ABSTRACT
Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer.
ABSTRACT
Objective:To investigate the radiosensitizing effect of three flavonoids on ovarian cancer SKOV3 cells under hypoxia. Methods:The SKOV3 cells were divided into normoxic group and hypoxic group. The hypoxic SKOV3 cellular model in vitro was es-tablished and tested by measuring the expression profile of HIF-1αprotein in SKOV3 cells. Colony-forming assay was used to detect the radiosensitivity of normoxic and hypoxic SKOV3 cells. The cytotoxicity and radiosensitizing effects of flavonoids were evaluated on the basis of cell death and MTT assay. Results:The Western blot results showed that the gray intensity ratio of HIF-1α/β-Actin in hypoxia group was significantly higher than that in normoxia group (1. 068>0. 117). Radiosensitivity of hypoxic SKOV3 cells in hypoxia group was significantly lower than that in normoxia group. The survival rate of SKOV3 cells was decreased with the increase of concentration. When the concentration was increased, D0 and Dqin chrysin group and quercetin group were significantly decreased (P0. 05). The overall radiobiological parameters of hypoxia group were higher than those of normoxia group. Conclusion: Hypoxia can induce the expression of HIF-1α in SKOV3 cells, which results in the decrease of radiosensitivity. Chrysin and quercetin can enhance the radiosensitivity of SKOV3 cells, and the enhancement is significant under hypoxia, while breviscapine is without such effect. The radiosensitizing effect may be achieved by the level decrease of HIF-1α in SKOV3 cells and inhibition of DNA damage repair.
ABSTRACT
Objective To study the effect of arsenic trioxide(As 2O 3) combin ed w ith radiation on the killing of ovarian cancer cells. Methods Using MTT and FCM to detect the cytotoxic and apoptosis at different As 2O 3 concentrations combined with 2 an d 8 ?Gy radiation on ovarian cancer cells(SKOV-3). Radiation survival curves were det ermined by cloning assay with 5?mol/L As 2O 3 combined with 2, 3, 4, 6, 8 and 12?Gy radiation. Curve was used to evaluate the effect of cell killing. Results ⑴ Inhibition of cell proliferation seemed more dependent on the increase of As 2O 3 concentration, ⑵Cell survival rate was lower in the combination of As 2O 3 an d r adiation than As 2O 3 alone, ⑶The apoptosis ratio was increased in 2?Gy and As 2O 3 with increase in As 2O 3 concentration, ⑷D q , D 2 value was decr eased in t he combined As 2O 3 and radiation than radiation only (D q: 1.44 vs 2.78, D 0: 0.85 vs 1.30, SF 2: 0.42 vs 0.87), with radiation enhancement ratio of 1.53 and 2.0 7 according to D 0 value and SF 2. Conclusions Arsenic trioxide is able to enhanc e radiation effect obviously ,especially at lower radiation dose.