Kashiwagi Korehito, Virgona Nantiga, Yamada Jin, Sato Ayami, Ota Masako, Yazawa Takuya, Yano Tomohiro
Faculty of Life Sciences, Toyo University, Itakura, Gunma 374-0192;
Exp Ther Med. 2011 Jul;2(4):719-724. doi: 10.3892/etm.2011.270. Epub 2011 May 12.
Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200-400 μg/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling.
恶性间皮瘤（MM）是一种侵袭性癌症，目前尚无有效的治疗方案。强制表达间隙连接（GJ）成分连接蛋白43（Cx43）可增加MM细胞对顺铂的敏感性。鲍曼-伯克蛋白酶抑制剂（BBI）可诱导多种肿瘤细胞中Cx43的恢复。在本研究中，我们检测了BBI通过诱导Cx43增强顺铂对MM细胞细胞毒性作用的能力。使用人MM H28细胞。通过WST-1试验评估细胞活力，通过荧光分析测定蛋白酶体活性。分别通过免疫印迹分析和实时RT-PCR测定蛋白质和mRNA水平。使用刮擦加载法评估由Cx43介导的GJ功能。BBI以剂量依赖性方式（200-400μg/ml）有效抑制H28细胞生长。与生长抑制同时，BBI处理可提高Cx43水平（mRNA和蛋白质）以及GJ功能。通过反义核苷酸处理敲低BBI诱导的Cx43几乎消除了生长抑制。BBI增强了顺铂对H28细胞的细胞毒性，反义核苷酸处理下调Cx43可消除BBI的增强作用。BBI诱导Cx43有助于Src失活并随后诱导Bax。此外，Src抑制剂（SU6656）也增强了顺铂对H28细胞的细胞毒性。这些结果表明，BBI通过抑制Src信号通路提高了顺铂对H28细胞的细胞毒性疗效。
