| Objective:In order to observe JH42-1has the role to inhibit cell proliferation and induce apoptosis of ovarian cancer SKOV3, we depicted this experiment, trying to explore the mechanism of action of JH42-1. Methods:Being cultured passaging, SKOV3cells were divided into three groups of negative control group, positive control group with a concentration of5μg/ml cisplatin treatment and the experimental group with different concentrations of JH42-1treatment. Under the microscope, we observed the cells’morphological changes. Cell proliferation was measured suppression by MTT (methylthiazolyltetrazolium) assay and Trypan blue exclusion test. Annexin V-EGFP/PI double labeling method of flow cytometry and fluorescence microscopy were detected the induction of apoptosis. Hoechst-33258staining of apoptotic cells observed by fluorescence microscopy, counting the rate of apoptosis. The immunofluorescence detected SKOV3cells in protein expression of PCNA and Caspase8. The expression of caspase8was detected by Western blot assay. Results: Observed under the inverted microscope:the cells in negative control group was wide fusiform or irregular polygon, full cell morphology, the most cell morphology, variation is small. Experimental group:JH42-1role24h,50,60and80umol/L group cell shrinkage round cell contour significantly enhanced, sizes, shape due to the rules. There were small bubble-like structures ranging from the number in the Cytoplasm and granular cytoplasm, membrane blebbing, and nuclear condensation. Processing after48h,40umol/L and above concentration group culture liquid appears in varying amounts of floating cells, and as the concentration is increased, the number of floating cells also increasing. Processing after72h,40,50,60and80umol/L, concentration group culture liquid floating cells was significantly increased, and even individual holes is difficult to find intact cells adherent.2. MTT assay:experimental group: JH42-1for48hours after each dose group (10,20,30,40,50,60,80μmol/L) of cell proliferation inhibition rate were22.09%,36.95%,40.74%,57.78%,63.80%,71.47%,73.41%respectively.24,48and72hours after treatment, the inhibition rate of cell proliferation manifested in a dose-and time dependencies. Larger dose group (60,80μmol/L), the inhibition of proliferation of the cells in each time period were significantly higher than that of the positive control group (p<0.05). Trypan blue dye experiment:in JH42-1treatment group, the survival rate of the cells decreased with the increase in the extension of time and concentration of the compounds, comparing with the results of the MTT assay.3. Flow cytometry:JH42-1treatment for24,48and72hours,30μmol/L cell apoptosis rate is21.63%,30.26%and36.08%, respectively. In50μmol/L dose group, the apoptosis rate was significantly greater than30μmol/L dose group and positive control group (p<0.05). Significantly increase with time, there is also dose-time-dependent.4. Double fluorescent staining:The experimental group showed apoptosis cells increased significantly compared with the negative control group. Hoechst-33258staining:Cell morphology was obvious apoptotic changes after JH42-1for48,72hours. In30μmol/L dose group apoptosis rate increased, and significantly increased apoptosis rate in40,50,60,80μmol/L each dose group.5. Immunofluorescence:pcna is located in the nucleus, which does not expressed in the cells of the blank control group, and was strongly positive expression in the negative control group. With the increase of the concentration of the compound, the experimental group concentrations of cell PCNA expression decreased gradually. Fluorescent cells positive rate were (95.46±0.68)%,(73.80±1.48)%,(57.15±2.37)%,(34.87±1.40)%,(24.89±1.48)%. Caspase-8in the cells of the control group did not express, which was Non-expression or low expression in the cells of the negative control group. The experimental group showed green fluorescence, The positive expression rate increased significantly with increasing doses JH42-1. The cells positive rate (1.84±0.35)%,(12.71±0.84)%,(26.02±1.01)%,(40.83±2.26)%,(58.35±1.24)%.6. Western-bloting:After JH42-1treated cells48h, caspase8protein expression was significantly higher. Comparing with the negative control group, the differences were statistically significant in the30,50umol/L concentration group (P<0.05). Conclusion:New compounds JH42-1can significantly inhibit human ovarian of serous cystadenocarcinoma SKOV3cells proliferation and induce apoptosis, depending on the Dose and time. The mechanism of induction may be raised Caspase-8and downregulation of PCNA. |
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