The Study Of Effects Of Parthenolide On Epithelial Ovarian Cancer Cell And Platinum-resistant Cell

Objective: Epithelial ovarian cancer is one of the most significant malignacies, because of lack of the early effective detection techniques, the 5-year survival rate is only 30% for all stages of ovarian cancer. However, most patients initially respond to the thoroughly cytoreductive surgery and platinum-based chemotherapy,the majority still experience the disease recurrence. Therefore, so much research has to focus on the discovery of the novel chemotherapeutics with lower toxicity and increased activity. It is beneficial for improving the life quality and reducing the mortality of patients with ovarian cancer.Parthenolide is one of the sesquiterpene lactones abundant in Tanacetum parthenium, used in the treatment of arthritis, migraine and fever for a long history in the past. It is well known that parthenolide is one of the potent nuclear factor kappa-B (NF-κB) inhibitors, which can suppress the activity of IκB kinase complex specifically and NF-κB. Recent studies have suggested that the anticancer effects of parthenolide through induction of apoptotic cell death in many human cancer cells, such as colon cancer, pancreatic carcinoma, hepatocellular carcinoma and multiple myeloma. Meanwhile, Parthenolide can enhance the sensitivity of a number of tumor cells to chemotherapeutics. But its role in ovarian cancer has not been illustrated.In this study, we investigated the effects of Parthenolide on the proliferation of SKOV3 and SKOV3/DDP.According to comparing the expression of the NF-κB before and after Parthenolide treated in the two cells, we determined the relationship of the NF-κB with ovarian cancer and drug resistance, discussed the effect of PAR on NF-κB in ovarian cancer. Meanwhile, we set out to study whether PAR sensitizes platinum-resistant ovarian cancer cell to Cisplatin in vitro. Methods: The human ovarian serous cancer cell line SKOV-3 and platinum-resistant ovarian serous cancer cell SKOV3/DDP were cultured in RPMI 1640 culture solution which contained 10% heat-inactivated FBS at 37℃,under a humidified 5% CO2 atmosphere,the two cells were applied to the experiment after they entered the logarithmic phase.1 SKOV-3 and SKOV3/DDP cells were treated by different concentrations of Parthenolide in different time courses, the cell morphological changes were observed by using the light microscope.2 MTT assay: Cell proliferation was measured by MTT assay. The cells were initiated in 96-well plates at the density of 5×10⁴/mL. SKOV-3 and SKOV3/DDP cells were treated respectively for different times (12h,24h,36h,48h) with various concentrations ( 0,6.25,12.5,25,50 and 0,25,50,100,200μmol/L ) of PAR with three replicate wells for every concentration. 0 ug/ml and DMSO were chosen as the control group and drug control group. Then the relative number of cells was determined by the MTT assay, in order to detect the effects of PAR.3 Immunohistochemistry: SKOV-3 and SKOV3/DDP cells were cultured routinely until 72 hour, then synchronization for 24 hour, after 25μmol/L PAR treated for 6 hour, dyeing and developing, using graphical analysis software to detect every shading value and comparing the expression of NF-kB among goups.4 FCM was used to set out whether PAR sensitizes platinum-resistant ovarian cancer cell to cisplatin. SKOV3/DDP cell were divided into four groups: Control group, PAR group, DDP group , PAR and DDP group.5 All statistical figures were analysed by SPSS 13.0 statistical software package.Results:1 SKOV-3 and SKOV3/DDP cells were treated with PAR in different concentrations, the cell morphological changes were observed under inverted phase contrast microscope. As time extended and concentration increased,the intensity and the proliferation of the cells were reduced significantly, The long shape of the cells became round gradually. The cell membrane also became imperfect. The round cells gradually increased. The volume of the cells became smaller generally , the cytoplasm condensed, the membrane got incomplete and morphology became irregular. The adhesion ability of the cells weakened obviously. In the end, these cells floated.2 SKOV-3 and SKOV3/DDP cells were treated with different concentrations of PAR, as the time extended and the concentration increased, the proliferation of two cells were reduced significantly when compared with the control group (p<0.05). The proliferation of the peak concentration of PAR group was significantly lower than the DMSO group (p<0.05).3 The immunohistochemistry results showed,in SKOV-3 cell, the shading value(0.3857±0.012)before treated by PAR was significantly lower compared with the shading value(0.4107±0.043)after treated (p<0.05). In SKOV3/DDP cell, the shading valu(e0.4207±0.017)before treated by PAR was significantly lower compared with the shading value ( 0.5313±0.035 ) after treated (p<0.05).Before treated by PAR, the expressions of NF-κB in karyon and cytoplasm of SKOV-3 cell were significantly lower than SKOV3/DDP cell, the shading value(0.3857±0.012)of SKOV-3 cell was significantly lower than the shading value(0.4207±0.017)of SKOV3/DDP cell(p<0.05). After treated by PAR, the expressions of NF-κB in cytoplasm were higher than karyon in both two cells. But the expressions of NF-κB in karyon and cytoplasm of SKOV-3 cell were still significantly lower than SKOV3/DDP cell, the shading value(0.4107±0.043)of SKOV-3 cell was significantly lower than the shading value(0.5313±0.035)of SKOV3/DDP cell(p<0.05).4 The results of FCM showed:The combination of Parthenolide and Cisplatin reduces the apoptosis of platinum-resistant ovarian cancer cell in addictive manner, blocks cell cycle of SKOV-3/DDP cell at S phase.â‘´The apoptosis rates of PAR group(3.25±0.91), DDP group (2.32±0.84)and PAR+DDP group(7.69±1.34)were all significantly higher than control group(p<0.05). The apoptosis rates of PAR group(3.25±0.91),DDP group(2.32±0.84)were both lower than PAR+DDP group(7.69±1.34),differences are significant statistically(p<0.05).⑵At G₀/G₁ phase, the numbers of SKOV-3/DDP cell in PAR group(53.2±1.00),DDP group(30.7±0.65)and PAR+DDP group(29.3±0.38) were significantly lower than control group(62.8±1.25)(p<0.05). At S phase, the numbers of SKOV-3/DDP cell in PAR group(26.1±1.56),DDP group(56.2±0.73)and PAR+DDP group(50.9±2.33) were significantly higher than control group(19±1.32)(p<0.05).Conclusion:1 PAR can inhibit the proliferation of SKOV-3 cell with time and dose effect. As the time extended, the inhibition effect of the same concentration of PAR on SKOV-3 cell was enhanced. At the same time, as the concentration increased, the inhibition effect of PAR on SKOV-3 cell was enhanced. After 50μmol/L PAR treated SKOV-3 cell for 48h, the inhibition of proliferation was 74.22%. The IC50 of 48h was 10.5μmol/L.2 PAR can inhibit the proliferation of SKOV-3/DDP cell with time and dose effect. As the time extended, the inhibition effect of the same concentration of PAR on SKOV-3/DDP cell was enhanced. At the same time, as the concentration increased, the inhibition effect of PAR on SKOV-3/DDP cell was enhanced. After 50μmol/L PAR treated SKOV-3/DDP cell for 48h, the inhibition of proliferation was 68.63%. The IC50 of 48h was 31.8μmol/L.3 The expression of NF-κB in karyon of SKOV-3/DDP cell was significantly higher than SKOV-3 cell, it demonstrated that NF-κB may be critical in ovarian cancer and platinum-resistant. PAR can inhibit the activity of NF-κB in SKOV-3 and SKOV-3/DDP cells.4 Parthenolide can sensitize platinum-resistant ovarian cancer cell to Cisplatin. Whether alone or cotreatment with DDP,PAR can block cell cycle of SKOV-3/DDP cell at S phase, maybe disturb DNA synthesis of SKOV-3/DDP cell to induce apoptosis.