Molecular basis of antifolate resistance.
ABSTRACT Folates play a key role in one-carbon metabolism essential for the biosynthesis of purines, thymidylate and hence DNA replication. The antifolate methotrexate has been rationally-designed nearly 60 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR) thereby achieving temporary remissions in childhood acute leukemia. Recently, the novel antifolates raltitrexed and pemetrexed that target thymidylate synthase (TS) and glycineamide ribonucleotide transformylase (GARTF) were introduced for the treatment of colorectal cancer and malignant pleural mesothelioma. (Anti)folates are divalent anions which predominantly use the reduced folate carrier (RFC) for their cellular uptake. (Anti)folates are retained intracellularly via polyglutamylation catalyzed by folylpoly-gamma-glutamate synthetase (FPGS). As the intracellular concentration of antifolates is critical for their pharmacologic activity, polyglutamylation is a key determinant of antifolate cytotoxicity. However, anticancer drug resistance phenomena pose major obstacles towards curative cancer chemotherapy. Pre-clinical and clinical studies have identified a plethora of mechanisms of antifolate-resistance; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of (anti)folates as well as in folate-dependent enzymes. These include inactivating mutations and/or down-regulation of the RFC and various alterations in the target enzymes DHFR, TS and FPGS. Furthermore, it has been recently shown that members of the ATP-binding cassette (ABC) superfamily including multidrug resistance proteins (MRP/ABCC) and breast cancer resistance protein (BCRP/ABCG2) are low affinity, high capacity ATP-driven (anti)folate efflux transporters. This transport activity is in addition to their established facility to extrude multiple cytotoxic agents. Hence, by actively extruding antifolates, overexpressed MRPs and/or BCRP confer antifolate resistance. Moreover, down-regulation of MRPs and/or BCRP results in decreased folate efflux thereby leading to expansion of the intracellular folate pool and antifolate resistance. This chapter reviews and discusses the panoply of molecular modalities of antifolate-resistance in pre-clinical tumor cell systems in vitro and in vivo as well as in cancer patients. Currently emerging novel strategies for the overcoming of antifolate-resistance are presented. Finally, experimental evidence is provided that the identification and characterization of the molecular mechanisms of antifolate-resistance may prove instrumental in the future development of rationally-based novel antifolates and strategies that could conceivably overcome drug-resistance phenomena.
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ABSTRACT: The impact of folate on health and disease, particularly pregnancy complications and congenital malformations, has been extensively studied. Mandatory folic acid fortification therefore has been implemented in multiple countries, resulting in a reduction in the occurrence of neural tube defects. However, emerging evidence suggests increased folate intake may also be associated with unexpected adverse effects. This literature review focuses on contemporary issues of concern, and possible underlying mechanisms as well as giving consideration the future direction of mandatory folic acid fortification. Folate fortification has been associated with the presence of unmetabolized folic acid (PteGlu) in blood, masking of vitamin B12 deficiency, increased dosage for anti-cancer medication, photo-catalysis of PteGlu leading to potential genotoxicity, and a role in the pathoaetiology of colorectal cancer. Increased folate intake has also been associated with twin birth and insulin resistance in offspring, and altered epigenetic mechanisms of inheritance. Although limited data exists to elucidate potential mechanisms underlying these issues, elevated blood folate level due to the excess use of PteGlu without consideration of an individual's specific phenotypic traits (e.g. genetic background and undiagnosed disease) may be relevant. Additionally, the accumulation of unmetabolized PteGlu may lead to inhibition of dihydrofolate reductase and other enzymes. Concerns notwithstanding, folic acid fortification has achieved enormous advances in public health. It therefore seems prudent to target and carefully monitor high risk groups, and to conduct well focused further research to better understand and to minimize any risk of mandatory folic acid fortification.
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ABSTRACT: Multidrug resistance (MDR) is a primary hindrance to curative cancer chemotherapy. In this respect, lysosomes were suggested to play a role in intrinsic MDR by sequestering protonated hydrophobic weak base chemotherapeutics away from their intracellular target sites. Here we show that intrinsic resistance to sunitinib, a hydrophobic weak base tyrosine kinase inhibitor known to accumulate in lysosomes, tightly correlates with the number of lysosomes accumulating high levels of sunitinib in multiple human carcinoma cells. Furthermore, exposure of cancer cells to hydrophobic weak base drugs leads to a marked increase in the number of lysosomes per cell. Non-cytotoxic, nanomolar concentrations, of the hydrophobic weak base chemotherapeutics doxorubicin and mitoxantrone triggered rapid lysosomal biogenesis that was associated with nuclear translocation of TFEB, the dominant transcription factor regulating lysosomal biogenesis. This resulted in increased lysosomal gene expression and lysosomal enzyme activity. Thus, treatment of cancer cells with hydrophobic weak base chemotherapeutics and their consequent sequestration in lysosomes triggers lysosomal biogenesis, thereby further enhancing lysosomal drug entrapment and MDR. The current study provides the first evidence that drug-induced TFEB-associated lysosomal biogenesis is an emerging determinant of MDR and suggests that circumvention of lysosomal drug sequestration is a novel strategy to overcome this chemoresistance.Oncotarget 12/2014; · 6.63 Impact Factor
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ABSTRACT: γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.Genes & Nutrition 12/2014; 10(1). DOI:10.1007/s12263-014-0444-0 · 3.42 Impact Factor