| Ovarian cancer is one of the most common malignant tumors of the female reproductive system. Due to its difficult early diagnosis, complex and diverse pathological type, the prognosis remains poor. Despite aggressive surgery and chemotherapy, the5-year survival rate still hovering around40%.In order to explore the new target of the treatment, we clarified the role and mechanism of PAF/PAFR signaling pathway in tumor invasion. We also observed the effects of gingkgolide B (GB), a specific PAFR inhibitor, on inhibition of nude mice ovarian cancer xenografts, and investigated the mechanisms of GB on reducing ovarian cancer risk in a BRCA1-mutant ovarian epithelial cell line. The study consists of the following three parts:Part1Effects and mechanisms of platelet-activating factor on invasion of ovarian cancer cells in vitroObjective To investigate the effects and possible mechanisms of PAF on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy.Methods (1) To determination of the optimal PAF concentration, ovarian cancer cell OVCA429was treated by0,0.1,1,10,100, and1000nM of PAF for6hours or24hours, respectively. To observe the different time point of protein changes, OVCA429were treated by100nM of PAF for0,5min、10min,30min,1hour or12hours, respectively. The total proteins of treated cells were extracted according to standard protocol. The expression of p38mitogen-activated protein kinase (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), transcription factor response element-binding protein (CREB), phosphorylated CREB (p-CREB) and matrix metalloproteinase-2(MMP2) were detected by western blot.(2)To verify the pathway involved in the PAF induction of the cancer cell invasion, we repeated the experiments by adding the inhibitors when treating cells with PAF. The inhibitors used were as follows, PAFR inhibitor-GB (100μM), p-p38MAPK inhibitor-SB203580(10μM)、 CREB binding protein (CBP)-CREB interaction inhibitor-217505(25μM).(3) The invasion abilities of cancer cells in above groups were determined by transwell assay.Results (1) Even a very low concentration of PAF (0.1nmol/L) could increase the expression of p-CREB and MMP2, while the most effective concentration of PAF was100nmol/L. The highest p-CREB protein expression was detected6hours after administration of100nmol/L PAF, as well as the expression of p-p38MAPK protein. Even12hours after treatment the p-p38MAPK protein could be detected, while there was no expression of p-CREB.(2) As compared with PAF, both in PAF+GB group and PAF+SB2035group, the expressions of p-p38MAPK, p-CREB and MMP2protein were decreased significantly; in PAF+217505group, although the expression of p-p38MAPK and p-CREB protein was significantly higher than the control group, the expression of MMP2protein was significantly lower; In PAF+SB203580+217505group, the expression of these three proteins were also significantly lower, but there was no significant difference as compared with the PAF+GB or PAF+SB203580group.(3) The results of transwell assay revealed that the PAF group had the largest number of transmembrane cells, while in PAF+GB group the number of transmembrane cells significantly reduced. In PAF+SB203580group, although the transmembrane cell number decreased, which was different significantly compared with control. In PAF+217505group, the invasion of cells was significantly decreased, which was lower than cells in PAF+SB203580group. PAF+SB203580+217505groups have a better inhibition of PAF-induced invasion effects, but which was still weaker than the PAF+GB group.Conclusion PAF could induce MMP-2expression and contributed to non-mucinous ovarian cancer cell invasion via activation of cyclic adenosine monophosphate (cAMP)-REB by phosphorylating of p38MAPK protein.Part2The effects of Ginkgolide B on treatment of ovarian cancer in vivoObjective To construct peritoneal ovarian cancer model in nude mice, monitoring the serum PAF levels, and to observe the anti-tumor effects of the PAFR inhibitor, GB, in vivo.Methods The expression of PAFR in SKOV3-luc cells was examed by immunofluorescence staining technique. The intraperitoneal ovarian cancer model was established be i.p. injection of SKOV3-luc cells in nude mice. The progression of tumors within the abdominal cavity of the animal was monitored by in vivo imaging system. The serums of animals in each group were obtained by taking100μl of blood through tail vein at the time point of before cancer injection,4weeks after injection and2weeks after treatment, respectively. Forty mice with intraperitoneal ovarian cancer were devided into four groups with10mice in each randomly4weeks after cell injection. All animals received treatment according to the following scheme:the negative control group (DMSO): DMSO100μl/d, the GB treatment group:GB10mg/d, the positive control group (CDDP):CDDP5mg/kg/w, GB+CDDP treatment group: GB10mg/d+CDDP5mg/kg/w, all treatments were intraperitoneally administered. The tumor progression was mornitored by in vivo imaging every3days since the begining of therapy. The tumor growth curves were drawn according to the fluorescence intensity in each group. The mice were sacrificed and the tumor tissues were obtained at the end of treatment. The tumor inhibitory rates were determined by calculating the average weight of tumor tissues in each group. The expressions of PAFR, Src, p-Src and Cyclin D1protein in tumor tissue were examed by Immunohistochemical staining.Results The high expression of PAFR was detected in SKOV3-luc cells by immunofluorescence staining. Four weeks after intraperitoneal injection of cancer cells, the diffuse distribution of tumor in the abdomen could be detected by in vivo imaging. The PAFR was detected in the tumor tissue as well by IHC. The concentration of PAF in mice serum was elevated (about6times)4weeks after cancer cell injection. The values did not change significantly after2weeks’ of treatment in all groups. The growth of tumor was similar in GB and CDDP treatment group (P>0.05), but it was significantly lower than that in DMSO group (P<0.001). In GB+CDDP group, the growth of tumor was much lower than that of the first two groups (P<0.001). The tumor inhibition rates in each group were GB48%, CDDP53.7%and GB+CDDP78.2%; respectively. The expression of Src was no difference in the tumor tissue of each group, while the expression of p-Src in GB or GB+CDDP group was lower than that of CDDP or DMSO group. The expresions of Cyclin D1in the tumor tissue were listed from weak to strong as GB+CDDP, CDDP, GB and DMSO groups, repectively.Conclusion The PAF level was elevated in the mice serum of intraperitoneal ovarian cancer model. The PAFR inhibitor GB could inhibit tumor progression.It had a synergistic effect on ovarian cancer when used in combination with CDDP. Our results indicate that GB could be a novel adjuvant agent to conventional therapy of ovarian cancer.Part3The study of cancer risk reduction by Ginkgolide B in BRCAl-mutant ovarian epithelial cellsObjective To explore whether Ginkgolide B might be effective agent to reduce ovarian cancer risk induced by BRCA1mutation by the assessment of protein expression pattern using antibody microarray technology.Methods A human ovarian surface epithelial cell line (HOSE636) was developed from a BRCA1mutant carrier who had prophylactic surgery but no cancer found. Cell line was characterized by gene mutation analysis, protein truncation assay and karyotype analysis. Cells were continuously treated with ginkgolide B (100μM) for7days, and DMSO was used as control. Protein lysates from the treated and non-treated cells were applied to antibody microarrays to determine up or down-regulated protein expression pattern after treatment. Selective altered proteins were validated by Western-Blot. Anti-cancer activities and the associated networking pathways in the BRCA1treated and untreated ovarian epithelial cells were analyzed using the Pathway Studio software.Results The HOSE636cell demonstrated two truncated BRCA1protein fragments rather than the intact protein. After ginkgolide B treatment,28proteins were shown to be consistently up regulated (1.5to15.5-fold), and22proteins were down regulated (1.5to28.3-fold). Our preliminary studies showed that25of the28up-regulated proteins were associated with anti-tumor activities such as P53, while all of the ginkgolide down-regulated proteins cells were positively linked to cancer transformation and metastasis such as β-catenin. Bioinformatic software analysis indicated that multiple mechanisms and signal pathways are involved in anti-cancer activities in BRCA1mutant cells by Ginkgolide B treatment. These pathways include cell proliferation (i.e. PU.1, paxillin, β-catenin, PLCbl, and DGKq), tumor suppression (i.e. P53), invasion (Fibronectin), DNA damage signaling and repair (P53, XPA, and SRPK1).Conclusion Our study demonstrated that ginkgolide B has anti-cancer activities in BRCA1mutant ovarian surface epithelial cells. Further studies including in vivo model are required to confirm the concept of using of Ginkgo as a chemopreventive agent in women who carry BRCA1mutations. |
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