| Objective: To detect the expression of HER2 in epithelial ovarian cancer tissues, to preliminarily explore the relationship related with age, pathological stage, grade of tumor cells, pathological type and lymph node metastasis; to preliminarily study how the new medicine Trastuzumab/Herceptin, directed by HER2, affects the increasing of the SKOV3 cells which overexpress HER2 protein, and how it affects the cells cycle, apoptosis and the expression of caspase-3 of the SKOV3 cells. To analysis the anti-tumor mechanism of trastuzumab, and to further explore the role of HER2 protein in the development of epithelial ovarian cancer. This study provides experiment basis for the targeted therapy of ovarian cancer.Method:1 Since February 2013 to February 2014, 56 cases of epithelial ovarian cancer were collected, which have been proved by the pathology analysis after surgery from gynaecology and obstetrics department of Hebei Medicine Science University fourth hospital. There were 40 ovarian serous lacteal head cystadenocarcinoma and 16 ovary mucin papillary cystadenocarcinoma patients, and none of them were treated by hormonotherapy, radiotherapy or chemotherapy. The patients’ age was from 35 to 75 years, mean 58.2 years. 20 patients with benign epithelial ovarian tumors were selected, including 12 ovarian serous lacteal head cystadenocarcinoma and 8 ovary mucin papillary cystadenocarcinoma patients, and 15 cases of normal ovary tissues were also selected. According to the FIGO standard, 21 patients were under stage I-II, 35 patients were under stage III-IV. The 56 cases of epithelial ovarian cancer including well differentiated(G1) 13 cases, moderately differentiated(G2) 21 cases and poorly differentiated(G3) 22 cases. 40 cases had Lymph node metastasis. The expression of HER2 was tested in EOC, benign epithelial ovarian tumors and the normal ovary tissues by immunohistochemistry(IHC) SP method. The amplification of HER2 gene was tested in EOC, and the normal ovary tissues with q RT-PCR method. The data was processed by SPSS13.0 software, and then divided by standard P<0.05 has a significant meaning to the statistics.2 The human epithelial ovarian cancer cell line SKOV3 was subcultured in incubator of 37℃, 5% CO2 and cryopreserved.3 The experiment was divided into control group(Herceptin was 0μg/l) and experimental groups of different concentrations(5, 10, 20, 40 and 80μg/l) of Herceptin. The proliferation of the SKOV3 cells was tested by MTS method, after the SKOV3 cell groomed 48 h with different concentrations of Herceptin. Inhibition rates of the corresponding concentration and IC50 were calculated then drew cells growth curve.4 The experiment was divided into control group(Herceptin was 0μg/l) and experimental group of different concentrations(10, 20 and 40μg/l) of Herceptin. After the SKOV3 cell groomed 48 h with different concentrations of Herceptin, the cell apoptosis of the SKOV3 cells were analyzed with Annexin V/PI staining method by FCM; the cell cycle of the SKOV3 cells were analyzed with the PI staining method by FCM; the expression of caspase-3 in the SKOV3 cells were tested by Western blot method, which increased in the early stage of the cells apotisis. Adopting SPSS13.0 software to analyse the statistics, and dividing them by P<0.05 is quite meaningful.Results:1 The positive expression rates of HER2 in malignance ovary tissues, benign tissues and normal tissues were 42.9%, 15.0%, 6.7%. The result showed that the expression of HER2 in malignant group was significantly higher than the benign group and normal control group(P<0.05), and there was little difference between the benign group and normal group(P>0.05). In EOC group, where the clinical stage and tumor grade increased, the expression rate of HER2 protein increased(P<0.05), and the expression rate of HER2 protein in lymph node metastasis-positive was higher than negative one(P <0.05).2 The result of q RT-PCR further showed that the expression of HER2 in EOC group was much higher than the one in normal group, and the result showed 2-ΔΔCт was 55.26 ± 2.00. Between malignant and normal group, the difference was statistically significant(P<0.05). The expression of HER2 was gradually increasing with the progress of EOC and/or the high grade(P<0.05).In EOC, the expression of HER2 in lymph node metastasis-positive group was significantly higher than negative one(P<0.05).3 The result of MTS method indicated that Herceptin could resist the proliferation of the SKOV3 cells. The OD figures were 0.622±0.019, 0.547±0.023, 0.410±0.020, 0.3350±0.016, 0.322±0.021 from the Herceptin(5, 10, 20, 40 and 80μg/l) groups, and The OD figures of the control group was 0.806±0.020,Each experimental group compared with the control group, and between all the experimental groups, the difference was statistically significant(P<0.05). The IC50 was 29.224μg/l by Logit method of statistics.4 The result of PI staining method(FCM) showed that:After the SKOV3 cell was groomed 48 h with different concentrations(10, 20, 40μg/l) of Herceptin, the G0/G1 phase cells increased significantly(54.300±1.776, 63.150±1.792, 68.800±1.120) with the control group(47.967±1.983)(P <0.05). While the G2/M(13.150±0.903, 10.835±0.776, 8.797±0.680) and S(32.417±1.745, 26.017±1.429, 22.483±1.402) phase cells in experimental groups decreased, compared with the control group G2/M phase(15.167±1.242)(P <0.05) and S phase(37.067±1.772)(P<0.05).5 The result of Annexin V/PI staining method(FCM) indicated that the apoptosis rate significantly increased with the concentration of Herceptin increased. In the experimental groups, the apotosis rates of the SKOV3 cell were 11.745±1.346, 19.905±1.793, 24.830±2.120. In control groups, the apotosis rate was 6.397±1.598. Each experimental group compared with the control group, and between all the experimental groups, the difference was statistically significant(P<0.05).6 The result of Western blot method showed that the expression of caspase-3 protein increased while the Herceptin density increased. In the experimental groups, the expression of caspase-3 protein in SKOV3 cells respectively was 0.432±0.023, 0.639±0.026, 0.683±0.019. In the control group(Herceptin for 0μg/l) the relative expressions of caspase-3 protein in SKOV3 cells was 0.348±0.020. The difference among all the experimental groups and the control group is meaningful to the statistics(P<0.05).Conclusion: The HER2 protein was highly expressed, and the HER2 gene relatively was highly amplified in EOC, and it closely related with clinical stage, tumor grade and lymph node metastasis. The result fully explained that the HER2 protein may play an important role in the development of EOC, and HER2 may be used as an index about poor prognosis in EOC. Herceptin could significantly inhibit proliferation of the SKOV3 cells, could stop the SKOV3 cells in stage G0/G1 and slow down the cells transforming to the S stage. The apotosis and the expression of caspase-3 protein of the SKOV3 cells were increased under Herceptin. Those showed that cycle arrest, induction of apoptosis may be one of the anti-tumor mechanisms of Herceptin, but specific anti-tumor mechanism of Herceptin needs further study. These results may provide us the experimental basis for the HER2 targeted therapy applying to the ovarian malignant tumor, and may provide a new target for the treatment of EOC. |
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