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  • We demonstrated that cells expressing less DHODH were less s


    We demonstrated that MK-4827 hydrochloride synthesis expressing less DHODH were less sensitive to DHODH inhibitors regardless of their oncogenic status. Based on the proliferation speed of cells, Kant et al. (1989), showed that slow-growing squamous cell carcinoma was more sensitive to BQR than fast-growing murine leukemia cells. Since then, no studies have been performed in different cell lines with respect to sensitivity to DHODH inhibition. In contrast to the observation of Kant and colleagues, our data showed that slow-growing breast cancer cells (MDAMB-436) and normal breast cells (W3.006) have lower expressions of DHODH enzyme and low sensitivity to all three DHODH inhibitors compared to the fast-growing breast cancer cells (T-47D and MDAMB-231). To strengthen our findings we also investigated the association between DHODH expression and the growth rate, as well as the sensitivity of a wide variety of cells in response to the DHODH inhibitors. Our results showed that there is a strong link between expression of DHODH, growth speed and sensitivity to inhibitors, independent of their oncogenic status. Earlier reports have stressed that the growth rate of tumors in breast cancer patients were very slow i.e. mean doubling time (DT) were reported to be approximately 100–300 days or even more [36], [37]. Several studies have shown significant effect of DHODH inhibitors on in vitro patient derived tumors [18], [38], [39]. Sykes and colleagues have reported recently that BQR is a promising tumour growth inhibitior in 4 patients' derived xenograft (PDX) models [40]. PDXs models are known to harbor bona fide tumour targets directly from the patient and the technique is gaining prominence for its clinically translatable potential in drug discovery, testing DHODH inhibitors in breast cancer PDX models would further be insightful. DHODH is the only enzyme in the pyrimidine biosynthesis that is located in the mitochondria rather than the cytosol. Mitochondria are intracellular organelles that are mainly associated with the electron transport chain (ETC) and generation of cellular energy. Mitochondrial bioenergetics plays a significant role in ROS production, which affects the activities of complexes I, III, and IV of the mitochondrial ETC [34], [35], [36], [37]. The functions of DHODH enzyme rely on the activity of complex III [42]. Oxidative stress can be beneficial or detrimental depending on the intensity, duration and context of the signalling. In this study, we demonstrated that treatment with DHODH inhibitors reduced the production of ROS in non-sensitive breast cancer cells while the ROS levels were maintained in sensitive breast cancer cells. The effect of DHODH inhibitors on ROS production was reported earlier in cancer cells where lower ROS production was observed with DHODH inhibitor treatment [22], [43]. Other than mitochondrial ETC, there are also other enzyme systems that contribute to the ROS pool, such as cytochrome P450, lipoxygenase, cyclooxygenase, the NADPH oxidase complex, xanthine oxidase, and peroxisomes [41]. Furthermore, DHODH inhibition could also cause activation of p53 which can have an impact on the level of ROS in the mitochondria [42]. Our current data suggests that DHODH inhibitors-mediated inhibition of proliferation is associated with maintenance of endogenous ROS level in sensitive cells (in contrast to reduction of ROS levels in non-sensitive cells). This observation is consistent with a previous study by Watabe and Nakaki [43], who reported that ROS production plays an essential role in apoptosis caused by ETC inhibitors by depleting the intracellular ATP level. To further prove this hypothesis, we demonstrated that DHODH inhibitors caused intracellular ATP depletion approximately 48 h after treatment. Interestingly, while ATP depletion was significant in the sensitive T-47D cell line, there was no significant depletion in non-sensitive MDAMB-436 cells even after prolonged treatment. This suggests that DHODH inhibitors-mediated effects require basal level of endogenous ROS in sensitive cells compared to non-sensitive cells, which is associated with significant intracellular ATP depletion. This is consistent with earlier study that showed direct depletion of ATP by ETC inhibitors does not enhance but requires endogenous basal level of ROS in SH-SY5Y cells [43].