| OBJECTIVE: Sodium Hydrogen Exchanger-1(NHE-1)plays an important role in regulating the proliferation and apoptosis of tumor cells to maintain the intracellular homeostasis,which are closely related to the generation of tumor resistance.This study aims to investigate the effects of NHE-1 inhibitor Cariporide with or without Adriamycin on the proliferation and apoptosis rate of human breast cancer cells,and the growth rate of transplanted tumor in nude mice to provide a new theoretical basis and treatment for multidrug resistance in tumor therapy.METHODS: The cytotoxicity of Cariporide and Adriamycin on MCF-7 and MCF-7 /ADR cells was determined by CCK8 assay at different concentration respectively,and the changes of IC50 value were analyzed after the combination therapy of CP and Adriamycin.Expression of protein NHE-1?CD44?MDR-1?Caspase3/9?NF-?B p65 were detected by western blot.The differential expression of nuclear transcriptional factor was analyzed by RT-PCR such as MDR1 and NF-?B.In vivo,the weight?longest diameter(a)and shortest diameter(b)of transplanted tumor in nude mice are measured respectively everytime before treatment,the volume of the tumor is calculated according to formula.Cell morphology and apoptosis are observed under light microscope.Iummunohistochemistry is used to measure expression of NHE-1and Caspase-3.RESULTS: CCK8 assay showed that CP and Adr decreased the proliferation of MCF-7 and MCF-7/ADR cells in a dose-time dependent manner.CP of 6?g/ml inhibited proliferation of MCF-7/ADR cells less than 10%.CP combined with Adr could significantly reduce the IC50 value of MCF-7/ADR cells to Adriamycin.The combined index was less than one by Compu Syn.The apoptosis rate of CP combined with Adr was significantly increased to(29.12 + 0.65)%,which was much higher than the other team after flow cytometry.So as to the mean fluorescence intensity of Adriamycin.Cell cycle analysis showed that the percentage of G0/G1 phase in the combined group reached to 68.32 ± 5.99%,which was significantly higher than that in the control group.Western blot showed that NHE-1 expression in drug resistant cells were higher than that in sensitive cells.After treated with the combinated group,the protein expression of NHE-1?CD44 ?MDR-1 and Bcl-2 was significantly lower than the other group(P<0.05);while the expression of Cleaved caspase3/9 was upregulated,P<0.05.RT-PCR results showed that the level of NF-?B?MDR-1 m RNA were decreased respectively after the combination.Human breast cancer cells tumor xenografts model in nude mice was established successfully.The body weights of all the groups were decreased gradually with no significant difference among all the groups.The combined group,which had larger necrosis areas and apoptotic cells,had smaller tumors size and smaller tumor volume(P<0.05)and the rate of growth was much more slowly than the other group.Tumors of the combined groups had a higher Cleaved Caspase-3 and Cleaved PARP protein expression(P<0.05)?less Bcl-2 and MDR-1(P<0.05)protein expression than the Adr-treated group and NS group.CONCLUSION: The combination of NHE-1 inhibitor CP and Adr not only significantly inhibited the proliferation of MCF-7/ ADR cells,but also effectively attenuate the growth of transplanted tumors in nude mice,which possibly by regulating NF-?B pathway,downregulating the expression of multidrug resistance associated protein and inducing cell apoptosis. |
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