Aaptamine, a spongean alkaloid, activates p21 promoter in a p53-independent manner Shunji Aoki a , Dexin Kong a , Hideaki Suna a , Yoshihiro Sowa b , Toshiyuki Sakai b , Andi Setiawan c , Motomasa Kobayashi a, * a Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan b Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan c Department of Chemistry, Faculty of Science, Lampung University, Jl. Prof. Dr. Sumantri Brodjonegoro No. 1, Bandar Lampung 35145, Indonesia Received 14 January 2006 Available online 3 February 2006 Abstract Aaptamine, a benzonaphthyridine alkaloid was isolated from a marine sponge on the guidance of a bioassay using the transfected human osteosarcoma MG63 cells (MG63luc + ). Aaptamine activated p21 promoter stably transfected in MG63 cells dose-dependently at the concentrations of 20–50 lM. Expression of p21 and its mRNA in the wild-type MG63 cells also increased by aaptamine-treatment. Furthermore, the cell cycle of MG63 cells was arrested at the G2/M phase within 48 h by the aaptamine-treatment. To analyze a respon- sive element of p21 promoter in the up-regulation of p21 by aaptamine, MG63 cells were transiently transfected with a series of the delet- ed or mutated promoter segments, and induction of luciferase with aaptamine treatment was examined by using these corresponding transfected cells. The activation of p21 promoter by aaptamine was led through acting Sp1 sites between À82 and À50 bp in a p53- independent manner. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Aaptamine; p21 promoter; MG63 cells; Sp1 sites; G2/M arrest Abnormal proliferation, which caused by the disruption of the cell cycle regulation, is a major undesirable property in tumor cells. Cell proliferation is mainly regulated by interactions between two families of proteins, cyclin-depen- dent kinase (CDK)–cyclin complexes and CDK inhibitors (CKIs). CDK–cyclin complexes promote the progression of cell cycle in an organized manner, while CKIs inhibit it for checking and repairing the damage on chromosome. P21 was originally identified as a target protein of p53 [1] and is known to be an inhibitor of CDK–cyclin complexes [2] to act as a negative regulator of the cell cycle progres- sion as a brake. The p21 expression is mainly controlled by diverse mechanisms in a p53-dependent manner. Due to the accumulating evidence that p53 is mutated in many human cancer cells [3,4], mutation of p53 has been recog- nized as one of the major events in carcinogenesis, while mutation of p21 is rarely observed in human tumors [5,6]. Therefore, the agents that induce an increase of p21 expression in a p53-independent manner might contribute to cancer prevention or treatment. The approach using such an agent has been termed ‘‘gene-regulating chemo- therapy or prevention’’ [7]. As a part of our discovery of bioactive substances from marine organisms, we have been searching for new antican- cer agents by using various screening bioassay methods. To date, we have found some interesting lead compounds such as long-chain acetylene alcohol (lembehyne A) [8,9], which induces neurite outgrowth of murine neuroblastoma Neuro 2A cells, sesquiterpene aminoquinones (smenospongine) [10,11], and pentacyclic guanidine alkaloid (crambescidin 800) [12], which induces erythroid differentiation of human chronic myelogenous leukemia K562 cells. Interestingly, all 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.119 * Corresponding author. Fax: +81 6 6879 8219. E-mail address: kobayasi@phs.osaka-u.ac.jp (M. Kobayashi). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 342 (2006) 101–106 BBRC