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Cancer metabolism and oxidative stress: Insights into carcinogenesis and chemotherapy via the non-dihydrofolate reductase effects of methotrexate.

ABSTRACT: Methotrexate has been in use as an anti-cancer agent for over 60 years. Though inhibition of dihydrofolate reductase is its best known mechanisms of action, its non-dihydrofolate reductase dependent mechanisms disrupt metabolic pathways resulting in a depletion of NAD(P)H and increasing oxidative stress. These mechanisms highlight a novel dependence of cancer cells on their metabolic abnormalities to buffer oxidative stress and chemotherapeutic agents interfere with these cellular abilities. Mitochondria appear to play a significant role in maintaining cancer cell viability and alterations in metabolism seen in cancer cells aid this mitochondrial ability. Further research is needed to understand the effects of other chemotherapeutic agents on these pathways.

SUBMITTER: Hess JA

PROVIDER: S-EPMC4661551 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Cancer metabolism and oxidative stress: Insights into carcinogenesis and chemotherapy via the non-dihydrofolate reductase effects of methotrexate.

Hess Joshua A JA Khasawneh Mohamad K MK

BBA clinical 20150207

Methotrexate has been in use as an anti-cancer agent for over 60 years. Though inhibition of dihydrofolate reductase is its best known mechanisms of action, its non-dihydrofolate reductase dependent mechanisms disrupt metabolic pathways resulting in a depletion of NAD(P)H and increasing oxidative stress. These mechanisms highlight a novel dependence of cancer cells on their metabolic abnormalities to buffer oxidative stress and chemotherapeutic agents interfere with these cellular abilities. Mit ...[more]

PMID: 26674389

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Metformin sensitises hepatocarcinoma cells to methotrexate by targeting dihydrofolate reductase.

Project description:Metformin, the first-line drug for type II diabetes, has recently been considered an anticancer agent. However, the molecular target and underlying mechanism of metformin's anti-cancer effects remain largely unclear. Herein, we report that metformin treatment increases the sensitivity of hepatocarcinoma cells to methotrexate (MTX) by suppressing the expression of the one-carbon metabolism enzyme DHFR. We show that the combination of metformin and MTX blocks nucleotide metabolism and thus effectively inhibits cell cycle progression and tumorigenesis. Mechanistically, metformin not only transcriptionally represses DHFR via E2F4 but also promotes lysosomal degradation of the DHFR protein. Notably, metformin dramatically increases the response of patient-derived hepatocarcinoma organoids to MTX without obvious toxicity to organoids derived from normal liver tissue. Taken together, our findings identify an important role for DHFR in the suppressive effects of metformin on therapeutic resistance, thus revealing a therapeutically targetable potential vulnerability in hepatocarcinoma.

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Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics.

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Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate.

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Influence of reduced folate carrier and dihydrofolate reductase genes on methotrexate-induced cytotoxicity.

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Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells.

Project description:Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line. Uptake experiments with [3H]MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line. Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line. To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells. Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells. Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme. Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX. These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects.

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In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.

Project description:Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

| S-EPMC2820606 | biostudies-literature

Lon Peptidase 2, Peroxisomal (LONP2) Contributes to Cervical Carcinogenesis via Oxidative Stress.

Project description:BACKGROUND Lon protease is responsible for degrading proteins injured by oxidation, and has 2 isoforms, located in mitochondria and peroxisomes. Recent research showed that Lon protease was upregulated in different types of human cancer, but the role of Lon peptidase 2, peroxisomal (LONP2) in cancer is not well understood. It is known, however, that in cancer biology, reduction-oxidation is one of the molecular mechanisms involved in tumorigenesis. MATERIAL AND METHODS Oncomine databases and tissue microarrays, initially using immunohistochemistry, were used to analyze LONP2 expression in cervical cancer. In order to uncover the biologic functions and mechanism(s) underlying LONP2 in cervical tumorigenesis, we downregulated the expression of LONP2 using 2 siRNAs transduced in HeLa and SiHa cells. CCK8 assays were performed to evaluate cell viability. Cell cycle and apoptosis assays were used to determine cell growth. Cell migration and invasion assays were used to study changes in cell migration and invasion capacity. Immunofluorescence and flow cytometry were performed to analyze the changes in ROS production. RESULTS We found that the expression of LONP2 was significantly upregulated in cervical cancer, and there was a significant association with pathology type, pathology grade, and clinical stage, but not with age or lymph node metastasis. Moreover, we demonstrated that knocking down LONP2 in HeLa and SiHa cells reduced cell proliferation, cell cycle, apoptosis, migration, invasion, and oxidative stress levels. CONCLUSIONS Our findings suggest that LONP2 promotes cervical tumorigenesis via oxidative stress and may be a potential biomarker and therapeutic target in cervical cancer.

| S-EPMC5846714 | biostudies-literature

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