| Objective:Long-term use of cisplatin in the treatment of ovarian cancer will produce obvious drug resistance.Rare earth lanthanum chloride has excellent anti-tumor effect,but the mechanism of action of rare earth lanthanum chloride in ovarian cancer cells resistant to cisplatin has not been clarified.Therefore,the purpose of this study was to investigate the effects of rare earth lanthanum chloride on ovarian cancer cells(human COC1 cell line,human ovarian cancer SKOV3 cell line)and chemotherapyresistant ovarian cancer cells(COC1/DDP cell line,SKOV3/DDP cell line)as well as the mechanism of protein expression,so as to provide reference for improving the clinical efficacy of ovarian cancer chemotherapy.Methods:1.Human ovarian cancer(COC1)cell lines,human ovarian cancer cisplatin-resistant cell lines(COC1/DDP)and human ovarian cancer(SKOV3)cell lines were selected as experimental cell lines,and SKOV3/DDP drug-resistant cell lines were obtained by growing SKOV3 in culture medium containing DDP and gradually increasing DDP concentration.1.Human ovarian cancer(COC1)cell lines,human ovarian cancer cisplatin-resistant cell lines(COC1/DDP)and human ovarian cancer(SKOV3)cell lines were selected as experimental cell lines,and SKOV3/DDP drug-resistant cell lines were obtained by growing SKOV3 in culture medium containing DDP and gradually increasing DDP concentration.2.The 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,and 5.0 mol/L lanthanum chloride was adopted for routine culture of COC1 cell line and COC1/DDP cell line and the co-culture by adding half of the cisplatin with known concentration.After 48 hours,the cell proliferation inhibition rate of each group was detected by Methyl Thiazolyl Tetrazolium(MTT)method.3.According to the concentration of lanthanum chloride added,the COC1 and COC1/DDP cells were divided into a control group(cell suspension + medium),a low-dose group(0.5 μmol/L lanthanum chloride),a medium-dose group(2 μmol/L lanthanum chloride),and a high-dose groups(5μmol/L lanthanum chloride).After the lanthanum chloride was added to culture for 48 hours,the morphological changes of COC1 and COC1/DDP cells treated with different concentrations of lanthanum chloride were observed under a transmission electron microscope(TEM).4.According to the cell processing method,COC1 and COC1/DDP cells were rolled into a control group(COC1 or COC1/DDP),a cisplatin group(COC1 or COC1/DDP+DDP),a lanthanum chloride group(COC1 or COC1/DDP+ Lanthanum chloride),and a cisplatin+lanthanum chloride group(COC1 or COC1/DDP+DDP+lanthanum chloride).Flow cytometry was used to detect the apoptosis rate of cells in each group,and Western blot was used to detect the the relative expression levels of excision repair cross complementing group 1(ERCC1),Ki67,cyclin-dependent kinases 6(CDK6),and Casitase B-lineage Lymphoma(c-Cbl)in COC1/DDP cells under different treatments.5.The ovarian cancer DDP resistant cell line(SKOV3/DDP)was induced by gradually increasing the concentration of DDP,and its morphological changes were observed under a microscope.6.DDP with different concentrations of 1,2,4,6,8,10,20,and 30 μg/m L was added to SKOV3 and SKOV3/DDP cells for culture for 24 hours.The cell growth inhibition rate(GIR)and resistance index(RI)of DDP on KOV3 and SKOV3/DDP cells were analyzed using cholecystokinin octapeptide(CCK8).7.The lanthanum chloride at concentrations of 5,10,20,40,60,80,160,and320 μg/m L was added to SKOV3/DDP cells,respectively,and then the the GIR of drug at different concentrations and different times was analyzed using the CCK method 36 and 48 hours after the culture.8.According to different cell processing methods,SKOV3 and SKOV3/DDP cells were divided into a control group(SKOV3 or SKOV3/DDP),a lanthanum chloride group(SKOV3 or SKOV3/DDP +10 μg/m L lanthanum chloride),a DDP group(SKOV3 or SKOV3/DDP).SKOV3/DDP +5 μg/m L DDP),and a lanthanum chloride + DDP group(SKOV3 or SKOV3/DDP +5 μg/m L DDP+10 μg/m L lanthanum chloride).After treatment,the cells were placed in an incubator and cultured for 7 days to calculate the number of cell clones in different experimental groups.Flow cytometry was used to detect the apoptosis rate of SKOV3 and SKOV3/DDP cells in each group,and Western blot was adopted to detect the relative expressions of ERCC1,Ki67,CDK6,c-Cbl,and Caspase-3 proteins in SKOV3/DDP cells under different treatments methods.Results:1.After cisplatin treatment,the proliferation rates of COC1 and COC1/DDP cells were greatly reduced,and all showed a concentration-dependent.The inhibitory concentration 50%(IC50)of the COC1 and COC1/DDP cell line was 2.11 μg/m L and22.86 μg/m L,respectively,and the drug resistance index was 10.53 times.2.The results of MTT detection of cell proliferation rate showed that lanthanum chloride had an obvious inhibitory effect on the growth of COC1 cells and COC1/DDP cells,and presented a concentration-dependent characteristic.Taking 1.5μmol/L as the key point,lanthanum chloride lower than this concentration showed reduced inhibitory effect of cell proliferation;and lanthanum chloride with higher concentration showed the enhanced inhibitory effect of cell proliferation,showing a concentration-dependence.Based on the above results,it was believed that when the concentration of lanthanum chloride was 1.5 μmol/L,its toxic and side effects on ovarian cancer cells alone were not obvious.In addition,the combination of lanthanum chloride and cisplatin on ovarian cancer cells inhibited the proliferation efficiency of the cells and increased the toxicity of the cells more obviously.Therefore,lanthanum chloride at a concentration of 1.5 μmol/L could be adopted to treat ovarian cancer cells to obtain the best cell sensitization effect.3.The COC1 and COC1/DDP cells in the control group and the low-dose group showed normal morphology,complete nuclear membrane,clear organelle and nucleus structure,no intracellular vacuoles,and no significant difference in cell morphology between the two groups.The COC1 and COC1/DDP cell lines in the medium-dose group showed slight shrinkage and a small amount of vacuoles appeared in the cytoplasm;and the cell status was slightly worse than that of the control group and the low-dose group.The chromatin of the COC1 and COC1/DDP cell lines in the high-dose group condensed and condensed into clumps,with irregular distribution and unclear borders;they gathered around the nuclear membrane and formed crescent or circular corpuscles,with concentrated cytoplasm,the loose endoplasmic reticulum fused with the cell membrane to form vacuoles;and the nucleus was lysed into fragments and apoptotic bodies were produced.In this group,cell apoptosis was more serious.4.The apoptotic rates of COC1 cells and COC1/DDP cells in the control group,cisplatin group,lanthanum chloride group,and cisplatin + lanthanum chloride group were 5.52 ± 0.92% and 4.60 ± 1.11%,25.40 ± 4.38% and 18.24 ± 2.81%,7.22 ±2.55% and 5.59 ± 1.73%,and 27.54 ± 4.03% and 29.12 ± 5.38%,respectively.Compared with the control group,the expression levels of ERCC1,Ki67,CDK6,and c-Cbl in COC1 and COC1/DDP cells in the cisplatin group and the cisplatin+lanthanum chloride group were greatly reduced(P< 0.01);and the levels of ERCC1,Ki67,CDK6,and c-Cbl proteins in COC1/DDP cells in the cisplatin+lanthanum chloride group were reduced greatly in contrast to the cisplatin group(P< 0.01).5.The SKOV3 cell line was cobblestone-like and showed a regular morphology.The SKOV3/DDP cell line induced by DDP showed a long spindle shape and was relatively flat.6.When SKOV3 and SKOV3/DDP cell lines were treated with the same concentration of DDP,the inhibitory rates of DDP on SKOV3 cells were much higher in contrast to that of SKOV3/DDP(P< 0.05).The IC50 of DDP to SKOV3 cells was4.54 μg/m L,the IC50 to SKOV3/DDP cells was 11.12 μg/m L,and the RI of SKOV3/DDP to DDP was 2.45.7.When the concentration of lanthanum chloride was lower than 40 μg/m L,the cell growth inhibition rate of lanthanum chloride on the SKOV3/DDP cell line was not statistically different(P> 0.05)when it was treated on SKOV3/DDP cell line for36 h and 48 h.When the concentration of lanthanum chloride was ≥ 40 μg/m L,the cell GIR of SKOV3/DDP cell line at the same concentration of lanthanum chloride for48 h was obviously higher than that of 36h(P< 0.05).8.Compared with the control group,the cell clone formation rate of the lanthanum chloride group,the DDP group,and the lanthanum chloride + DDP group were decreased observably,showing statistically obvious differences(P< 0.05);the cell clone formation rate of the DDP group was greatly different from that of the control group(P< 0.01);that in the lanthanum chloride + DDP group was different greatly(P< 0.001),which was much lower in contrast to that of the DDP group(P<0.01).9.In contrary to the control group,the apoptosis rate of SKOV3/DDP cells in the lanthanum chloride group,the DDP group,and the lanthanum chloride + DDP group increased visibly.Compared with the control group,the apoptosis rate of SKOV3/DDP cells in the lanthanum chloride group was not statistically different(P>0.05).The apoptosis rates of SKOV3/DDP cells in the DDP group and the DDP+lanthanum chloride group were higher than that the rate in the control group(P< 0.05),and the DDP+lanthanum chloride group showed higher apoptosis rate of SKOV3/DDP cells in contrast to the DDP group(P< 0.05).The control group showed the much higher expressions of ERCC1,Ki67,CDK6,and c-Cbl proteins in contrast to the DDP group and the lanthanum chloride + DDP group(P< 0.05);the DDP group and the lanthanum chloride + DDP group showed obviously lower levels in ERCC1,Ki67,CDK6,and c-Cbl protein than the lanthanum chloride group(P< 0.05);and the expressions of these four proteins in the lanthanum chloride + DDP group were decreased in contrast to the DDP group(P< 0.05).The control group showed the much lower expression of Caspase-3 protein than the lanthanum chloride group,the DDP group,and the lanthanum chloride + DDP group(P< 0.05);the Caspase-3 in the DDP group and the lanthanum chloride + DDP group was obviously higher than that in the lanthanum chloride group(P< 0.05);and it was higher in the lanthanum chloride + DDP group in contrast to the DDP group(P< 0.05).Conclusion:1.Low concentration of lanthanum chloride exerted no visible inhibitory effect on the growth of ovarian cancer COC1,COC1/DDP,SKOV3,and SKOV3/DDP cells;2.The co-culture of lanthanum chloride and cisplatin could effectively enhance the inhibitory effect of cisplatin on the proliferation of ovarian cancer cells and promote the apoptosis of ovarian cancer cells;3.The potential mechanism of lanthanum chloride increasing the sensitivity of ovarian cancer cells to cisplatin may be related to the down-regulation of ERCC1,Ki67,CDK6,and c-Cbl protein expression and the up-regulation of Caspase-3.4.Lanthanum chloride could down-regulate the expression of ERCC1 protein in SKOV3/DDP cells,block the DNA repair function of ERCC1 gene,and increase the activation of Caspase-3 protein expression and increase cell apoptosis in SKOV3/DDP cells.In summary,this study could provide reliable experimental basis for the clinical application of lanthanum chloride as an anticancer drug.However,further research was needed to reveal the mechanism involved in the synergistic effect of lanthanum chloride and cisplatin. |
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