Human Epithelial Ovarian Cancer SKOV3 Research Articles

Role of ovarian tumor stem-like cells sorted from human epithelial ovarian cancer SKOV3 cells in vasculogenic mimicry formation

To isolate tumor stem-like cells from human epithelial ovarian cancer SKOV3 cells and explore their role in the formation of vascularization mimicry (VM). SKOV3 cells were passaged to the 7th generation by suspension culture in serum-free medium, and the percentages of CD133- and CD117-positive cells in the 1st, 3rd, 5th and 7th generations were analyzed using flow cytometry. The proliferative activity of the cells sorted from the 7th generation SKOV3 cells was assessed with colony formation assay. A three-dimensional cell culture model was established to compare the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2 (MMP-2) and MMP-9 in the two groups were detected using real-time PCR and Western blotting. Some SKOV3 cells formed typical cell spheres with suspension growth in serum-free medium and were passaged to the 7th generation. Flow cytometry revealed that the percentage of CD133-positive cells increased with cell passaging. The cloning efficiency of the sorted cells was significantly higher than that of the parental SKOV3 cells (50.33% vs 5.33%, P < 0.001). The VM formation ability of the sorted cells was stronger than that of the parental SKOV3 cells in the three-dimensional cell culture system. RT-PCR and Western blotting showed that the expression levels of MMP-2 and MMP-9 were significantly higher in the 7th passage cells than in the parental cells (P < 0.05). The sorted cells from SKOV3 cells cultured in serum-free medium exhibit biological properties of tumor stem cells with strong VM formation ability, suggesting their role in VM formation.

Effect of resveratrol on cisplatin chemotherapy sensitivity of ovarian cancer cells

Objective To investigate the effect and mechanism of resveratrol on cisplatin chemotherapy sensitivity of human epithelial ovarian cancer SKOV3 cells. Methods The cultured in vitro human ovarian cancer SKOV3 cells in logarithmic growth phase were treated with resveratrol (20 μg/ml), cisplatin (40 μg/ml) and resveratrol (20 μg/ml) + cisplatin (20 μg/ml) respectively, and the blank group was set, n=10. After 48 hours, the cellular growth inhibition rate was calculated by methyl thiazolyl thiazolium (MTT), cell cycle and apoptosis rate were analyzed by flow cytometry, the expressions of Bax mRNA and Bcl-2 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of cleaved Caspase-3 protein was detected by Western blotting. Results The cellular growth inhibition rate of SKOV3 cells in resveratrol + cisplatin group was significantly increased than that in cisplatin group [(53.0±8.1)% vs. (37.5±6.5)%, P=0.017], the proportion of G0-G1 phase cells in resveratrol + cisplatin group was significantly higher than that in cisplatin group [(61.2±3.4)% vs. (54.9±2.8)%, P=0.017], the proportion of G2-M phase cells in resveratrol + cisplatin group was significantly lower than that in cisplatin group [(5.1±0.5)% vs. (8.6±0.9)%, P=0.008], the apoptosis index in resveratrol + cisplatin group was significantly increased than that in cisplatin group [(59.3±7.4)% vs. (43.6±6.0)%, P=0.015], the ratio of Bax/Bcl-2 in resveratrol + cisplatin group was significantly increased than that in cisplatin group (4.6±1.8 vs. 3.3±1.4, P=0.026), and the expression of cleaved Caspase-3 protein in resveratrol + cisplatin group was significantly increased than that in cisplatin group (0.47±0.15 vs. 0.38±0.12, P=0.011). Compared with blank group, the G0-G1 phase in cell cycle of resveratrol group was significantly increased [(46.5±2.4)% vs. (37.8±1.9)%, P=0.023], the expression of Bcl-2 mRNA was significantly decreased [(32.9±11.2) ×10-3vs. (44.8±13.0)×10-3,P=0.028], the ratio of Bax/Bcl-2 was significantly increased (2.1±0.8 vs. 1.5±0.6, P=0.019), and the expression of cleaved Caspase-3 protein was significantly increased (0.26±0.10 vs. 0.08±0.03, P<0.001). Conclusion Resveratrol can effectively enhance the effect on human epithelial ovarian cancer SKOV3 cells treated by cisplatin, which is perhaps related to its effects of blocking the cell cycle G0-G1 and altering the expression of apoptosis-related genes and proteins. Key words: Ovarian neoplasms; Cisplatin; Drug therapy; Apoptosis; Resveratrol

Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells.

Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5′-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of ovarian cancer.

Downregulation of MACC1 expression enhances cisplatin sensitivity in SKOV-3/DDP cells.

The aim of this study was to investigate the correlation between MACC1 expression and resistance to cisplatin (DDP) in DDP-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP). MACC1 mRNA and protein expression levels in SKOV-3 and SKOV-3/DDP cells were detected by reverse transcriptase polymerase chain reaction and western blot. The SKOV-3/DDP cells were divided into 5 groups: control, shVect (transfected with p-super-EGFP-1 plasmid), pshMACC1 (transfected with psuper-EGFP-shMACC1 plasmid), PD (pretreated with 20 μM PD98059), and combined (transfected with psuper-EGFP-shMACC1 plasmid and pretreated with 20 μM PD98059) groups. Cisplatin sensitivity and cell apoptosis in SKOV-3/DDP cells were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. ERK1/2 and p-ERK1/2 expression was determined by western blot. MACC1 mRNA and protein expression levels in SKOV-3/DDP cells were 2.66 ± 0.54 and 1.95 ± 0.45 times those seen in SKOV-3 cells (P < 0.05). Cisplatin sensitivity of pshMACC1 group was much higher than that in the control and shVect groups. Cisplatin-induced cell apoptosis rates increased significantly in the pshMACC1, PD, and combined groups, compared to the control and shVect groups. Moreover, the apoptosis rate was the highest in the combined group among the 5 groups (IC50 = 20.836 ± 0.629 μM). p-ERK1/2 expression decreased significantly in the pshMACC1, PD, and combined groups (this decrease was the most obvious in the combined group). In conclusion, downregulation of MACC1 expression could enhance cisplatin sensitivity and decrease drug resistance in SKOV- 3/DDP cells.

Effect of Diallyl Trisulfide on Human Ovarian Cancer SKOV-3/DDP Cell Apoptosis

To investigate the effects of diallyl trisulfide (DT) on apoptosis of cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP), and the role of p53 upregulated modulator of apoptosis (PUMA). SKOV-3/DDP cells were randomly divided into control, DT, DPP and DPP+DT groups, which were treated with DT or combined DT and DDP. All cells were incubated for 48 h. and apoptosis rates were assessed by flow cytometry. mRNA and protein expression of PUMA, Bax and Bcl-2 was determined by RT-PCR and Western blot assays, respectively. Compared with control group, the apoptosis rates of SKOV-3/DDP cells in DT groups were obviously increased, with dose-dependence (P < 0.05), the mRNA and protein expressions of PUMA, Bax also being up-regulated (P < 0.05), while those of Bcl-2 were down-regulated (P < 0.05). Compared with DT groups, the apoptosis rate in the DDP+DT group was significantly increased (P < 0.05). After knockdown of PUMA with specific siRNA, the apoptosis rate of SKOV-3/DDP cells was obviously decreased (P < 0.05). DT can promote the apoptosis of SKOV-3/DDP cells with PUMA playing a critical role.

Evaluation of characteristics of CD44+CD117+ ovarian cancer stem cells in three dimensional basement membrane extract scaffold versus two dimensional monocultures

BackgroundCancer stem cells (CSCs) are thought to be capable of surviving conventional chemotherapeutic treatments because the cells have more resistant to anticancer drugs than common cancer cells. Most in vitro studies in experimental cancer cells have been done in a two-dimensional (2D) monocultures, while accumulating evidence suggests that cancer cells behave differently when they are grown within a three-dimensional (3D) culture system.ResultsThe CD44+CD117+cells isolated from human epithelial ovarian cancer SKOV-3 cell line using magnetic-activated cell sorting were found to grow faster than the SKOV-3 cells in the 3D culture and in the nude mice. Anticancer drugs 5FU, docetaxel, cisplatin, and carboplatin were seen to inhibit growth of the CD44+CD117+ cells by 50% in the 2D culture with IC50 concentration, whereas, in the 3D culture, the four drugs inhibited the cell growth by only 34.4%, 40.8%, 34.8% and 21.9% at 3D one, respectively. Effect of paclitaxel on the CD44+CD117+cell viability indicated that fewer cells underwent apoptosis in 3D culture than that in 2D one. In addition, anticancer drugs markedly increased the expression of ABCG2 and ABCB1 of CD44+CD117+cells in 3D culture.ConclusionOur assay demonstrated that human epithelial ovarian cancer CD44+CD117+cells possessed the properties of CSCs that exhibited more chemoresistance in the 3D culture than that of in 2D one. The 3D culture provides a realistic model for study of the CSC response to anticancer drugs.

CURCUMIN INDUCED CHANGES IN TP53 AND CYCLIN D1 ENHANCE CYTOTOXIC EFFECT OF CISPLATIN IN HUMAN OVARIAN CANCER CELLS

Curcumin, the active ingredient of tumeric, has anti-proliferative and chemo-sensitizing properties in other cancer cells. Curcumin, through the actions of the tumor suppressor protein TP53, can arrest the cell-cycle in the G2/M phase leading to the release of cytochrome c from mitochondria causing apoptosis, or by down-regulation of cyclin D1 preventing apoptosis. The objective of this experiment was to determine effects of curcumin on cell proliferation and apoptosis in human epithelial ovarian cancer SKOV-3 cells. It was hypothesized that the anti-proliferative properties of curcumin would enhance the TP53-mediated apoptotic effect of cisplatin. Cultured SKOV-3 cells were treated with curcumin (0, 10, or 20 μM) for 4, 8, 16 and 24 h at 37°C and expression of TP53 and cyclin D1 was determined by Western blot analysis. Viability of similarly treated cells was determined by trypan blue staining. Incidence of apoptosis was detected by TUNEL assay. Effects of curcumin and cisplatin (0.25 μg/ml) on the numbers of metabolically active cells were determined by MTT assay. Data were analyzed by GLM methods of SAS. Curcumin at 10 and 20 μM decreased (P < 0.001) numbers of viable cells. Curcumin at 20 μM, but not 10 μM, increased (P < 0.001) expression of TP53. Expression of cyclin D1 was decreased (P < 0.001) with 10 and 20 μM curcumin. Although curcumin decreased numbers of viable cells, numbers of cells with positively-stained apoptotic nuclei were small < 10%. Cisplatin or curcumin at 10 or 20 μM alone decreased (P < 0.001) numbers of metabolically active cells. Numbers of cells were further decreased (P < 0.05) when cells were treated with curcumin in combination with cisplatin, when compared to curcumin or cisplatin alone. The reduced number of metabolically active cells following treatment with curcumin may be due to the arrested cell cycle mediated through an increased TP53 and decreased cyclin D1 expression. Lack of a robust apoptotic effect suggests that curcumin, while possibly lytic at high doses, at low doses may primarily arrest the cell cycle facilitating an increased cytotoxic effect of cisplatin. (poster)