The Effects Of Bigelovin On Human Epithelial Ovarian Cancer And Peripheral Blood Mononuclear Cells

Objective: Epithelial ovarian cancer (EOC) is a common malignant tumor in female reproductive system. To date, due to lack of effective early detection techniques, chemoresistance and postoperative recurrence, the 5-year survival rate is still about 30%. Despite advances in cytoreductive surgery and the use of the most effective chemotherapy (currently consisting of carboplatin/ paclitaxel), EOC is still the leading cause of death from gynecological malignancy in China. The platinum drugs can cause serious adverse reactions including bone marrow suppression which leads to secondary infection, hemorrhage and aplastic anemia. Chemoresistance and postoperative recurrence are also seriousproblems of ovarian cancer. The majority of patients will eventually relapse in 3 years and die from chemoresistant diseases. So a new kind of chemotherapy drug which has low toxicity and high activity may be an important methord to solve the problems.Sesquiterpene lactones (SQLs) are a class of naturally occurring plant terpenoids of Asteraceae family, known for their various biological activities, such as phytotoxic, antiprotozoal, antimicrobial, anti-inflammatory, and cytot- oxicity against different tumor cell lines. At present, the studies of SQLs turn to anti-tumor and reversing resistance. Bigelovin is one of SQLs and widely exists in Compositae family, which can inhibit cells growth in many human tumors, such as lung cancer, hormone dependent prostatic cancer, breast cancer and so on. Until now, here have not been any reports about the roles of bigelovin in human ovarian serous cancer cell line of cisplatin resistance and peripheral blood mononuclear cells.The present study was conducted to investigate the effects of bigelovin on the proliferations and cell cycle distributions of human epithelial ovarian cancer SK-OV-3 cells, SK-OV-3/DDP cells and peripheral blood mononuclear cells (PBMC) in order to find a new chemotherapy drug which has low toxicity and high activity. It provided experimental basis for overcoming chemoresistance and recurrence of ovarian cancer and resonable methods for screening anti-cancer drugs.Methods:1 SK-OV-3 and SK-OV-3/DDP cell cultures in vitro: The SK-OV-3 and SK-OV-3/DDP cells were cultured in RPMI 1640 solution contain 10% heat-inactivated FBS at 37℃with 5% CO2. Cells were applied to the experiment when they entered logarithmic phase.2 Separate PBMC by density-gradient centrifugation: Heparinized blood was collected and PBMC were separated by density-gradient centrifugation over Ficoll-Hypaque, using Alamar blue assay. PBMC were washed and resus- pended in RPMI-1640 medium containing 20% fetal bovine serum and 2% phytohemagglutinin at 37℃with 5% CO₂. After 24h culture, cells were treated with the test compounds.3 Inverted microscope analysis: We stimulated SK-OV-3, SK-OV-3/DDP and PBMC cells with bigelovin at different concentrations in different time courses, and observe the cell morphological changes by inverted microscope.4 MTT assay4.1 Choose the best inoculum densities in different time courses: SK-OV-3, SK-OV-3/DDP were seeded in 96-well plates at concentrations of 1×10⁴/mL, 2×10⁴/mL, 3×10⁴/mL, 4×10⁴/mL, 5×10⁴/mL, 6×10⁴/mL, 7×10⁴/mL, 8×10⁴/mL cells in 200μL RPMI-1640 medium containing 10% FBS. The absorbances were measured after incubating for 24h, 48h and 72h. We drew the inoculum density-absorbance curves of two cell samples in different time courses and chose the best inoculum density.4.2 Choose the best means of inoculum: Take SK-OV-3 treated with 8 mg/L cisplatin(DDP) for example, SK-OV-3 cells were seeded in a 96-well plate at concentrations of 1.5×10⁴/mL, 3×10⁴/mL, 6×10⁴/mL. DDP were added to each well after cells adherence and 8 mg/L is served as final concentration. Three equal 96-well plates were made and incubated respectively for 24h, 48h and 72h. We measured their absorbances which were compared in the same time courses and chose the best means of inoculum.4.3 We computed IC50, resistance index and performance ratio after detecting the effects of bigelovin and DDP on proliferations of three cell samples using the means in Table 1 and Table 2, then evaluated the efficacies of bigelovin on ovarian cancer cells.5 PI labed FCM detected the cell cycle phases of SK-OV-3 and SK-OV-3/DDP in different experimental groups. The Cells were grouped as follows: SK-OV-3 was treated with 0μM, 2μM, 4μM, 8μM bigelovin for 24h. SK-OV-3/DDP was treated with 0μM, 4μM, 8μM, 16μM bigelovin for 72h.6 All statistical analysises were performed with SPSS 13.0 statistical software package.Results:1 Inverted microscope analysis: SK-OV-3 and SK-OV-3/- DDP are adherent cell. SK-OV-3 is fusiform and has clear boundaries. SK-OV-3/DDP is polygonal and the boundaries are unclear.PBMC is suspension cell which can mild adhere but do not change their shapes. After SK-OV-3 and SK-OV-3/DDP were treated with bigelovin, the cells had begun to appear characteristics of apoptosis gradually. There were not obvious changes in PBMC until bigelovin treating for 72h. With the increased concentration and prolonged time, the inhibiting effects increased significantly.2 MTT assay2.1 The best inoculum densities in different time courses: The best inoculum densities of SK-OV-3 in 24h, 48h and 72h were respectively chosen 6×10⁴/mL, 3×10⁴/mL and 1.5×10⁴/mL. The best inoculum densities of SK-OV-3/DDP in 24h, 48h and 72h were respectively chosen 8×10⁴/mL, 5×10⁴/mL and 3×10⁴/mL. The best inoculum density of PBMC in 72h was chosen 50×10⁴/mL.2.2 The best means of inoculum: In the same time courses,the inhibition ratioes of DDP with the best inoculum densities were different from non-optimal ones(P<0.05). The inoculum density of cells had an effect on the inhibition ratio. The standard deviations of inhibition ratioes were minimum with the best inoculum densities in three time courses, which were compared with non-optimal inoculum densities. Inhibition ratioes using the best inoculum densities can much better stand for the real inhibiting actiones of DDP. So the best inoculative means was using the best inoculum density in each time course.2.3 The effects of drugs on SK-OV-3, SK-OV-3/DDP and PBMC2.3.1 DDP: The IC50 of SK-OV-3 treated with DDP for 24h, 48h and 72h were 14.622mg/L, 7.999mg/L and 2.176mg/L, respectively. The IC50 of SK-OV-3/DDP treated with DDP for 24h, 48h and 72h were 23.375mg/L, 13.695mg/L and 4.899mg/L, respectively. The IC50 of PBMC treated with DDP for 72h were 14.922mg/L. The resistance index of SK-OV-3/DDP to DDP was 1.854 which was less than the primary one. The 72h performance ratioes of DDP on SK-OV-3 and SK-OV-3/DDP were 6.858 and 3.046, respectively. The inhibitory effects of DDP were in time-dose dependence.2.3.2 Bigelovin: The IC50 of SK-OV-3 treated with bigelovin for 24h, 48h and 72h were 6.643μM, 2.176μM and 0.940μM, respectively. The IC50 of SK-OV-3/DDP treated with bigelovin for 24h, 48h and 72h were 70.352μM, 44.573μM and 15.933μM, respectively. The IC50 of PBMC treated with bigelovin for 72h was 21.933μM. The inhibitory effect of bigelovin on SK-OV-3/DDP was valid only after 72h. The resistance index of SK-OV-3/DDP to bigelovin was 8.634 times than DDP. The 72h performance ratioes of bigelovin on SK-OV-3 and SK-OV-3/DDP were 23.333 and 1.377, respectively. The inhibitory effects of bigelovin were in time-dose dependence.3 The cell cycle phases of SK-OV-3 and SK-OV-3/DDP which were detected by PI labed FCM showed: When cells were treated by bigelovin, the percentage of G₂/M stage increased. As the concentration increased, the percentages of G₂/M and S+G2/M stages increased significantly. Conclusion:1 When screening anti-cancer drugs in vitro by MTT, the inoculum density of cells have an effect on the inhibition ratio. The best inoculative means is using the best inoculum density in each time course.2 The normal ovarian cells and normal endometrial cells are unsuited as normal control when screening anticarcinogens of the ovarian cancer in vitro. PBMC is a logical choice for such experiments.3 The effect of bigelovin is better than DDP on DDP sensitive EOC cell line. In the future, we can perform experiments in vivo to make certain the effect of bigelovin on EOC.4 The effect of bigelovin is poor on DDP resistant EOC cell line. There is cross resistance between bigelovin and DDP. Bigelovin can not treat DDP resistant EOC alone.5 Bigelovin arrests cell cycles of SK-OV-3 and SK-OV-3/DDP at G2/M phase, which may be the mechanism of bigelovin inhibiting proliferations of ovarian cancer cells.6 Bigelovin may be a potential sensitizer of chemotherapy.