The Synergistic Effects Of4-HPR Combined With Cisplatin On The Proliferation And Apoptosis Of Human Ovarian Carcinoma SKOV-3

SECTION ONE:THE SYNERGISTIC EFFECTS OF4-HPR COMBINED WITH CISPLATIN ON THE PROLIFERATION AND APOPTOSIS OF HUMAN OVARIAN CARCINOMA SKOV-3IN VITROobjective:N-4-hydroxyphenyl retinode (4-HPR) is a synthetic amide analogue of ATRA. It was reported that4-HPR could inhibit the growth of ovarian cancer cells in vitro. Cis-platinum (DDP) is a first choice chemical agent used in ovarian cancer chemo-therapy. To study the proliferation and apoptosis effects of combinative application of4-HPR and DDP on the human ovarian carcinoma SKOV-3Methods:Human ovarian carcinoma SKOV-3cell lines were interfered by4-HPR groups, DDP groups and combinative groups of4-HPR and DDP. The inhibitory effects of4-HPR and DDP on the proliferation SKOV-3were evaluated by MTT assay. The median-effect principle was used to analyze the mechanisms of the inhibitory effect after exposure to the combinative application of4-HPR and DDP. Cell apoptosis was detected by flow cytometry. Datas were shown as means±SEM by using SPSS software(version13.0; Chicago, USA). Significant differences were considered at P<0.05.Results:MTT:1. It showes time-dose dependent when the two drugs used alone.2. DDP:There are statistical differences only between DDP20μM and DDP10μM in each adjacent drug group after48hours. There are statistical differences from DDP2.5μM with their adjacent drug groups after72hours.3.4-HPR:There are statistical differences only between4-HPR20μM and4-HPR10μM in each adjacent drug group after48hours. There are statistical differences from4-HPR10μM to4-HPR20μM with their adjacent drug group after72hours.4. After the combination of two drugs, all the groups after48and72hours show CI<1except4-HPR1μM+DDP5μM after48hours.HOECHST:1. Both DDP and4-HPR have a dose-dependent manner when used72hours.2. DDP with4-HPR in combination, according to the calculation of the moderate effective concentration, Both4-HPR5μM+DDP5μM group and4-HPR10μM+DDP5μM group show CI<1, In other words, the combination of two drugs produces a synergistic effect.Streaming double staining method:1. Both DDP and4-HPR have a dose-dependent manner when used72hours.2. DDP with4-HPR in combination, according to the calculation of the moderate effective concentration, the three groups of drug combination group were CI<1, in other words, the combination of two drugs produces a synergistic effect.Streaming single dye detection method:DDP with the combination of4-HPR group:DDP by reducing the proportion of cells in G1phase, inhibit the proliferation of SKOV-3cells through the cell cycle arrest in the G2phase;4-HPR arrest the cell in the S and G2phase. Two drugs have additive effect.Conclusion:4-HPR5μM+DDP5μM and4-HPR10μM+DDP5μM significantly inhibited proliferation of ovarian cancer SKOV-3cells after72hours, and the effect is synergistic. The intensity increased with the concentration of4-HPR increased. SECTION TWO:THE SYNERGISTIC EFFECTS OF4-HPR COMBINED WITH CISPLATIN ON THE PROLIFERATION AND APOPTOSIS OF HUMAN OVARIAN CARCINOMA SKOV-3IN VIVOObjective. To establish the tumor-bearing nude mice models for human ovarian cancer, exam the effect of the application of4-HPR, DDP and combination of both4-HPR and DDP on transplanted tumor for further discussion of its mechanism of action.Methods. In preparation of the tumor-bearing mice model, the human epithelial ovary cancer cell line SKVO-3which we fostered in advance was vaccinated to the nude mice using the cell suspension method.30mice were subcutaneously vaccinated with4-HPR using the trocar tissue block inoculation method. They were subsequently divided into groups and treated with medicine separately according to the formation of the tumor:(1) Control group:physiological saline;(2)4-HPR group:8mg/kg;(3) Cisplatin group:3mg/kg;(4) Combined treatment group:3mg/kg of cisplatin6hours after8mg/kg of4-HPR, both treated with intraperitoneal injection,0.2ml each time, twice a week for3weeks, the size of the tumor was measured every week and the tumor formation curve was graphed, tumor was removed for histopathological analysis; the expression of caspase-3was detected by immunohistochemical S-P method; the apoptosis rate and the periodic change of the cells were also detected.Results. The mouse which was vaccinated with2.5×1010per mouse dosage cell suspension was successfully produced,2-3days after being subcutaneously vaccinated with SKVO-3tissue block, the ridge disappeared, tiny tumor nodules could be observed, the vaccination was successful. By the end of the treatment, the volumes of transplanted tumor were:4937.72±563.28mm3,3726.34±462.32mm3,2687.78±631.91mm3and1532.69±723.84mm3, the inhibition rates were:0.00%,30.21%,42.78%and61.37%. The sizes of the transplanted tumor in groups which were treated were significantly smaller than the size of the one in the control group; the transplanted tumor size in the combined treatment group was smaller than the one in single treatment groups, all differences have statistical significance(p<0.05); the histopathological analysis of the tumor tissue: according to the section view, the majority appeared to be round and tough grey surface vessels were observed. Through the light microscope, the tumor in control group demonstrated the general characters of malignancy and the distinctive structure of glandular cavity. Punctiform, flake necrosis emerged in in experimental groups with the infiltration of inflammatory cells, this phenomenon was most significant in the combined treatment group. Caspase-3which was examined by the immunohistochemical method for control group,4-HPR group, Cisplatin group and combined treatment group the IHS was:2.78±1.33,4.15±0.76,5.22±0.62and6.37±1.25. The differences among groups have statistical significance. The flow cytometry illustrated that the cell apoptosis rate in all groups gradually raised to6.43±2.31%,14.75±4.32%,21.54±1.47%and25.76±3.42%, G0/G1period cells reduced while the amount of G2/M period cells increased, and S period cells remained the same amount.conclusion.The4-HPR inhibition of SKVO-3cells proliferation illustrated its dependence on time and dosage, and the combined treatment of4-HPR and cisplatin demonstrated enhanced suppression on the formation of SKVO-3cells.4-HPR can block SKVO-3cells in the G2/M period and lead them to apoptosis and the effect is more efficient with cisplatin.4-HPR has the potential to improve the caspase-3expression and increase the sensitivity of SKVO-3cells to cisplatin.