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.