochondrial apoptotic pathway [44]. In addition, p53 can induce a transcription-independent apoptosis through a direct interaction with the Bcl-2 family proteins [45]. However, the role of mitochondrial ETC activity in the induction of p53 response remains ambiguous. Deficiency in pyrimidine is critical for the induction of p53 in response to ETC complex III inhibitors such as LFM, BQR and 4SC-101(29). In agreement with Khutornenko and colleagues [29] our data shows that DHODH inhibitors induce the expression of p53 in cancer cells. The correlation between p53 and NF-kB signalling was reported in an earlier study that showed treatment with doxorubicin activated NF-κB in p53 mutant cancer cells but not wild type cells [46]. In agreement with this report, we observed the induction of p65/NF-κB expression in p53 mutant T-47D cells. Our data strongly suggests the involvement of p53 and NF-kB signalling in response to DHODH modulation. However, further work is needed to elucidate the precise mechanisms of action. In our studies, we found that the DHODH inhibitors increased the expressions of STAT6 and p65/NF-κB in sensitive breast cancer cells. These signalling molecules could interact directly with each other in vitro and in vivo by cooperatively binding to their associated DNA focus on  and synergistically activating transcription [25]. Previous study reported that the over-expression of STAT6 can lead to growth inhibition in breast cancer cells (MCF7) via IL-4 activation [27]. In addition, over-expression of p65/NF-κB in breast cancer cells can also enhance the pro-apoptotic pathway by repressing the anti-apoptotic properties of NF-κB [47]. Most studies have concluded that p65/NF-κB can be an activator and repressor of its target genes, depending upon the manner in which it is induced, types of tumour, context and environmental conditions [46], [48]. There have been reports of strong crosstalk between p65 and p53 in mutant cells [49], such as T-47D, which could explain our focus on  observations on the increase in p65 with DHODH inhibitors treatment. Another study reported that over-expression of p65 in MCF7/ADR cells reduced their tumorigenic ability in nude mice [50]. Our data suggests that the augmentation of p53, p65 and STAT6 in T-47D is interlinked with pyrimidine deficiency triggered by DHODH inhibition. DHODH inhibitors showed relatively high S-phase arrest in T-47D cells compared to MDAMB-436 cells. This is in agreement with previous study where LFM arrested 143B cells and other sensitive cell lines at S-phase of the cell cycle progression [21], [51]. p53-null cells but not wild type cells treated with hydroxyurea, showed arrest in mitosis [52]. Nevertheless, the effect of DHODH inhibition on cell cycle arrest in cells with different p53 status requires further investigation. Apart from the potential factors discussed above on the response of breast cancer cells to DHODH inhibitors, some additional mechanisms in the cells may affect sensitivity of tumour cells against DHODH inhibitors; such as the expression level of breast cancer resistance protein (BCRP/ABCG2), P-glycoprotein (Pgp), multidrug resistance-associated protein 1 (MRP1) and lung cancer resistance protein (LRP) [53], [54]. BCRP or ABCG2 is an ATP binding cassette (ABC) membrane transporter which affects the absorption of anticancer drugs into the cells [55], [56]. The expression of this protein in cancer cells may directly cause impounding of drugs within cytoplasmic vesicles, followed by expelling the drug out of the cells [53], [57], [58]. The potential role of BCRP/ABCG2 in determining the efficacy of DHODH inhibitors has not been investigated in our study. However, earlier publications have suggested a possible role of LFM as the substrate for the efflux transporter which inhibits BCRP/ABCG2 mediated methotrexate transport [59]. Unfortunately, to date, no other evidence was found regarding the efflux pump effect on breast cancer cells related to LFM or other DHODH inhibitors. Of particular interest, it is unknown whether LFM is a genuine substrate for multidrug resistant (MDR) proteins, or whether over-expression of specific MDR proteins i.e. Pgp or BCRP/ABCG2 confers non-sensitivity of the studied cells to DHODH inhibitors. In addition, other than the study of MDR protein involvement in drug resistance mechanisms in MCF7 breast cancer cells; the role of these proteins towards resistance mechanisms in T-47D, MDAMB-231 and MDAM-436 cells is not clear [60], [61], [62], [63]. Study of the molecular relationship between the expressions of drug efflux transport proteins could be insightful to understand any resistance mechanism in breast cancer cells in response to DHODH inhibitor treatment.