Study Of Keap1-Nrf2 Pathway On Endometrial Cancer Cell Proliferation Metastasis And Drug Resistance

Objective:To investigate the effect of Keap1-Nrf2 pathway on the cell proliferation,metastasis,and drug resistance of endometrial cancer cells.Methods:(1)The protein levels of Keap1,Nrf2 as well as its downstream genes were assayed in KLE,Ishikawa cell lines(which represent type ? endometrial cancer)and ARK2,SPEC-2 cell lines(which represent type ? endometrial cancer).Real-time PCR was used to assay the mRNA expression of Keap1 and Nrf2 genes in ARK2 cells which had the highest expression of Nrf2 protein and KLE cells which had the lowest expression of Nrf2.MTT assay was performed to detect the sensitivities of these two cell lines to paclitaxel and cisplatin with different concentrations.Nrf2 gene silencing was performed on KLE and ARK2 cells,and the effect of silencing Nrf2 gene on tumor cell resistance was evaluated.(2)Keap1 was overexpressed in ARK2 cell line with a lower Keap1 expression and higher drug resistance.Real-time PCR,Western blot,MTT assay,nude mouse tumor formation experiment,colony formation assay and transwell assay were performed to evaluate the effect of overexpression of keap1 on the expression of Nrf2 and its downstreamgene,cell proliferation,invasion,migration and drug resistance of ARK2 cells.Results:(1)Compared with normal endometrial tissue,the expression of Keap1 protein was decreased in each cell line of endometrial cancer,while the expression of Nrf2 protein was increased,indicating that the signaling pathway was significantly activated.There was no significant difference in mRNA expression of Nrf2 between ARK2 and KLE cells,but the expression of Keap1 gene in ARK2 cells was lower than that of KLE cell line both mRNA and protein expression.The expression of Nrf2 protein in the ARK2 cell line was significantly higher than that in the KLE cell line.Keap1 expression was lower in ARK2 cells than KLE cells.ARK2 cells which had a higher expression of Nrf2 protein showed higher resistance to chemotherapeutic drugs than KLE cells which had a less expression of Nrf2 protein.(2)Overexpression of Keap1 in ARK2 cell lines could significantly down-regulate expression of Nrf2 protein and of its downstream genes ABCC2 and g-GCS;MTT assay,nude mouse tumor formation experiment,colony formation assay and transwell assay showed overexpression of Keap1 could decrease the cell proliferation,migration and invasion of ARK2 cells,and enhanced its sensitivity to paclitaxel and cisplatin.Conclusions:(1)The Keap-Nrf2 signaling pathway was significantly activated in endometrial cancer,and the activation of this signaling pathway in type ? endometrial cancer cells was higher than that of type ? endometrial cancer cells.Keap1 regulated Nrf2 protein expression based on post-transcriptional,and a low expression of Keap1 and high expression of Nrf2 protein was associated with a high drug resistance in tumor cells.There might be a “threshold” of the activation level of Keap1-Nrf2 signaling pathway in tumor cells,when the activation level was lower than the “threshold”,the sensitivity of tumor cells to chemotherapeutic drugs was at the same level,which was shown to be more sensitive to chemotherapeutic drugs;when the degree was above the "threshold",the tumor cells showed a markedly elevated resistance to chemotherapeutic drugs.(2)Overexpression of Keap1 gene could effectively down-regulate the expression of Nrf2 protein and its downstream genes in ARK2 cells,reduce the growth,migration and invasion of tumor cells,and enhance the sensitivity of tumor cells to chemotherapeutic drugs.