Guo Kun, Kang Nan Xiao, Li Yan, Sun Lu, Gan Lin, Cui Feng Jie, Gao Mei Dong, Liu Kun Yin
Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China.
BMC Cancer. 2009 Mar 31;9:100. doi: 10.1186/1471-2407-9-100.
During the process of metastasis, cells are subjected to various apoptotic stimuli. Aberrant expression of apoptotic regulators often contribute to cell metastasis. Heat shock protein 27(HSP27) is confirmed as an apoptosis regulator, but its antiapoptotic mechanism in metastatic hepatocellular carcinoma (HCC) cells remains unclear.
Levels of HSP27 protein and its phosphorylation in Hep3B, MHCC97L to MHCC97H cells with different metastatic potentials were determined by western blot analysis. MHCC97H cells were transfected with specific small interference RNA (siRNA) against HSP27. The in vitro migration and invasion potentials of cells were evaluated by Transwell assay. The apoptosis ratio of MHCC97H cells was analyzed by TUNEL staining and Flow Cytometry. Alteration of signal transduction pathway after HSP27 knockdown in MHCC97H cells was evaluated through a Human Q Series Signal Transduction in Cancer Gene Array analysis. Nuclear NF-kappaB contentration and endogenous IKK activity were demonstrated by ELISA assay. The association of IKKalpha, IKKbeta, IkappaBalpha with HSP27 and the association between IKKbeta and IKKalpha in MHCC97H cells were determined by co-immunoprecipitation assay followed by western blot analysis.
HSP27 protein and its phosphorylation increased in parallel with enhanced metastatic potentials of HCC cells. siRNA-mediated HSP27 knockdown in MHCC97H significantly suppressed cells migration and invasion in vitro and induced cell apoptosis; the prominently altered signal transduction pathway was NF-kappaB pathway after HSP27 knockdown in MHCC97H cells. Furthermore, inhibition of HSP27 expression led to a significant decrease of nuclear NF-kappaB contentration and endogenous IKK activity. In addition, HSP27 was associated with IKKalpha, IKKbeta, IkappaBalpha in three HCC cells above. ELISA assay and western blot analysis also showed a decrease of the association between IKKbeta and IKKalpha, the association between phosphor-HSP27 and IKK complex, and an increase of total IkappaBalpha but reducing tendency of phosphor-IkappaBalpha when HSP27 expression was efficiently knocked down in MHCC97H cells.
Altogether, these findings revealed a possible effect of HSP27 on apoptosis in metastatic HCC cells, in which HSP27 may regulate NF-kB pathway activation.
在转移过程中，细胞会受到各种凋亡刺激。凋亡调节因子的异常表达通常会促进细胞转移。热休克蛋白27（HSP27）被确认为一种凋亡调节因子，但其在转移性肝癌（HCC）细胞中的抗凋亡机制仍不清楚。
通过蛋白质印迹分析测定不同转移潜能的Hep3B、MHCC97L至MHCC97H细胞中HSP27蛋白及其磷酸化水平。用针对HSP27的特异性小干扰RNA（siRNA）转染MHCC97H细胞。通过Transwell实验评估细胞的体外迁移和侵袭潜能。通过TUNEL染色和流式细胞术分析MHCC97H细胞的凋亡率。通过人类癌症基因阵列Q系列信号转导分析评估MHCC97H细胞中HSP27基因敲低后信号转导通路的变化。通过ELISA检测证明核NF-κB浓度和内源性IKK活性。通过免疫共沉淀实验和蛋白质印迹分析确定MHCC97H细胞中IKKα、IKKβ、IκBα与HSP27的关联以及IKKβ与IKKα之间的关联。
HSP27蛋白及其磷酸化水平随着HCC细胞转移潜能的增强而平行增加。siRNA介导的MHCC97H细胞中HSP27基因敲低显著抑制细胞体外迁移和侵袭并诱导细胞凋亡；MHCC97H细胞中HSP27基因敲低后，显著改变的信号转导通路是NF-κB通路。此外，抑制HSP27表达导致核NF-κB浓度和内源性IKK活性显著降低。此外，HSP27与上述三种HCC细胞中的IKKα、IKKβ、IκBα相关。ELISA检测和蛋白质印迹分析还显示，当MHCC97H细胞中HSP27表达被有效敲低时，IKKβ与IKKα之间的关联、磷酸化HSP27与IKK复合物之间的关联降低，总IκBα增加，但磷酸化IκBα呈下降趋势。
总之，这些发现揭示了HSP27对转移性HCC细胞凋亡的可能影响，其中HSP27可能调节NF-κB通路的激活。
