title: Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest.
abstract: Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: S
authors.initials: S
authors.lastname: Raz
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Sheban
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: N
authors.initials: N
authors.lastname: Gonen
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: M
authors.initials: M
authors.lastname: Stark
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: B
authors.initials: B
authors.lastname: Berman
authors.affiliation: Department of Biology, The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D015687:Cell Hypoxia; D049109:Cell Proliferation / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D013997:Time Factors; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology
chemical_list: D000600:Amino Acids, Dicarboxylic; D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D011480:Protease Inhibitors; D059005:Topoisomerase II Inhibitors; D005492:Folic Acid; C040947:oxalylglycine