title: The plant alkaloid cryptolepine induces p21WAF1/CIP1 and cell cycle arrest in a human osteosarcoma cell line.
abstract: We previously established a bioassay method to screen for compounds that activate the promoter activity of p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases, in a p53-independent manner. As an activator of p21(WAF1/CIP1) promoter activity, we isolated cryptolepine (CLP: 5-methyl indolo (2,3b)-quiniine), an indoloquinoline alkaloid, from the traditional Ayurvedic medicinal plant Sida cordifolia. We show here that CLP induces the expression of p21(WAF1/CIP1) with growth arrest in p53-mutated human osteosarcoma MG63 cells. Four micromolar of CLP completely inhibited the growth of MG63 cells and caused G2/M-phase arrest. CLP up-regulated the expression of p21(WAF1/CIP1) at both mRNA and protein levels in a dose-dependent manner. Using several mutant p21(WAF1/CIP1) promoter constructs, we found that the CLP-responsive element is an Sp1 site at -82 relative to the transcription start site of the p21(WAF1/CIP1) promoter. These findings suggest that CLP arrests the growth of MG63 cells by activating the p21(WAF1/CIP1) promoter through the specific Sp1 site in a p53-independent manner. In addition, CLP-mediated cell cycle arrest was reduced by the knockout of the p21(WAF1/CIP1) gene in human colon cancer HCT116 cells, suggesting that the cell cycle arrest by CLP was at least partially mediated through the induction of p21(WAF1/CIP1) expression. Although we need further study of chemotherapeutic effect in vivo, these results raise the possibility that CLP might be a suitable chemotherapeutic agent for treatment of osteosarcoma.
authors.affiliation: Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Taka-aki
authors.initials: TA
authors.lastname: Matsui
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sowa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Murata
authors.forename: Koichi
authors.initials: K
authors.lastname: Takagi
authors.forename: Ryoko
authors.initials: R
authors.lastname: Nakanishi
authors.forename: Shunji
authors.initials: S
authors.lastname: Aoki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yoshikawa
authors.forename: Motomasa
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Tomoya
authors.initials: T
authors.lastname: Sakabe
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Kubo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sakai
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D026121:Indole Alkaloids / Q000494:pharmacology*; D008156:Luciferases; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011804:Quinolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D016329:Sp1 Transcription Factor / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism