Mohamad Fairus A K, Choudhary B, Hosahalli S, Kavitha N, Shatrah O
Aurigene Discovery Technologies (M) Sdn. Bhd., Level 2, Research Management and Innovation Complex, University of Malaya, 50603 Kuala Lumpur, Malaysia; Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Aurigene Discovery Technologies (M) Sdn. Bhd., Level 2, Research Management and Innovation Complex, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Biochimie. 2017 Apr;135:154-163. doi: 10.1016/j.biochi.2017.02.003. Epub 2017 Feb 11.
Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion which subsequently leads to cell cycle arrest at S-phase.
二氢乳清酸脱氢酶（DHODH）是原核生物和真核生物中嘧啶从头生物合成的关键酶。嘧啶生物合成的从头途径在癌细胞增殖中至关重要。来氟米特是一种已获批准的DHODH抑制剂，已广泛用于治疗关节炎。同样，布喹那钠是另一种DHODH抑制剂，当与氟尿嘧啶联合使用时，在MC38结肠癌细胞中显示出抗肿瘤作用。尽管DHODH抑制剂在癌症治疗中具有潜在作用，但其作用机制仍不清楚，有待进一步阐明。在此，我们评估了DHODH抑制剂对敏感和不敏感乳腺癌细胞中ATP和ROS产生的影响。随后，在敏感的T-47D细胞和不敏感的MDAMB-436细胞中评估了DHODH抑制剂对细胞周期以及对p53、p65和STAT6等信号分子的影响。还在一组癌细胞系中研究了DHODH蛋白表达、增殖速度与对DHODH抑制剂敏感性之间的相关性。对DHODH抑制剂敏感的T-47D细胞和MDAMB-231细胞似乎将ROS产生维持在接近内源性ROS水平，而在不敏感的MDAMB-436细胞和W3.006细胞中则观察到相反的情况。此外，与不敏感的MDAMB-436细胞相比，我们在高度敏感的T-47D细胞和MDAMB-231细胞中观察到约90%的细胞内ATP消耗。敏感细胞中p53、p65和STAT6信号分子有明显的过表达，这可能参与介导细胞周期进程中的S期阻滞。目前的研究表明，DHODH抑制剂在表达高水平DHODH酶的细胞中最有效。这些抑制剂对细胞增殖的抑制似乎伴随着ROS产生以及ATP消耗。观察到的信号分子表达增加可能是由于嘧啶耗竭，随后导致细胞周期在S期停滞。
