Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis

Tonkinson, John L.; Marder, Philip; Andis, Sherri L.; Schultz, Richard M.; Gossett, Lynn S.; Shih, Chuan; Mendelsohn, Laurane G.

doi:10.1007/s002800050608

Cited by 68 publications

(42 citation statements)

References 16 publications

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“…Drugs interacting at different sites in the cell cycle might potentiate the therapeutic response. In this study, FACS analysis demonstrated that pemetrexed caused an S-phase arrest, as previously detected in different tumour cell lines (Tonkinson et al, 1997(Tonkinson et al, , 1999Giovannetti et al, 2005). In contrast, enzastaurin alone increased the percentage of cells in the G1 phase, whereas in the combination the cells accumulated in G2/M phase.…”

Section: Discussionmentioning

confidence: 51%

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

et al. 2008

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Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKCb inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription -PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3b and Akt phosphorylation, which was additionally reduced by drug combination (À58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (450% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients.

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Section: Discussionmentioning

confidence: 51%

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

et al. 2008

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“…Secondly, raltitrexed and MTX exert their cytotoxic effects by blocking cells in the S phase. 1,23,24) Thus, MTX might enhance the cytotoxicity of raltitrexed by blocking cells in the Sphase, in which the cells are most sensitive to raltitrexed.…”

Section: Discussionmentioning

confidence: 99%

Schedule‐dependent Synergism and Antagonism between Raltitrexed (“Tomudex”) and Methotrexate in Human Colon Cancer Cell Lines in vitro

Kano

Akutsu

Tsunoda

et al. 2001

Japanese Journal of Cancer Research

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The folate-dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed ("Tomudex"), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (IC 80 and IC 50 ) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule-dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.Key words: Raltitrexed -Methotrexate -Synergism -Isobologram -Colon cancerThe folate-dependent enzymes are attractive targets for cancer chemotherapy because of their critical role in the synthesis of the nucleotide precursors of DNA. Methotrexate (MTX), the classical antifolate, is one of the oldest and still most commonly used anti-cancer agents. 1) Although the precise cytotoxic mechanism of MTX remains controversial, the main target of MTX is considered to be dihydrofolate reductase. The inhibition of this enzyme results in a lack of tetrahydrofolate coenzyme, which is required for the de novo synthesis of thymidylate, purines, and methionine. Thymidylate is required for the synthesis and repair of DNA, and inhibition of thymidylate synthesis is considered to be the major cytotoxic mechanism of MTX.Currently, several new folate analogs with unique biochemical properties are being tested for clinical application.2) Raltitrexed ("Tomudex") is a promising new agent that targets thymidylate synthase, the enzyme responsible for the final step of thymidylate synthesis.2, 3) Like MTX, this agent relies on the reduced folate carrier for cellular...

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“…Pemetrexed is a new multitarget antifolate metabolite that acts on the enzymes thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT), 4 thereby depleting nucleotide pools and blocking DNA synthesis. 5,6 Pemetrexed is transported into the cell via the reduced folate carrier and the folate binding protein. After being transported into the cell, Pemetrexed is polyglutamated by the enzyme folyl-polyglutamyl synthase.…”

Section: Introductionmentioning

confidence: 99%

Pemetrexed acts as an antimyeloma agent by provoking cell cycle blockade and apoptosis

et al. 2007

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Multiple myeloma (MM) is a malignancy of terminally differentiated B lymphocytes, characterized by accumulation of a monotypic plasma cell population in the bone marrow, monoclonal immunoglobulin in serum and/or urine and osteolytic lesions. Despite recent advances in the treatment, MM remains an incurable disease. This calls for an effort to develop novel therapeutics in order to eradicate the disease. Here we have evaluated the potential antimyeloma action of Pemetrexed, an antifolate drug that has shown promising results in other neoplastic diseases. Pemetrexed had a potent antimyeloma effect on cell lines sensitive and resistant to conventional therapeutic agents, and was also efficient on fresh cells from patients and in a murine MM model. Furthermore, Pemetrexed abrogated the protective action on MM cell death of several growth factors produced by the bone marrow microenvironment. Mechanistic studies indicated that Pemetrexed provoked this action by a combined effect that included cell cycle blockade, probably by p21 upregulation, and induction of apoptosis through caspase-dependent and -independent mechanisms. These data, together with the fact that Pemetrexed is already licensed for the therapy of other neoplastic diseases, opens the possibility for the inclusion of Pemetrexed in the therapeutic armamentarium to battle MM.

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Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis

Abstract: These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.

Cited by 68 publications

References 16 publications

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Schedule‐dependent Synergism and Antagonism between Raltitrexed (“Tomudex”) and Methotrexate in Human Colon Cancer Cell Lines in vitro

Pemetrexed acts as an antimyeloma agent by provoking cell cycle blockade and apoptosis

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