| Objective: In this study,a drug-resistant subpopulation of esophageal squamous cell carcinoma cell EC109 cell line was constructed to investigate the function of ouabain on the inhibition of Wnt/?-catenin entrance into the nucleus both in xenograft tumor in nude mice and in CDDP resistant EC109/CDDP esophageal cells.Methods:1.Establishment of cisplatin-resistant human esophageal squamous carcinoma cell line(EC109/CDDP)by intermittent pulse administration of CDDPThe EC109 cells were induced by a pulse method of moderate dose of cisplatin administration to establish a CDDP-resistant human esophageal squamous carcinoma cell line.The growth inhibition rate of the above two cells was measured by MTT assay,and the half inhibition concentration(IC50)was calculated;the apoptosis rate of both cells was detected by Annexin V-FITC/PI;CDDP resistance in EC109 and EC109/CDDP were determined by CCK-8;the cell cycle distribution of the two cells was detected by Fluorescence Activated cell sorter(FACS);the invasive ability of the two cells was detected by Transwell assay.2.Effect of ouabain on esophageal carcinoma EC109/CDDP cells by inhibiting the entry of Wnt/?-catenin into the nucleus.The growth inhibition rates of EC109/CDDP cells treated by ouabain,CDDP,and ouabain combined with CDDP were detected by MTT assay.The effect of ouabain,CDDP and ouabain combined with CDDP on the apoptosis and cell cycle of EC109/CDDP cells were detected by FACS;the expression levels of MDR1,P-gp and Bcl-2 in EC109/CDDP cells were detected by q RT-PCR and Western blot.The plasmids of TOPFlash,FOPFlash and luciferase reporter gene system were used to explore Ouabain's regulation of ?-catenin transcription.3.Reversal effect of Ouabain on esophageal carcinoma EC109/CDDP-resistant xenograft in nude mice.The xenograft tumors in nude mice were established and randomly divided into four groups with six in each group: CDDP group(3 mg/kg),ouabain group(0.1 mg/kg),CDDP(3 mg/kg)plus ouabain group(0.1 mg/kg),and the control group(PBS).After the tumor volume reached approximately 50 mm3,CDDP and Ouabain were injected intraperitoneally to right side of the nude mice once or twice a day for 2 weeks.During the experiment,the mental and dietary status of the nude mice were observed and recorded daily.The weight and size of the tumors were measured at the same time,and the tumor volume was calculated.Through the approximate volume of the tumor as the ordinate,and the number of days of growth as the abscissa,the test data was taken to establish the tumor growth curve.The approximate volume of the tumor was equal to half of the product of the longest diameter and square of the shortest diameter of the tumor.One day after the drug was discontinued,the subjects were sacrificed by cervical dislocation.The tumors were weighed and recorded,and the data related to tumor weight inhibition rate were calculated.The expression of ?-catenin was detected by Western-blot.Results:1.Establishment of the resistant cell line of the human esophageal squamous cell carcinoma induced by cisplatin and its resistance analysis.The resistant cell line of human esophageal squamous cell carcinomaresistant to 0.5 ?g/ml CDDP was obtained by intermittent pulse of the same concentration(1 ?g/ml)of CDDP.MTT assay results showed that the IC50 values of EC109 and EC109/CDDP on CDDP were significantly different(P<0.05);FACS results showed that cell cycle distribution of cisplatin-resistant cells was longer in G2/M phase than that of wild-type cells.There was no significant difference in G0/G1 phase and S phase.Annexin V-FITC/PI assay showed that the apoptosis rate of cisplatin-resistant cells was significantly lower than that of wild-type cells whentreated with CDDP(P<0.05);It was shown that EC109/CDDP cells still had stronger invasiveness after cisplatin treatment,which was stronger than wild-type cells in Transwell assay(P<0.05).2.Effect of ouabain on the resistance of resistant EC109/CDDP cell line of esophageal squamous cell carcinomaby inhibiting the entry of Wnt/?-catenin into the nucleus.The results of MTT assay showed that both wild-type EC109 and resistant EC109/CDDP cells were sensitive to ouabain with IC50 values of 14.38 n M and 43.02 n M,respectively.Compared with the group treated with 5?M CDDP alone,the cell apoptosis significantly increased by FACS in the combination group of 5?M CDDP and 10 n M or 20 n M ouabain,whose differences were statistically significant(P < 0.05).In the reversal assay of the effect of ouabain on EC109/CDDP,when treated with 0-20 n M ouabain,the concentration-dependenceof IC50 of EC109/CDDP cells on CDDP was reduced,and the IC50 of EC109/CDDP cells on CDDP was significantly different when treated with 1 n M Ouabain and 20 n M Ouabain.In detail,in these experiments when using the highest concentration of Ouabain(20 n M),IC50 of EC109/CDDP cells on CDDP was effectively reduced from 36.53?M to 3.39?M,indicating that 20 n M of Ouabain increased the sensitivity to CDDP by 10.78 fold.Induction of 20 n M verapamil in the positive control group increased the sensitivity to CDDP by 13.84 fold.The expression of MDR1/P-gp in EC109/CDDP cells was detected by q RT-PCR and Western blot.The results showed that compared with EC109 cells,the expression of MDR1/P-gp in EC109/CDDP cells increased significantly.The MDR1 m RNA levels were reduced in a dose and time dependent manner after ouabain treatment.After 24 h of culture,the expression level of MDR1 decreased significantly with increasing concentration of ouabain.Western blot results showed that as the concentration of ouabain increased,the expression level of P-gp protein gradually decreased.When the concentration of ouabain was 20 n M,the expression level of P-gp was the lowest.The expression of Bcl-2 in EC109/CDDP cells was analyzed by q RT-PCR and Western blot.The results showed that compared with EC109,the expression of Bcl-2 in EC109/CDDP cells was significantly increased.Compared with EC109/CDDP cells that had not been treated with ouabain,the levels of Bcl-2 m RNA in cells incubated with 1 n M,5 n M,10 n M,and 20 n M ouabain for 24 hours were significantly decreased,and gradually decreased with the increasing dose.After treated with 20 n M ouabain,the levels of Bcl-2 m RNA decreased in a time-dependent manner.At the same time,Western blot analysis showed that Bcl-2 protein was over expressed in EC109/CDDP cells.TOPFlash luciferase assays and Western blots showed that ?-catenin was activated,and more ?-catenin was translocated into the nucleus in EC109/CDDP cells compared with EC109 cells,which may be related to the effective suppression of ouabain on the one hand.On the other hand,ouabain induced apoptosis of EC109/CDDP through caspase-3 and caspase-9 pathways.However,ouabain failed to induce apoptosis when the expression of ?-catenin was reduced by si RNA.3.Reversal effect of Ouabain on cisplatin resistance in esophageal carcinoma EC109/CDDP xenograft of nude miceA cisplatin-resistant xenograft model of human esophageal squamous cell carcinoma was successfully established in nude mice and all the tumors were successful.The treatment with CDDP combined with Ouabain had a good inhibitory effect on tumors.After 15 days of the combination therapy,the tumor volume was about 8 folds of that before treatment(P<0.05).In addition,compared with the control group,the inhibitory rates of CDDP group,and the group of CDDP combined with ouabain were 14.18% and 65.26%.group control with the Compared,treatment of CDDP combined with ouabain significantly inhibited tumor growth(P<0.05).Western blot results showed that the expression of ?-catenin was significantly down-regulated in the group of CDDP combined with Ouabain compared with the CDDP group or Ouabain group alone.Conclusion:1.In this experiment,a cisplatin-resistant cell line of human esophageal squamous cell carcinoma(EC109/CDDP)was successfully established.2.Ouabain had a potent effect on reversing multidrug resistance,which was dependent on ?-catenin.Ouabain may block the activity of ?-catenin and prevent the transfer of ?-catenin from the cytoplasm to the nucleus,thereby to up-regulate the sensitivity of resistant EC109/CDDP to CDDP,which indicated that ouabain may be a potential candidate to reverse the chemotherapy resistance of tumors.3.A xenograft model of EC109/CDDP cells was established in nude mice;Ouabain could reverse the cisplatin resistance of EC109/CDDP xenografts in nude mice.Ouabain enhanced the reversal effect of CDDP on EC109/CDDP possibly by inhibiting the Wnt/?-catenin signaling pathway. |
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