| Objective: Ovarian carcinoma is very late when it was been discovered because of lacking the effective diagnostic methods. The mortality rate of ovarian carcinoma is the first one in the malignant tumor of gynecology. At present, operation is the main method for the patients of ovarian carcinoma. But it is the very important way that we use multiple methods such as chemotherapy,radiotherapy...etc. Among these methods, many courses of chemotherapy is important .Especially it is significant for those patient of ovarian carcinoma in later period. It is easy for the cell of ovarian carcinoma to emerge the resistance during the courses of chemotherapy. At present almost doctors choose Cisplatin(CDDP) as the first choosing drug; meanwhile, they choose some other drugs as the compatibility. Among these compatibility drugs, topoisomerase (topoII) inhibitor is often used, such as Etoposide(VP-16), adrianycin, novobiocin…etc. Some studies suggested the saitivity of cancer cell for drugs depend on the expression of topoIIα. The reduction of the expression of is one of reasons that result in the resistance in studying cell lines and clinical samples, meanwhile, discovered that the expression of topoII mRNA decrease, the activity of topoII promoter depress and the level of NF-Y and Sp1 is low. There are GC box and several inversed CCAAT boxes in human topoIIαand topoIIβpromoters. Transcription factor NF-Y and SP1can bind specific ICBs and GC of topoII promoters respectively to regulate the expression of topoIIαandβ. And NF-Y and SP1 have cooperation. Our previous studies suggest that CDDP can cooperate with VP-16 by up-regulating the expression of topoIIαand topoIIβin Ovarian carcinoma cell line SKOV3. Meanwhile ,we discovered the expression of topoII mRNA increased in SKOV3 cells after cisplatin treatment. This cooperation possibly due to provid target for (topoII ) inhibitor by CDDP up-regulating the expression of topo IIαand topoIIβ.In this study, our objective is to approach the role of transcription factor NF-Y and SP1 in CDDP up-regulating the expression of topoII .Western blot was used to determine the expression of NF-Y and SP1 after different concentration drugs treatment, to demonstrate whether up-regulation of topoII is relevant to the expression of NF-Y and SP1.Methods: 1 Cell cultureHuman ovarian serous cystadenocarcinoma SKOV3 cells were freezed in our gynecology laboratory. SKOV3 cells were cultured in 1640 medium containing 10% fetal bovine serum,4 mmol/ml L-glutamine,1 mol/L HEPES,100U/ml Penicillin,100μg/ml Streptomycin , all of the cells were cultured at 37°C in a humidified chamber containing 5% CO2.2 Drug concentration and groups According to the peak serum concentration(PSC), we divided these cells into six groups. We dealed with the first group cells by 1/10 PSC of CDDP(0.3μg/ml), the second group cells were dealed with by one time PSC of CDDP(3μg/ml), the third were by ten time PSC of. The other three groups cells were dealed with by VP-16 which in PSC combined with different concentration of CDDP(1/10 PSC,PSC,10 PSC)respectively.3 Nuclear extracts and Western Blot Analysis3.1 Preparation of Cellular ExtractsOvarian carcinoma cells were collected at 24h after different concentration of drugs treatment for target protein determinations, every groups were washed twice by PBS, nuclear extracts were produced at 4℃for 10 mins by lysing cells.The procedure of nuclear extracts refer to the description.The cellular debris was removed from the extract by centrifugation, and the supernatant was stored at–70℃for pre-emergency. The protein concentrations of nuclear extracts were determined using the methods of G-250.3.2 Western Blot AnalysisExtracted protein from every groups ovarian carcinoma cells combined with different concentrations drugs treatment were separated on 8 or 10%SDS-PAGE gels at 4℃. Proteins were transferred to a cellulose nitrate membrane using a semidry blotter in 110 mA current flow for 3-3.5 hours at 4℃. Western blot analysis was performed with an appropriate dilution of each antibody.First, confining liquid was used at 4℃overnight; second, add to rabbit antihuman NF-YA and Sp1 polyclone antibody respectively at 4℃overnight; third, add to goat antirabbit IgG antibody for 2 hours ordinary temperature.the reaction completed followed by DAB or ECL visualization using enhanced chemiluminescence. And the expression of transcription factor NF-Y and sp1 were assayed by Western blot on protein level, respectively.4 Data were analysed using the Satistical Package of the SPSS14.0. Statistical analysis was performed using univariate analyses, one-way ANOVA, experimental data use mean and standard deviation ( X±S). Western blot analysis use HPIAS-1000 by determing optical density and peak value areas of straps. A P value of less than 0.05 was considered as significant.Results: Western blot analysis showed that the expression of transcription factor NF-Y and Sp1 protein.1.Expression of NF-Y: increased with concentration, It was found that the expression of NF-Y in groups with different concentrations CDDP significantly increased compared with the control group (G3:2.39±0.09;G4:2.64±0.03;G5:3.01±0.12 vs control group: 1.99±0.02 respectively), there was a significant difference, P<0.01.And the expression of NF-Y protein have a orderly reinforcement to accompany with the concentration increasing of CDDP, P<0.01. However, the expressions of NF-Y decreased by Western blot on protein level in VP-16 groups(1.45±0.12vs 1.99±0.02), P<0.01.NF-Y protein in CDDP and VP-16 groups compared with control group was a significant difference (G6:2.11±0.02;G7:2.13±0.04;G8:2.17±0.02 vs control group: 1.99±0.02 respectively), P<0.05.2. Expression of SP1 protein: SP1 protein increase after different concentration CDDP by western blot analysis compared with control group (G3:0.85±0.03;G4:0.90±0.02;G5:0.93±0.02 vs control group:0.66±0.02),P<0.01, but there were no significant difference in every groups with different concentration CDDP ,P>0.05. The expression of SP1 decreased by Western blot on protein level in VP-16 groups(0.38±0.03 vs 0.66±0.02) ,P<0.01.SP1 protein in CDDP and VP-16 groups compared with control group increased(G6:0.74±0.05;G7: 0.72±0.04;G8:0.76±0.03 vs control group:0.66±0.02),there was significant difference, P<0.05.Conclusion: The expression of NF-Y and SP1 were enhanced after cisplatin treatment in ovarian carcinoma cell SKOV3 in vitro. CDDP can up-regulate the expression of topoIIαand topoIIβby NF-Y and Sp1 binding specific GC and ICBs of topoII promoters, respectively, this provides the target for topoII inhibitor.It is very significant for decreasing the resistance of CDDP and clinical doctors to find a most effective plan of chemotherapy. |
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