Objective: To examine the expressions of IKKε protein in the specimens and cells of epithelial ovarian cancer and investigate the effect of IKKε inhibitor on cell proliferation and apoptosis. Methods: (1) A total of 118 cases of patients with the median age of 59 who have accepted surgical treatment due to ovarian cancer in the First Affiliated Hospital of Dalian Medical University from January 2006 to April 2013 were selected. Twenty cases of patients with the median age of 55 who have accepted hysterectomy and salpingo-oophorectomy due to uterine leiomyoma during the same period were selected as the control. The expressions of IKKε protein were detected by immunohistochemistry in normal ovarian tissues and epithelial ovarian cancer specimens, and the relationship between the expressions of IKKε and the clinical features of patients was analyzed. IKKε protein was determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 treated with or without IKKε inhibitor. The cellular proliferation and apoptosis of ovarian cancer cells after 48 hours treatment of IKKε inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical results showed that IKKε was highly expressed in epithelial ovarian cancer specimens with the expression rate 66.1% (78/118), compared with normal ovarian tissue with the expression rate 35.0% (7/20), which exhibited statistically significant difference (χ(2)=6.993, P=0.008). The expression of IKKε protein was correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, the level of CA(125) in preoperative serum and distribution of the tumor (P<0.05), but no correlation with age, histological type, the incidence pattern, and tumor size (all P>0.05). (2) IKKε was widely overexpressed in different levels in SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 cells, and the expression of IKKε decreased as the increase of the concentration of IKKε inhibitor (0.1 and 0.5 μmol/L) in OV2008, C13, A2780S, and A2780CP cells after 48 hours treatment. Different concentrations of IKKε inhibitor (0.05, 0.1, 0.5, 1, 5, 10, and 25 μmol/L) significantly inhibited the proliferation of OV2008, C13, A2780S, A2780CP, and SKOV3 cells in a concentration-dependent manner (P<0.05), and the half maximal inhibitory concentration (IC(50)) was 0.43, 0.86, 0.10, 0.19, and 0.24 μmol/L, respectively. The cell apoptotic rate of OV2008, C13, A2780S, A2780CP, and SKOV3 cells was significantly increased after 48 hours treatment of IKKε inhibitor with the concentration of 0.1 and 0.5 μmol/L (P<0.05). Conclusions: The IKKε protein in epithelial ovarian cancer specimens and cells is overexpressed. IKKε inhibitor could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner. Together, the result indicated that IKKε may be a candidate target for the treatment of ovarian cancer in future.
目的： 探讨卵巢上皮性癌（卵巢癌）组织和细胞中IKKε蛋白的表达，以及抑制其表达后对卵巢癌细胞增殖、凋亡的影响。 方法： （1）选取2006年1月至2013年4月大连医科大学附属第一医院经手术治疗且经病理检查证实为卵巢癌的石蜡组织标本118份，患者中位年龄为59岁；另取同期因子宫肌瘤行子宫及卵巢切除术且术后经病理检查证实为正常卵巢组织标本20份作为对照，患者中位年龄为55岁。免疫组化SP法检测卵巢癌和正常卵巢组织中IKKε蛋白的表达，并分析IKKε蛋白表达与卵巢癌患者临床病理特征之间的关系。（2）选择卵巢癌细胞系SKOV3、OV2008、C13、A2780S、A2780CP、OV4、OV5、OV8和CAOV3细胞，蛋白印迹法检测卵巢癌细胞中IKKε蛋白的表达，以及IKKε抑制剂对卵巢癌细胞IKKε蛋白表达的影响；四甲基偶氮唑蓝（MTT）比色法和流式细胞仪分别检测IKKε抑制剂对卵巢癌细胞增殖和凋亡的影响。 结果： （1）免疫组化SP法检测显示，卵巢癌、正常卵巢组织中IKKε蛋白阳性表达率分别为66.1%（78/118）、35.0%（7/20），两者比较，差异有统计学意义（χ(2)=6.993，P=0.008）。卵巢癌组织中IKKε蛋白的表达与手术病理分期、病理分化程度、术前血清CA(125)水平和肿瘤分布（包括单侧卵巢、双侧卵巢）均明显相关（P<0.05），而与年龄、病理类型、发病模式分型[包括Ⅰ型（即低级别卵巢癌）、Ⅱ型（即高级别卵巢癌）]和肿瘤直径均无明显相关性（P>0.05）。（2）蛋白印迹法检测显示，9种卵巢癌细胞即SKOV3、OV2008、C13、A2780S、A2780CP、OV4、OV5、OV8和CAOV3细胞中均表达IKKε蛋白，其表达强度略有差异；IKKε抑制剂（分别为0.1、0.5 μmol/L）作用后，OV2008、C13、A2780S和A2780CP细胞中IKKε蛋白的表达强度分别与IKKε抑制剂作用前比较均明显减弱。MTT比色法检测显示，不同浓度（分别为0.05、0.1、0.5、1、5、10、25 μmol/L）IKKε抑制剂作用后，OV2008、C13、A2780S、A2780CP和SKOV3细胞增殖的抑制率随着IKKε抑制剂浓度的增加均逐渐增高，呈明显浓度依赖性（P<0.05）；其50%抑制浓度（IC(50)）分别为0.43、0.86、0.10、0.19、0.24 μmol/L。流式细胞仪检测显示，不同浓度（分别为0.1、0.5 μmol/L）IKKε抑制剂作用后，OV2008、C13、A2780S、A2780CP和SKOV3细胞凋亡率分别与IKKε抑制剂作用前比较均明显增高（P<0.05）。 结论： IKKε蛋白在卵巢癌组织和细胞中均呈高表达；IKKε抑制剂下调IKKε蛋白表达后可抑制卵巢癌细胞增殖并诱导卵巢癌细胞发生凋亡，提示IKKε有望成为卵巢癌分子治疗的新靶点。.
Keywords: Apoptosis; Cell proliferation; I-kappa B kinase; Immunohistochemistry; Ovarian neoplasms.