| ObjectiveThe study intends to observe the effect and machanism of GRP78on resisitance of cisplatin in ovarian carcinoma,to provide new tagets and ideas for the effective reversal of DDP resistance in ovarian cancer.MethodsThe GRP78of SKOV3cells and SKOV3/DDP cells were detected by RT-PCR and Western Blot.Then the both cells were treated by regulatory factor BAPTA-AM and A23187,that were used decreased and increased GRP78,respectively. The expressions of GRP78、PI3K、AKT、p-AKT in SKOV3and SKOV3/DDP cells were detected by RT-PCR and Western Blot. And growth inhibition rate and the sensitivity to cisplatin of both cells were accessed by MTT. The application of flow cytometry analysed the apoptosis rates of cell and distributed characteristics of cell cycles after downing and upregulating of GRP78expression,repectively,to determine GRP78involved in the effect and mechanisms of cisplatin resistance in ovarian cancer.Results1. The expressions of GRP78in ovarian cancer and sensitivity to cisplantin:GRP78were expressed in SKOV3cells and SKOV3/DDP cells.But the expressions of GRP78in SKOV3/DDP cells were higher than that of SKOV3cells. And the expression of GRP78mRNA were(0.5464±0.04048),(0.7267±0.05508) in SKOV3and SKOV3/DDP cells, respectively. And the expression of GRP78proteins were (0.4937±0.03147),(0.6913±0.05633)in both cells,respectively.Th e expression of GRP78mRNA and protein in SKOV3/DDP cells were siganificantly higher compared with SKOV3cells(t=0.01,P (0.05;t=5.304,P=0.006).It was known the GRP78high expressions were related with cisplatin resistance in ovarian cancer.2. The effect of GRP78expression regulated in ovarian cancer on sensitivity to cisplatin:The expressions of GRP78mRNA and protein in two cells were decreased which were detected after treating by BAPTA-AM. The results were as follows:the SKOV3cells (0.3200■0.01000),(0.310±0.02000);SKOV3/DDP cells(0.4400±0.03464),(0.3500±0.04000).And the results of control groups were as follows:SKOV3cells (0.5133±0.03512),(0.5033±0.03215);SKOV3/DDP cells (0.6300±0.04359),(0.6467±0.07506).Their results were siganificantly lower when they were compared with the control groups (SKOV3cells t=8.286, P=0.014; t=6.526, P=0.023; SKOV3/DDP cells t=9.127, P=0.012;t=5.130,P=0.036).The expressions of GRP78mRNA and protein in two cells were increased which were detected after treated by A23187. The results of expressions of GRP78mRNA and protein in two cells after added drug were as follows:the SKOV3cells (0.7600±0.07211),(0.7233±0.03512);SKOV3/DDP cells(0.8733±0.03786),(0.8500±0.02646).Their results were siganificantly higher than the control groups(SKOV3cells t=7.529, P=0.017;t=7.750, P=0.016; SKOV3/DDP cells t=5.518, P=0.031;t=9.165, P=0.012).After interference by BAPTA-AM,the sensitivity to DDP of SKOV3and SKOV3/DDP cells increased37.15%,52.91%.And after interference by BAPTA-AM,the sensitivity to DDP of two cells reduced54.86%,27.72%.Resistance to cisplatin in ovarian cancer related to GRP78.The suppression of GRP78expression can increase the drug sensitivity of ovarian cancer to cisplatin.3. The effect of GRP78expression regulated in ovarian cancer on the expression of PI3K、AKT、p-AKT:(1)The gene levels of PI3K and AKT were reduced when GRP78were downed by BAPTA-AM,respectively.The results were as follows:SKOV3cells(0.2600±0.03606),(0.3867±0.04041),SKOV3/DDP cell s(0.3767±0.03055)、(0.4500±0.0100).And the results of control groups are as follows:SKOV3cells (0.4367±0.03215)、(0.6167±0.03512);SKOV3/DDP cells (0.5600±0.04359)、(0.7067±0.01528).They were lower than control groups and have significance (SKOV3cells t=8.082,P=0.015;t=23.000,P=0.002;SKOV3/DD P cells t=12.618, P=0.006;t=17.665, P=0.003)The protein levels of PI3K、AKT、p-AKT were also decreased when GRP78were downed by BAPTA-AM,respectively. The results were as follows:SKOV3 cells(0.4033±0.04163)、(0.3611±0.03464)、(0.3600±0.04359), SKOV3/DDP cells[(0.4933±0.02517)、(0.5000±0.04163)、(0.4267±0.04041). And the results of control groups were as follows:SKOV3cells (0.5267±0.04726)、(0.5433±0.04619)、(0.4600±0.0100) and SKOV3/DDPcells (0.6500±0.03606)、(0.6800±0.02646)、(0.5767±0.03215).The results were lower than control groups and had significance(SKOV3cells t=10.262,P=0.009;t=27.5, P=0.001;t=5.000, P=0.038);SKOV3/DDP cells t=17.764, P=0.003;t=4.750, P=0.042;t=12.990, P=0.006)。(2) The gene levels of PI3K and AKT were increased when GRP78were upregulated by A23187,respectively.The results were as follows:SKOV3cells SKOV3cells (0.6867±0.02082)、(0.7200±0.01732),SKOV3/DDP cells (0.7467±0.04041).(0.8467±0.03055). And the results of control groups were as follows: SKOV3cells (0.4367±0.03215)、(0.6167±0.03512);SKOV3/DDP cells (0.5600±0.04359).(0.7067±0.01528).They were higher than control groups and had significance(SKOV3cells t=7.529, P=0.017;t=7.750, P=0.016; SKOV3/DDP cells t=5.518, P=0.031; t=9.165, P=0.012)The protein levels of PI3K. AKT. p-AKT were also increased when GRP78were upregulated by A23187,respectively. The results were as follows:SKOV3cells (0.6633±0.07506).(0.6567±0.03512)、(0.5367±0.03055),SKOV3/DDP cells (0.7200±0.03000).(0.8300±0.04000)、(0.6900±0.02000). The results were higher than control groups and had significance (SKOV3cells t=4.799, P=0.041; t=9.430, P=0.011; t=4.6, P=0.044; SKOV3/DDP cells t=4.583, P=0.044; t=15, P=0.004; t=9.430, P=0.011)。GRP78could regraduate the expression levels of PI3K. AKT. p-AKT in both cells.So GRP78may involved in cisplatin resistance of ovarian cancer through PI3K/AKT signal transduction pathway.4. The effect of GRP78expression regulated in ovarian cancer on cell apotosis and cell cycle:The rates of cell apoptosis were27.42%,21.91%after SKOV3and SKOV3/DDP cells treated by BAPTA-AM,respectively. The results were obviously higher when they were compared with control group (19.57%,12.74%),respectively. The rates of cell apoptosis were12.21%,7.22%after SKOV3and SKOV3/DDP cells treated by A23187,respectively.The results were obviously lower than control group,respectively. And the rate of apoptosis in SKOV3cells is higer than the rate of apoptosis in SKOV3/DDP cells.The cell ratio in G1phase was gradually increased and the cell ratio in S and G2phase were gradually reduced along with decreasing GRP78in both cells. So decreased expression of GRP78may arrest cell cycles of SKOV3and SKOV3/DDP cells during G1phase and increase the lethality of cisplatin on ovanan cancer.Conclusions1. GRP78high expression were related to cisplatin resistance in ovarian cancer The suppression of GRP78expression can increase the drug sensitivity of ovarian cancer to DDP.So GRP78may provide new tagets and ideas for the effective reversal of DDP resistance in ovarian cancer.2. GRP78may involved in cisplatin resistance of ovarian cancer through regulating PI3K/AKT signal transduction pathway,anti-apoptosis and arresting cell cycle. |
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