XIAP Antisense Oligodeoxynucleotides Induces Apoptosis In Hep-2 Cells And Sensitizes The Cells To Chemotherapy (Cisplatin)

Objective By transfecting XIAP ASODN into Hep-2 cells, we explore the effect of XIAP ASODN on apoptosis and chemotherapy sensitivity.Methods Firstly we culture Hep-2 cells under the conditions of 37℃,5﹪CO₂, 20﹪O₂ and 95﹪humidity. Cells in the logarithmic phase of cell growth were selected to proceed with the following experiments.1 Transfection of XIAP ASODN into Hep-2 cells was carried out by Lipofectamine2000. The experiment is devided into four groups: 100,200,400,600nmol/L. And the cells were observed under the fluorescence inversphase microscope after transfected 6 hours.2 Measure the inhibition rate of cells for 24h received DDP in different concentration by MTT. Measure apoptosis and the expression of XIAP protein and mRNA in Hep-2 lines by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry3 Measure the inhibition rate of transfection with DDP by MTT. The cells in the logarithmic phase of cell growth were selected to be transfected with Lipofectamine2000 mediated XIAP ASODN. The experiment is devided into several groups as follows: blank group replaced with RPMI-1640 medium, Lipofectamine2000 for control, XIAP ASODN transfection for control increased gradually following the raise of ASODN concentration (100,200,400,600nmol/L), XIAP SCODN transfection for control increased gradually following the raise of SCODN concentration (100,200,400,600 nmol/L) These four groups have two congditions: with-DDP group and non-DDP group. Each group consists of three parallel cells. Before transfection, the cells were cultured under the conditions of 37℃,5﹪CO2 ,20﹪O2and 95﹪humidity till the covering rate had been to 85﹪-90﹪. The chems groups need add 3ug/ml DDP every cell 6h after transfected. we add MTT (5ug/ml) 20ul every well 12,24,36,48hours after transfected respectively , cultured under the same conditions. we remove the medium carefully and add formazan solution, vibrate 10 minutes and measure optical density(OD)with enzyme-linked analyzer.4 Observe the changes of XIAP mRNA pre-and-after transfection with DDP by RT-PCR. As above, cells in the logarithmic phase of cell growth are selected. Also the experiment is devided into four groups: blank group, Lipofectamine2000 for control, 200nmol/ml XIAP ASODN, 200nmol/ml XIAP SCODN, These four groups have two congditions also: with-DDP groups and non-DDP groups. Each group consists of three parallel cells. After 36h transfection, we collect 1.7×10⁷ cells and extract total RNA with Trizol method. The same quantity of total RNA was taken to proceed with RT in a 20ul reaction system according to the manusfacture's instruction, and then amplified in a 5ul reaction system. Electrophoresis was used to analyse the products, and observe under ultraviolet light.5 Measure apoptosis and XIAP protein in the condition of transfection with DDP by flow cytometry: The group is the same with RT-PCR. Each group consists of three parallel cells. The cells in the logarithmic phase of cell growth were selected to be transfected with Lipofectamine2000 mediated XIAP AS-SCODN, and cultured under the conditions of 37℃,5﹪CO2,20﹪O2and 95﹪humidity, after 36h transfection, we collect every group cells and make single cell suspension, then measure with flow cytometry.Results1 Observing with fluorescence inversphase microscope, we find changes of cells after transfection, such as reducing of cell volume, anomaly form, shrinking, shrinking of nucleolus, cytoplast roughness and fewer cell division. However the untreated cells are in integrity form and well growth. The cells transfected with XIAP ASODN tagged with FITC for 6h are showing green fluorescence, the green fluorescence mainly distributes in the nucleolus and less in cytoplasm. we find the average efficiency of transfection at final concentration of 100nmol/L, 200nmol/L, 400nmol/L and 600nmol/L were (95±2.1)﹪,(98±1.3)﹪,(96±2.6)﹪,(93±3.0)﹪. Basing on the ANOVA analysis, there was no different in statistic between groups (P>0.05), there has no transfected in blank group.2 All concentrations of DDP can inhibit proliferation and show a dose- and time- dependent manners, the difference was significant(P<0.05). The inhibition rate of 3ug/ml DDP has a significant increase, 5ug/ml DDP was the highest (16.72±0.81)﹪. Between 2ug/ml and 3ug/ml group has a significant differences(P<0.01). The apoptosis rate increased gradually following the raise of C-DDP's concentration (1,2,3,4,5ug/ml), 5ug/ml DDP was the highest (11.38±0.42)﹪. Between 2ug/ml and 3ug/ml group has a significant differences(P<0.01). The fluorescence index (FI) indicating the expression level of XIAP protein decreased inorder along with the increasing of DDP concentration. Basing on the correlation analysis, there was positive correlation between the cell apoptosis rate and the fluorescence index(FI)(r=-0.984,P<0.01). We use 3ug/ml DDP to the follow study.3 The inhibition rate of Hep-2 cell lines pre-and-after transfection under the condition of DDP and non-DDP by MTT.The result shows that all concentrations of XIAP ASODN(100nmol/L-600nmol/L)can inhibit proliferation and show a dose- and time- dependent manners. All concentrations of XIAP ASODN groups with 3ug/ml DDP were detected at different stage of time, the inhaibitation rate higher than the only transfected XIAP ASODN group and only with DDP group. the difference was significant(P<0.05).4 The effects of XIAP ASODN on the Cisplatin (DDP) induced apoptosis in Hep -2 cell lines The result by flow cytometry shows that the apoptosis rate in chemotherapy combine with XIAP ASODN group is significantly higher than the chemotherapy group(P<0.01). There was no different in statistic between SCODN group and the blank group (P>0.05). And also no different in statistic between chemotherapy combine with SCODN group and the chemotherapy group (P>0.05).5 The effects of XIAP ASODN combine with Cisplatin (DDP) on the expression of XIAP mRNA by RT-PCR. The results showed that the products of RT-PCR were detected in the position of 802bp. Compare with the blank group, Lipfectamine2000 group, SCODN group, in the ASODN group, the light of the line is less brighter than above three groups. This shows that the relative expression level of XIAP mRNA in Hep-2 cells decreased significantly, and the chemotherapy combine with XIAP ASODN group decreased seriousely. But the relative expression level of XIAPmRNA both in the blank group and SCODN group had no change.6 The effects of XIAP ASODN combine with Cisplatin (DDP) on the expression of XIAP protein.The result by flow cytometry shows that the fluorescence index(FI) in ASODN group is lower than the blank group, Lipfectamine2000 group and SCODN group (P < 0.05), chemotherapy combine with XIAP ASODN group is significantly lower than the chemotherapy group (P<0.01). There was no different in statistic between chemotherapy combine with SCODN group and chemotherapy group (P>0.05). Basing on the Pearson correlation analysis, there was positive correlation between the cell apoptosis rate and the fluorescence index(FI)(r=-0.976,p<0.01).Conclusion Transfecting XIAP ASODN into Hep-2 cells can inhibit cells proliferation, induce apoptosis and enhance the sensitivity of Hep-2 cells to chemotherapy.