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

Abstract

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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