The Transcriptional Mechanisms Of NRF-2 On Mitochondrial Function And Cell Biological Behaviour In Human Cancer Cells

Nuclear respiratory factors (NRFs), including NRF-1 and NRF-2, play a great role in the control of mitochondrial function and biogenesis by regulating mitochondrial (mt) DNA transcription, replication and helping to coordinate the synthesis of respiratory chain subunits. Our previous studies had strongly indicated that the level of NRFs paralleled that of mitochondrial respiratory chain complex under normal and functionally altered states. One of the prominent characteristic of rapidly growing tumor cells is their tendency to carry out high rates of glycolysis and low oxidative phosphorylation (OXPHOS) even under aerobic conditions. However, it is unknown whether the deficient OXPHOS is resulted from disability of NRFs in transcriptional regulation on mitochondrial function.To determine the potential transcriptional mechanisms of NRF-2 in tumor cells, we established the monoclonal antibody (MAb) against the human NRF-2αprotein and investigated its distribution in human carcinomas by immunohistochemistry. Using lipofectamine, NRF-2αgene was transfected into HeLa cells to establish a cell model (HeLa-EGFP-NRF-2α) stably expressing NRF-2α. Then, expression of genes related to mitochondria metabolism and the mtDNA copy number were analyzed by RT-PCR and Western blot. Mitochondrial membrane potential (MMP) was detected by Mito Tracker staining and cytochrome C oxidase (COX) activity detected by histochemistey, and followed image analysis respectively. Effects in cell growth, apotosis and chemotherapy sensitivity of in HeLa-EGFP-NRF-2αcells and in a xenograft model were analyzed by performing MTT, growth curve, flow cytometry, TUNEL or Hoechst DNA staining. The xenograft sections were also analyzed by hematoxylin and eosin staining, histochemistry, immunohistochemistry and immunofluorescnce.Immunohistochemical analysis found that the expression of human NRF-2αprotein was down-regulated in hepatocellular carcinoma, cervical squamous cell carcinoma and intraepithelial neoplasia tissues comparing with tumor-adjacent or normal tissues. Increased expression of peroxisome proliferator-activated receptor gamma coactivetor-1 (PGC-1), mitochondrial transcription factor A (TFAM), Cytochrome C oxidase (COX) and Cytochrome C (Cyt C) were found in HeLa-EGFP-NRF-2 cells, accompanied with increased mtDNA copy number, MMP and ATP production.Up-regulation of NRF-2αresulted in sensitivity to cisplatin-induced cell death which was associated with the inhibition of cell proliferation and increased G1 phase arrest. The result was further confirmed in the animal model, which showed that the HeLa-EGFP-NRF-2 cells inoculated in nude mice grew significantly slower than the control cells after cisplatin treatment.Our results demonstrated for the first time that the expression of human NRF-2αprotein was down-regulated in hepatocellular carcinoma, cervical squamous cell carcinoma and intraepithelial neoplasia tissues, indicating that the regulation of NRF-2 on mitochondria function might be deficient in these tumors. Overexpression of NRF-2αprotein in HeLa cells led to G1 phase arrest in cell cycle, up-regulated mitochondrial respiratory chain protein, promoted OXPHOS and increased sensitivity to cisplatin-induced cell apoptosis. The in vivo data are consistent with the results obtained in vitro and support the suggestion that the overexpression of NRF-2αcan sensitize the tumor to the action of the cisplatin. Thus, NRF-2αcan be served as a potential target for developing new therapeutics which can enhance the antitumor activity of chemotherapy. The present study has established foundation for further profound understanding of NRF-2 in oncogenesis and anticancer research.