title: Potent in vitro and in vivo antitumor effects of MDM2 inhibitor nutlin-3 in gastric cancer cells.
abstract: The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. However, its mutation rate is relatively low in gastric cancer compared with other cancers. In this study, we investigated the mechanisms underlying the antitumor effects of nutlin-3, an inhibitor of human homolog of murine double minute 2 (MDM2). MDM2 is a negative regulator of p53. Four gastric cancer cell lines with wild-type p53 (wt p53) and three with mutant-type p53 (mt p53) were analyzed for MDM2 and MDM4 expression by immunoblotting, and for their gene amplification by quantitative real-time PCR. Moreover, the viability of cells exposed to nutlin-3 was examined by WST-8 assay, and the expression of p53 and its downstream genes was analyzed by immunoblotting. Nutlin-3 stabilized p53 and increased the expression of p21(WAF1) and Noxa, and cleaved poly (ADP)-ribose polymerase regardless of the pre-expression levels of MDM2 and MDM4 in gastric cancer cells with wt p53. Flow cytometry revealed that nutlin-3 arrested the cell cycle in G(1) phase and induced apoptosis in the cell lines. These nutlin-3 effects were not observed in the cell lines with mt p53. Nutlin-3 exerted additive or synergistic cytotoxicity in combination with 5-fluorouracil or cisplatin in most cell lines with wt p53. An in vivo antitumor effect of nutlin-3 alone and its additive augmentation by 5-fluorouracil were confirmed in an MDM2 overexpressed xenograft tumor model. Nutlin-3 showed potent antitumor activity against human gastric cancer cells with wt p53 and shows promise as a single agent and in combination with conventional anticancer drugs.
authors.affiliation: Department of Gastroenterology and Hepatology, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Endo
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamato
authors.forename: Sachiko
authors.initials: S
authors.lastname: Hirai
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Moriwaki
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Fukuda
authors.forename: Hideo
authors.initials: H
authors.lastname: Suzuki
authors.forename: Masato
authors.initials: M
authors.lastname: Abei
authors.forename: Ichiro
authors.initials: I
authors.lastname: Nakagawa
authors.forename: Ichinosuke
authors.initials: I
authors.lastname: Hyodo
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D018628:Gene Dosage; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000032:analysis; D010879:Piperazines / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000235:genetics; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
chemical_list: D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D007093:Imidazoles; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D051736:Proto-Oncogene Proteins c-mdm2; D002945:Cisplatin; D005472:Fluorouracil