title: Mechanism(s) of antitumor action in protracted infusion of low dose 5-fluorouracil and cisplatin in gastric carcinoma.
abstract: The therapeutic efficacy of low dose administration of 5-fluorouracil (5-FU) and cisplatin (CDDP) (low dose FP) has been reported in patients with advanced and recurrent gastric carcinoma. Mechanism(s) by which low dose FP exerts antitumor effect is not entirely clear. We investigated mechanism(s) of the therapeutic efficacy in combination with 5-FU and CDDP in terms of signal transduction pathways leading to apoptosis. Using two human gastric carcinoma cell lines, MKN28 and MKN45, antitumor effect in combination treatment with 5-FU and CDDP was assessed by MTT 5-day assay. The significant antitumor effect was determined with more than 50% growth inhibition compared to control cells. Enhancement of antitumor effect in the combination treatment was analyzed using isobologram. Apoptotic cell death was assessed by DNA ladder formation assay, and expression of apoptosis-related genes was detected by Western blotting. Concentration of free platinum and 5-FU was measured by high-pressure liquid chromatography (HPLC), and dihydropyrimidine dehydrogenase (DPD) activity and total folate levels were assessed by enzyme immunoassays. Antitumor effect in single treatment with 5-FU was not observed significantly with the concentration from 1 to 5 microM in vitro. In contrast, antitumor effect in combination treatment with 5-FU and CDDP showed a synergism with the concentration of CDDP from 1.5 to 3 microM. Single treatment with CDDP also did not show significant antitumor effect with the concentration from 1.5 to 3 microM. The enhancement in the synergistic effect by CDDP was dose-dependent. Any free platinum treated with low dose CDDP was not detected into gastric carcinoma cells, however, treatment with CDDP induced a receptor signaling pathway, that is mediated by Fas but not DR4. It may directly activate caspase 3 leading to apoptosis. Although the receptor signaling pathway in apoptosis was not observed by 5-FU, Bax-induced cytochrome c and caspase 3 was also observed in a receptor-independent pathway by 5-FU and CDDP. Total folate levels by cotreatment with CDDP was increased to 1.5-fold compared to 5-FU alone, whereas DPD activity and 5-FU concentration were not changed by cotreatment of CDDP in vivo. The enhancement of antitumor effect by low dose FP can be explained as follows: i) low dose treatment with CDDP induces apoptotic cell death through a receptor signaling pathway even in absence of free platinum into cells; ii) increased folate level by CDDP and a non-receptor signaling pathways by 5-FU contribute to apoptotic cell death in gastric carcinoma.
authors.affiliation: Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan. rkim@ipc.hiroshima-u.ac.jp
authors.forename: Ryungsa
authors.initials: R
authors.lastname: Kim
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Tanabe
authors.forename: Hideki
authors.initials: H
authors.lastname: Inoue
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toge
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis; D002478:Cells, Cultured; D002945:Cisplatin / Q000008:administration & dosage*; D003574:Cytochrome c Group / Q000096:biosynthesis; D053938:DNA Fragmentation; D042943:Dihydrouracil Dehydrogenase (NADP); D004305:Dose-Response Relationship, Drug; D005472:Fluorouracil / Q000008:administration & dosage*; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D010088:Oxidoreductases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000188:drug therapy*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000096:biosynthesis
chemical_list: D000970:Antineoplastic Agents; C494826:BAX protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D005492:Folic Acid; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002945:Cisplatin; D005472:Fluorouracil