The In Vitro And In Vivo Study Of Growth Inhibition Of Human SKOV3 Ovarian Cancer Cells By Brucea Javanica Fruit Oil Emulsion And Its Combination With Cisplatin

Ovarian cancer is a common malignant tumor of gynecology that seriously threats the health and lives of women. Injection of the effective components of the traditional Chinese medicine Brucea Javanica fruit oil emulsion (BJFOE) can cycle-non-specifically block the growth of tumor cells. BJFOE also enhances the immune system, improves cellular immunity, shows low toxicity or side-effects, and is thus widely used in clinical practice as a traditional Chinese medicine. In this study, we performed experiments to better understand the antitumor effect of BJFOE and mechanisms of this activity. We employed the MTT method to measure the growth inhibitory effects of different concentrations of BJFOE and cisplatin, as well as their combination, on a human ovarian cancer cell line SKOV3. We used the transmission electron microscopy to detect the morphology change of apoptotic cells and the flow cytometry (FCM) to determine apoptotic rates of SKOV3 cells subjected to these treatments. By using RT-PCR and Western blot, we also analyzed the gene expression of NF-kappa B/p65, caspase-3 and its downstream apoptosis-associated proteins Bcl-2 and Bax in SKOV3 cells treated with BJFOE, cisplatin and their combination. In addition, we established a nude mice model of ovarian cancer derived from SKOV3 cells. We intraperitoneally injected the ovarian cancer model mice with BJFOE, cisplatin and their combination to observe growth inhibition effects on the tumors derived from SKOV3 cells. We also examined the counts of white blood cells and platelet and ALT, LDH, CR for the liver and kidney functions of these mice for evaluating side-effects of these treatments. We used electron microscopy to analyze the pathological and morphological changes of the tumor tissues and employed Western blots to measure the expression levels of MRP-1/CD9 and integrina-5 in transplanted tumors of these nude mice. The main results of this study are summarized as follows:1. The survive and proliferation of SKOV3 cells were significantly suppressed by BJFOE, which was positively dependent on the time and dosage of the treatment. The medium and low concentrations (25 and 50μg/mL) of BJFOE led to the early apoptosis, while high concentrations (100μg/mL) caused necrosis of SKOV3 cells.2. Under the same treatment time, the combination of BJFOE and cisplatin showed significantly higher inhibition to SKOV3 than these two drugs alone. The inhibitory effect on SKOV3 proliferation of these treatments increased correlating with the prolongation of treatment time. Measured by FCM, the apoptosis of SKOV3 cells induced by the combination of BJFOE and cisplatin was shown significantly higher than that induced by BJFOE and cisplatin alone. Correlated with the prolongation of time, the apoptotic population of SKOV3 cells induced by each treatment increased gradually.3. Reduced expression of NF-κB/p65 and Bcl-2 and increased expression of caspase-3 and Bax were observed associating with the appearance of apoptosis of SKOV3 cells induced by the treatment of BJFOE, cisplatin and their combination.4. Established a nude mouse model of SKOV3 cell-derived ovary cancer and delivered antitumor drugs to the model mice by intraperitoneal injection. The treatment of BJFOE, cisplatin and their combination to the orthotopic transplantation tumors of these nude mice (n=8 of each treatment) had the inhibitory rates of 23.56±3.67%,42.57±3.45% and 48.36±3.08%, respectively. Compared with cisplatin, BJFOE demonstrated weaker side-effects on white blood cells, platelets, as well as liver and kidney functions (represented by ALT, LDH, CR) of these nude mice.5、Examined the pathological morphology of tumor tissues of nude mice by light and electron microscopy and determined protein expression levels of MRP-1/CD9 and integrina5 by Western blot. By examining histologic slices, we found that correlated with the increased inhibitory effects of each treatment on SKOV3 cells, fused regions of tumor cells were observed in morphologies of degeneration to necrosis, and apoptotic bodies were clearly visible. Different expression levels of integrina-5 and MRP-1/CD9 in tissues from mice among each treatment were also detected. Compared with the control group, treatments of BJFOE and cisplatin significantly increased the expression of integrina-5 and MRP1/CD9 (P< 0.05).In conclusion, this study, for the first of time, proposed an approach of intraperitoneal injection of BJFOE and its combination with cisplatin to treat the transplanted ovarian cancer in nude mice. We employed in vitro and in vivo studies on the growth inhibitory effects of BJFOE and its combination with cisplatin on the ovarian cancer SKOV3 cells and orthotopic transplantation tumors in the nude mice mouse. Our data provide experimental evidences and action mechanisms for the clinical application of BJFOE and give insights for ovarian cancer therapies.