Matsui Taka-aki, Sowa Yoshihiro, Murata Hiroaki, Takagi Koichi, Nakanishi Ryoko, Aoki Shunji, Yoshikawa Masayuki, Kobayashi Motomasa, Sakabe Tomoya, Kubo Toshikazu, Sakai Toshiyuki
Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Int J Oncol. 2007 Oct;31(4):915-22.
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.
我们之前建立了一种生物测定方法，用于筛选能够以不依赖p53的方式激活细胞周期蛋白依赖性激酶的强效抑制剂p21（WAF1/CIP1）启动子活性的化合物。作为p21（WAF1/CIP1）启动子活性的激活剂，我们从传统的阿育吠陀药用植物匙叶伽蓝菜中分离出了一种吲哚喹啉生物碱隐丹参酮（CLP：5-甲基吲哚并（2,3b）喹啉）。我们在此表明，CLP在p53突变的人骨肉瘤MG63细胞中诱导p21（WAF1/CIP1）的表达并导致生长停滞。4微摩尔的CLP完全抑制了MG63细胞的生长并导致G2/M期停滞。CLP以剂量依赖性方式在mRNA和蛋白质水平上调p21（WAF1/CIP1）的表达。使用几种突变的p21（WAF1/CIP1）启动子构建体，我们发现CLP反应元件是相对于p21（WAF1/CIP1）启动子转录起始位点-82处的一个Sp1位点。这些发现表明，CLP通过以不依赖p53的方式通过特定的Sp1位点激活p21（WAF1/CIP1）启动子来阻止MG63细胞的生长。此外，人结肠癌HCT116细胞中p21（WAF1/CIP1）基因的敲除降低了CLP介导的细胞周期停滞，这表明CLP引起的细胞周期停滞至少部分是通过诱导p21（WAF1/CIP1）表达介导的。尽管我们需要进一步研究其体内化疗效果，但这些结果增加了CLP可能是治疗骨肉瘤的合适化疗药物的可能性。
