Raltitrexed In Combination With5-FU In The Treatment For Colon Cancer In Vitro

Colon cancer is a common malignant tumors of the digestive system.Although surgery is considered to be the preferred treatment for colon cancer,systemic chemotherapy is an important method for the distant metastases orlocally advanced unresectable patients in order to prolong their survival.Evidence-based medicine indicate that postoperative adjuvant and neoadjuvantchemotherapy are important for patients in increasing long-term survival andremovel rate.Raltitrexed contains glutamic acid residues in its structure. It is carried byreduced folate methotrexate cell membrane carrier (RFC) into cells, and thenplays poly glutamic acid with the catalytic help of foly poly glutamylatesynthase (FPGS). It plays the anti-cancer effects by affecting the biosynthesisof DNA by combining to the folic acid binding site of Thymidylate synthase.This new cytotoxic anticancer drug was originally developed by Zeneca andlisted in the UK in1996. It is mainly used for the treatment of advancedcolorectal cancer,breast cancer and other solid tumors. In addition, the drughas similar anti-tumor effect with fluorouracil, but it has long half-life of198hours, takes only15minutes in use, brings convenience to patients, reducesgreatly the suffering caused by continuous intravenous, and has fewer adversereactions.5-fluorouracil (5-Fu) is first used of anticancer drugs in clinic duringthymidylate synthase inhibitors and it is the basis for colon cancer. Ittransforms into dUMP and combines to the corresponding binding sites ofThymidylate synthas to leads to cell damage and death by affecting thebiosynthesis of DNA, and thus produce anti-tumor effect. But it has many sideeffects, such as its short half-life,5-10minutes, continuous intravenous injection in clinic, poor tolerance of patients, drug resistance during long-termapplication, harmful to cells in high dose.When we use two chemotherapy drugs together, it is additive effects if theefficacy equals to that of the two drugs alone. Otherwise it is synergistic effectsif the efficacy proceeds that of two drugs alone. It is antagonistic effects if theefficacy is less than that of either of them.5-fluorouracil and raltitrexed combine to the different binding sites ofThymidylate synthase througe different ways, and the mechanism of them arenot identical, so the combination of the two drugs may produce additive orsynergistic effect, increases the inhibition of tumor cells.There are less reports about the combination of5-fluorouracil andraltitrexed at home and abroad. It is not sufficient of combination use of thetwo drugs in the treatment of colon cancer.Objective: To provide a theoretical and experimental basis for thecombination of raltitrexed with5-fluorouracil for the treatment of colon cancerthrough research the inhibition of raltitrexed, different exposure to raltitrexedand5-fluorouracil to human colon carcinoma cell line HCT-8and SW480.Method:1.Experiments were divided into raltitrexed group,5-Fu group,simultaneous exposure to raltitrexed and5-Fu group, sequential exposure toraltitrexed followed by5-Fu group, sequential exposure to5-Fu followed byraltitrexed group, control group.2.HCT-8、SW480cells were seeded in96-well plates and were allocated tocontrol group, raltitrexed group,5-Fu group, and raltitrexed in combinationwith5-Fu group. For the groups containing with raltitrexed and raltitrexed havedifferent drug concentration. The growth state of cells was observed undermicroscopy and the inhibition rates of each group were measured with MTTassay. 3.Detecting cell cycle distribution of HCT-8and SW480by Propidiumiodide (piodize, PI) single staining.4.Detecting the inhibition of raltitrexed to HCT-8and SW480by platecolony forming assay.Results：1.The states of the growth of cells in each group under the microscopeafter treatment:(1)The growth status of HCT-8、SW480cells were larger, poorrefractive index, poor growth status and not obvious increase in number inraltitrexed group than that in the control group;(2)5-fluorouracil group wassimilar to raltitrexed group and part of cells were poor;(3)The cells ofsimultaneous exposure to raltitrexed and5-Fu group and sequential exposure toraltitrexed followed by5-Fu group were smaller, poor refractive index, poorgrowth status and decrease in number;(4)The cells of control group were well.With the increase of time and concentrations of raltitrexed, it enhance theinhibition to cancer cells.2.The inhibition of simultaneous exposure to raltitrexed and5-Fu groupand sequential exposure to raltitrexed followed by5-Fu group to cancer cells ishigher than the separate group and sequential exposure to5-Fu followed byraltitrexed group.3.HCT-8and SW480cells appears the phenomenon of cell cycle arrest,mainly in G1and S phase, after adding raltitrexed.4.HCT-8cells after the effect of raltitrexed, raltitrexed in combination of5-fluorouracil, sequential exposure to raltitrexed followed by5-Fu group andsimultaneous or sequential exposure to raltitrexed and5-Fu group cloned few,about50%. sequential exposure to5-Fu followed by raltitrexed group clonedmore, about100%.Conclusion: We observed that raltitrexed can inhibit cell growth of HCT-8and SW480, and present a concentration-time dependent model. sequential exposure to raltitrexed followed by5-Fu group and simultaneous exposure toraltitrexed and5-Fu group can inhibit cell growth synergistically or addiative.HCT-8and SW480cells appears the phenomenon of cell cycle arrest,mainly in G1and S phase.Sequential exposure to raltitrexed followed by5-Fu group andsimultaneous exposure to raltitrexed and5-Fu group have higher inhibition rateto colon cell in by plate colony forming assay.