| Objectives Ovarian cancer is seriously harmful to women's health, the mortality rate ranks first in gynecological malignant tumor, among which the most common one is epithelial ovarian cancer. Epithelial ovarian cancer develops rapidly, with high metastasis and chemotherapy resistance, which are difficult to overcome in the treatment. Research results show that brain-derived neurotrophic factor/Tyrosine receptor kinase B signal transduction pathway is involved in the occurrence and development of many kinds of tumor cells by activating PI3K/Akt pathway, RAS/MAPK pathway and other signaling pathways, which is closely related to the proliferation, drug resistance, invasion, migration, tumor angiogenesis and other processes. TrkB and BDNF are over expressed in epithelial ovarian cancer, which are involved in proliferation, invasion, migration and anoikis resistance of ovarian cancer, and related to the stages and prognosis. Using tyrosine kinase inhibitor K252 a to block the BDNF/TrkB signal pathway can decrease the proliferation and anoikis resistance of ovarian cancer cells, but the related mechanism was less studied. The aim of this study was to explore the effect of BDNF/TrkB signal pathway inhibitor K252 a on proliferation, apoptosis, invasion, migration, drug resistance and anoikis resistance of SKOV3 cells, then to investigate the possible mechanism in this process.Methods 1 Choose SKOV3 cell lines to study; 2 Experiments were divided into 10 groups: 1~5 groups were added 0 nmol/l, 10 nmol/l, 100 nmol/l, 1000 nmol/l and 2000nmol/l K252 a to culture for 24h; 6~10 groups were added 0 nmol/l, 10 nmol/l, 100 nmol/l, 1000 nmol/l and 2000nmol/l K252 a to culture for 48h; 3 The level of phosphorylated TrkB protein in each group was measured by western blot; 4 Cell proliferation was assessed by MTT assay; 5 Cell apoptosis rate was detected by FCM; 6 Cell invasion and migration ability was determined by Transwell assay; 7 The sensitivity of cisplatin was determined by MTT assay; 8 Establish anoikis pattern in vitro, cell apoptosis rate was measured by FCM; 9 The relative expression level of c-myc, Fas, Bcl-2, LPA2, NF-?B m RNA were measured by Real time-PCR;Results 1 The result of western blot: The level of phosphorylated TrkB protein of each group were 0.1846±0.0161,0.1713±0.0096,0.1595±0.0199, 0.0955±0.0281, 0.0648±0.0210, 0.1744±0.0172,0.1625±0.0060,0.1011±0.0165,0.0878±0.0115, 0.0301±0.0069 respectively, the result showed that the level of phosphorylated TrkB protein was affected by incubation time and K252 a concentration. The relative expression of phosphorylated TrkB protein was the lowest after cultured by 2000nmol/l K252 a for 48 h, which was significantly different from other groups(P<0.05). Therefore, 2000nmol/l K252 a to culture for 48 h was chosen for the following experiments. 2 MTT assay result: The OD490 of control and experimental group were 0.749±0.078 and 0.419±0.026 respectively, the OD490 of experimental group was significantly lower than control group(P<0.05), the result showed that K252 a can significantly inhibit the proliferation of SKOV3 cells. 3 The result of apoptosis: Apoptosis rate of control and experimental group were(4.180±0.838)% and(9.610±0.966)% respectively, experimental group was significantly higher than that of control group(P<0.05), the result showed that K252 a promote the apoptosis of SKOV3 cells. 4 The result of cell invasion and migration: Invasive cells in control and experimental group were 86.074±2.465 and 41.895±2.766 respectively, migrating cells were 102.434±8.299 and 63.712±7.027 respectively, invasion index were 84.029% and 65.757% respectively. The number of cells in experimental group was significantly reduced compared with control group(P<0.05), the result showed that K252 a inhibit the invasion and migration ability of SKOV3 cells. 5 The result of cisplatin sensitivity: The OD490 of control, K252 a, cisplatin and cisplatin+K252a group were 0.739±0.065, 0.410±0.020, 0.372±0.008 and 0.292±0.027 respectively, the OD490 of K252 a group, cisplatin group and cisplatin+K252a group were significantly reduced compared with the control group(P<0.05), the OD490 of cisplatin+K252a group was significantly lower than the cisplatin group(P<0.05), the result showed that K252 a raise cisplatin sensitivity of SKOV3 cells. 6 The result of anoikis resistance ability: Apoptosis rate of control group was(14.937±1.998)%, apoptosis rate of experimental group was(36.100±2.632)%, the apoptosis rate of experimental group was significantly higher than that of control group(P<0.05), the result showed that K252 a decrease anoikis resistance ability of SKOV3 cells. 7 The result of Real-time PCR: The relative expression level of Bcl-2, c-myc, LPA2, NF-?B and Fas m RNA in control group were 1.0876±0.3237, 1.0341±0.3044, 1.0191±0.2274, 1.0258±0.2640, 0.8477±0.1186 respectively, the relative expression level of Bcl-2, c-myc, LPA2, NF-?B and Fas m RNA in experimental group were 0.4952±0.1501, 0.4540±0.1918, 0.5522±0.1284, 0.2954±0.1594, 1.9384±0.5408 respectively, compared with control group, the expression level of Bcl-2, c-myc, LPA2, NF-?B in experimental group was reduced, and the expression level of Fas was increased(P<0.05).Conclusions 1 K252 a resist ovarian cancer in vitro by blocking the BDNF/TrkB signal transduction pathway; 2 K252 a block the BDNF/TrkB signal transduction pathway to anti ovarian cancer by regulating the m RNA expression of c-myc, Fas, Bcl-2, LPA2, NF- ?B. |
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