An Exploration On The Drug-resistant Reverse Effects And Related Mec Hanism Of Lanthanum Chloride Against The Cisplatin-resistant Cells SKOV3/DDP In Human Ovarian Cancer

AIM: culture the SKOV3 cell, induce the birth of cisplatin resistant strains SKOV3/DPP, research about the reverse effects and related mechanism of rare earth lanthanum chloride against the cisplatin resistance on ovarian cancer.METHODS:1. Induce the birth of the SKOV3/DPP cell strain, start from the low density of cisplatin, then increase it gradually, lead to the appearance of the cisplatin resistance on ovarian cancer SKOV3/DPP cell, invert them into the microscope and examine the morphology differences between these two cells. Use the CCK8 method to measure growth inhibiting rate of cisplatin against these two cells, and obtain IC50, calculate the drug-resistance index of the strains.2. Use the CCK8 method to test the proliferation of SKOV3/DDP cells and their activities after a certain gradient density of lanthanum chloride has acted on them upon different time, and select the density of non chemotherapeutic lanthanum chloride drug. research the reverse effects of resistance that the lanthanum chloride in a nontoxicity density works upon the SKOV3/DDP cells.4. Use the clone formation test to check the changes upon proliferation ability of different experiment groups; Check the cell apoptosis ratio of different experiment groups through flow cytometry method; Check the expression level of Caspase-3、ERCC1 protein in different experiment cell groups by western blot;the test is divided to four groups: blank control group, 5μg/ml cisplatin alone group, 10μg/ml lanthanum chloride alone group and the group of 10μg/ml lanthanum chloride combining with 5μg/ml cisplatin.RESULTS:1. In about 5 months, successfully induce SKOV3 cisplatin cells through gradually increase of the density of DDP from a low level, they have the ability to proliferation, grow slower than the SKOV3 cells; when the cell growth comes to 60-70% density, the shape of SKOV3 cells is pebble-style, regular form and uniform in size, while the SKOV3/DDP cells are prolate or spindle, relatively flat, polygonal with a slightly low refractivity.2. the RI of SKOV3/DDP upon DDP is2.09 times. Use different density of cisplatin to work on SKOV3 cells and SKOV3/DPP cells, the IC50 of DDP against SKOV3 is4.96μg/ml, while DDP against SKOV3/DDP is10.37μg/ml.3. Lanthanum chloride could restrain the outside proliferation of SKOV3/DDP cells When in low density(<10μg/ml) the restraint effect is not significant, moreover as time goes by the restraint ratio will not rise apparently; while in a relatively high density, the restraint ratio would correspondingly increase when the density of lanthanum chloride goes up, and the ratio will slightly rise given more time. The test proves: the lanthanum chloride at 10μg/ml or lower has no evident cytotoxic effect upon SKOV3/DDP cells, its restraint ratio smaller than 5%.4. 10ug/ml lanthanum chloride and DDP combination can strengthen DDP cytotoxic effect of SKOV3 / DDP cells.After 10μg/ml lanthanum chloride(put in to action 12 hours in advance)combining with different density of DDP(1、2、4、6、8、16、32μg/ml) works for 24 hours, the IC50 of DDP against SKOV3/DDP is7.25μg/m L, compared to10.37μg/m L of the IC50 when use DDP to stimulate alone for 24 hours, the former is significantly lower.5. Clone formation test: 10ug/ml lanthanum chloride and DDP combination can effectively inhibit SKOV3/DDP cell clone formation. The clone formation rates are(11.20±0.52)%,(8.73±0.50)%,(0.96±0.15)%,0.respectively refer to the blank control group, 10μg/m L lanthanum chloride alone group, 5μg/m L DDP alone group, and the group of 10μg/m L lanthanum chloride combining with 5μg/m L DDP; and compared to the blank control group, the amount of SKOV3/DDP cells are less in the two later groups.6. Check the cell apoptosis ratio through flow cytometry method: 10ug/ml lanthanum chloride and DDP combination can increase SKOV3 / DDP cells apoptosis. theapoptosis ratios of the blank control group, lanthanum chloride10μg/m L alone group, cisplatin5μg/m L alone group, and the group of lanthanum chloride10μg/m L combining with cisplatin 5μg/m L are 19.2%,23.5%,29.1%,39.2%.respectively. Compared to the blank control group, the ratio of cisplatin5μg/m L alone group rises, the ratio of lanthanum chloride10μg/m L alone group rises slightly, while the ratio of the combined group increases significantly.7. The expression level of Caspase-3,ERCC1 protein in SKOV3/DDP cells: there is a rise of expression of ERCCI protein in the cisplatin5μg/m L alone group, a marked decrease in the combined group; these two groups both are significantly different from the blank control group(P<0.05) respectively, and there is a marked difference between these two(P<0.05). In SKOV3/DDP cells, compared to the blank control group, the expression of Caspase-3 protein of the cisplatin5μg/m L alone group has statistic difference,and the expression of Caspase-3 protein of the cell group of lanthanum chloride10μg/m L combining with cisplatin 5μg/m L enjoys aevident rise, the difference has statistic meaning(P<0.05).CONCLUSION:1.,Start from the low density of cisplatin can induce the birth of the SKOV3/DPP cell strain.2.A certain concentration of lanthanum chloride can inhibit the proliferation of SKOV3 / DDP cells,No cytotoxicity lanthanum chloride increases the sensitivity of SKOV3 / DDP cells for DDP.3.When DDP role in SKOV3/DDP cells, the expression of ERCC1 protein will increasing. While at the same time add the concentration of lanthanum chloride can inhibit the expression of ERCC1 protein. the DNA repair function will blocked.the increasing of cleaved Caspase-3 make cells apoptosis.