Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 μmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 μmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 μmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.
目的： 探讨有丝分裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）信号通路在卵巢上皮性癌（卵巢癌）组织和细胞中的表达及其临床意义。 方法： （1）收集2004年1月—2013年12月大连医科大学附属第一医院收治的术后病理检查证实为卵巢癌的104例患者的石蜡标本，采用免疫组化SP法检测卵巢癌组织中磷酸化ERK1/2（p-ERK1/2）蛋白的表达，并分析其与各临床病理特征的关系。（2）蛋白印迹（western blot）法检测MAPK激酶（MEK）抑制剂——司美替尼（AZD6244）作用前后不同卵巢癌细胞（包括SKOV3、OV2008、C13、A2780S、A2780CP、OVCAR4、OVCAR5、OVCAR8和CAOV3细胞共9种）中p-ERK1/2、ERK1/2蛋白的表达，四甲基偶氮唑蓝（MTT）比色法检测AZD6244作用后不同卵巢癌细胞的增殖情况，流式细胞仪检测AZD6244作用后不同卵巢癌细胞的凋亡率及细胞周期比例。 结果： （1）免疫组化SP法检测显示，p-ERK1/2蛋白的阳性表达率与卵巢癌患者的年龄、病理类型、手术病理分期、术前血清CA(125)水平均无明显相关性（P>0.05）。进一步分析发现，在不同病理类型的卵巢癌组织中，p-ERK1/2蛋白的阳性表达率，高级别浆液性癌、黏液性癌及子宫内膜样癌均低于低级别浆液性癌（LGSC），分别比较，差异均有统计学意义（P<0.05）；而透明细胞癌与LGSC比较，差异则无统计学意义（P=0.196）。（2）western blot法检测显示，p-ERK1/2、ERK1/2蛋白在9种卵巢癌细胞中均有不同程度的表达；不同浓度（5、10 μmol/L）的AZD6244分别作用于OVCAR5和OVCAR8细胞后，均可抑制p-ERK1/2、细胞周期蛋白D1（cyclin D1）、半胱氨酸天冬氨酸蛋白酶3（caspase-3）、聚二磷酸腺苷核糖聚合酶（PARP）蛋白的表达，且AZD6244作用后可伴有PARP裂解片段的出现。MTT比色法检测显示，不同浓度（0、2.5、5、10、25、50、100 μmol/L）的AZD6244作用后，OVCAR5、OVCAR8和A2780S细胞增殖的抑制率均随着AZD6244浓度的增高而降低，呈显著的浓度依赖性（P<0.05）。流式细胞仪检测显示，不同浓度（0、5、10 μmol/L）的AZD6244作用后，OVCAR5、OVCAR8和A2780S细胞的凋亡率均随着AZD6244浓度的增高而增高，呈显著的浓度依赖性（P<0.01）；OVCAR5和OVCAR8细胞的G(0)/G(1)期比例显著增高，S期比例显著降低，呈显著的浓度依赖性（P<0.05）。 结论： 作为MAPK/ERK信号通路的活化形式和功能单位，p-ERK1/2蛋白在卵巢癌组织和细胞中均有不同程度的表达，MEK抑制剂可抑制卵巢癌细胞的增殖、诱发卵巢癌细胞的凋亡。提示，MAPK/ERK信号通路可能在卵巢癌发生、发展中起一定作用，抑制该信号通路有望成为临床治疗卵巢癌的新靶点。.
Keywords: Apoptosis; Cell proliferation; Mitogen-activated protein kinases; Ovarian neoplasms; Phosphorylation.