title: Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells.
abstract: 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.
authors.affiliation: 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. Electronic address: turqoise47@siswa.um.edu.my.
authors.forename: A K
authors.initials: AK
authors.lastname: Mohamad Fairus
authors.affiliation: Aurigene Discovery Technologies (M) Sdn. Bhd., Level 2, Research Management and Innovation Complex, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: bhavesh_c@aurigene.com.
authors.forename: B
authors.initials: B
authors.lastname: Choudhary
authors.affiliation: Institute of Transdisciplinary Health Sciences and Technology (TDU) #74/2, Jarakabande Kaval, Post Attur via Yelahanka, Bangalore, 560 064 Karnataka, India. Electronic address: hosahalli.s@gmail.com.
authors.forename: S
authors.initials: S
authors.lastname: Hosahalli
authors.affiliation: Aurigene Discovery Technologies Limited, 39-40, KIADB Industrial Area, Electronic City Phase II, Hosur Road, Bangalore, 560100 Karnataka, India. Electronic address: kavitha_n@aurigene.com.
authors.forename: N
authors.initials: N
authors.lastname: Kavitha
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: shatraho@um.edu.my.
authors.forename: O
authors.initials: O
authors.lastname: Shatrah
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001943:Breast Neoplasms / Q000201:enzymology* / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D044925:Oxidoreductases Acting on CH-CH Group Donors
keywords: ATP; Breast cancer; Dihydroorotate dehydrogenase; Normal cells and reactive oxygen species production