Nagasawa Tomoaki, Matsushima-Nishiwaki Rie, Toyoda Hidenori, Matsuura Junya, Kumada Takashi, Kozawa Osamu
Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
Department of Gastroenterology, Ogaki Municipal Hospital, Ogaki, Gifu 503-8502, Japan.
Oncol Rep. 2014 Sep;32(3):1291-5. doi: 10.3892/or.2014.3278. Epub 2014 Jun 23.
A small heat shock protein (HSP), HSP20 (HSPB6) is ubiquitously expressed in various tissues and has several functions. We previously reported that the expression of HSP20 protein in human hepatocellular carcinoma (HCC) cells is inversely proportional to the progression of HCC. In addition, we showed that HSP20 is associated with phosphoinositide 3-kinase (PI3K) and inhibits the proliferation of HCC cells via suppression of the AKT signaling pathway. However, the relationship between HSP20 and apoptosis in HCC has not yet been elucidated. To clarify whether HSP20 is implicated in the apoptosis of HCC cells, in the present study, we examined the effect of HSP20 on caspases, the central regulators of apoptosis, using human HCC-derived HuH7 cells that are transfected with wild-type human HSP20 (HSP20-overexpressing cells). The cleavage of caspase-3 and caspase-7 in HSP20-overexpressing cells was enhanced compared with the empty vector-transfected cells (control cells). In addition, the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) in HSP20-overexpressing cells was also strengthened. We further investigated the direct targets of HSP20 focusing on Bcl-2 family proteins in the HSP20-overexpressing cells. HSP20 proteins in the cells were coimmunoprecipitated with Bax. On the contrary, Bad, Bcl-2 and Bcl-xL were not coimmunoprecipitated with HSP20. These findings strongly suggest that HSP20 directly associates with Bax and stimulates caspase cascade in human HCC cells.
小分子热休克蛋白（HSP）HSP20（HSPB6）在各种组织中普遍表达并具有多种功能。我们之前报道过，HSP20蛋白在人肝癌（HCC）细胞中的表达与HCC的进展呈负相关。此外，我们还表明HSP20与磷酸肌醇3激酶（PI3K）相关，并通过抑制AKT信号通路来抑制HCC细胞的增殖。然而，HSP20与HCC细胞凋亡之间的关系尚未阐明。为了阐明HSP20是否与HCC细胞凋亡有关，在本研究中，我们使用转染了野生型人HSP20的人HCC来源的HuH7细胞（HSP20过表达细胞），研究了HSP20对凋亡的核心调节因子半胱天冬酶的影响。与空载体转染细胞（对照细胞）相比，HSP20过表达细胞中半胱天冬酶-3和半胱天冬酶-7的切割增强。此外，HSP20过表达细胞中核聚（ADP-核糖）聚合酶（PARP）的切割也得到加强。我们进一步在HSP20过表达细胞中聚焦于Bcl-2家族蛋白研究HSP20的直接靶点。细胞中的HSP20蛋白与Bax进行了共免疫沉淀。相反，Bad、Bcl-2和Bcl-xL未与HSP20进行共免疫沉淀。这些发现强烈表明，HSP20直接与Bax结合并刺激人HCC细胞中的半胱天冬酶级联反应。
