The Nrf2/PGC1α Pathway Regulates Antioxidant and Proteasomal Activity to Alter Cisplatin Sensitivity in Ovarian Cancer

Drug resistance remains a barrier in the clinical treatment of ovarian cancer. Proteasomal and antioxidant activities play important roles in tumor drug resistance, and increasing evidence suggests the existence of an interaction between antioxidant and proteasomal activities. However, the mechanism of the synergistic effects of proteasomal activity and antioxidation on tumor drug resistance is not completely clear. In this study, we compared two ovarian cancer cells, A2780 and SKOV3 cells. Among them, SKOV3 cell is a human clear cell carcinoma cell line that is resistant to platinum. We found that compared with the findings in A2780 cells, SKOV3 cells were less sensitive to both proteasomal inhibitor and cisplatin. Proteasomal inhibition enhanced the sensitivity of A2780 cells, but not SKOV3 cells, to cisplatin. Notably, the Nrf2-mediated antioxidant pathway was identified as a resistance mechanism in proteasome inhibitor-resistant cells, but this was not the only factor identified in our research. In SKOV3 cells, PGC1α regulated the antioxidant activity of Nrf2 by increasing the phosphorylation of GSK3β, and in turn, Nrf2 regulated the transcriptional activity of PGC1α. Thus, Nrf2 and PGC1α synergistically participate in the regulation of proteasomal activity. Furthermore, the Nrf2/PGC1α pathway participated in the regulation of mitochondrial function and homeostasis, further regulating proteasomal activity in SKOV3 cells. Therefore, exploring the roles of PGC1α and Nrf2 in the regulation of proteasomal activity by antioxidant and mitochondrial functions may provide new avenues for reversing drug resistance in ovarian cancer.


Introduction
Ovarian cancer is the most deadly gynecological malignancy, and drug resistance has become a major challenge in its treatment in recent years. Resistance to chemotherapy in tumor cells is related to multiple factors, including the repair of DNA damage, blockade of drug-induced apoptosis, reactive oxygen species-(ROS-) mediated redox state, and protein degradation [1][2][3]. Current research suggests that exploring the mechanisms of the interaction between different signals can shed light on the mechanisms of tumor drug resistance. Proteasomal activity plays important roles in tumorigenesis and chemotherapy resistance, and abnormally elevated proteasome levels are found in a variety of cancers [4][5][6]. The proteasome can eliminate oxidative and damaged proteins to reduce oxidative stress damage, and enhanced antioxidant capacity can promote the expression of 20S and 19S proteasome subunits [7,8]. These findings suggest the existence of an interaction between proteasomal activity and redox function, and exploring this relationship will be more helpful in solving the problem of chemotherapy resistance in ovarian cancer.
The ubiquitin-proteasome system (UPS) maintains cell homeostasis by regulating proteins involved in signal transduction and cell cycle pathways [9]. Inhibition of the proteasome can cause the accumulation of proapoptotic proteins and induce tumor cell apoptosis. However, recent clinical studies found that some tumors are not sensitive to proteasome inhibitors. Proteasome inhibitor-resistant tumors generally have higher expression levels of antioxidant genes [10]. The nuclear factor E2-related factor 2 (Nrf2, gene name NFE2l2)-mediated antioxidant stress pathway was identified as a resistance mechanism in the proteasome inhibitorresistant phenotype. Nrf2 can regulate the transcriptional activity of proteasome mature protein, which can promote resistance to proteasome inhibitors [11]. This suggests that Nrf2-mediated antioxidant activity may be related to proteasome-mediated tumor resistance.
Nrf2 is an important element of the antioxidant response element (ARE) transcription complex, and it can regulate the expression of various protective genes. Nrf2-and antioxidant-related gene expression is elevated in drugresistant tumor cells, and its activity is regulated by a variety of factors, including transcription and posttranscription modification. The dissociation of Nrf2 and Kelch-like ECHassociated protein 1 (Keap1) enables Nrf2 to enter the nuclear and regulate the transcription of the oxidase gene [12,13]. What's more, glycogen synthase kinase 3β (GSK3β) can inhibit Nrf2 by preventing nuclear accumulation of the CNC-bZIP factor, and evidence has provided that GSK3β interferes with Nrf2 transactivating activity and nuclear exclusion of the Nrf2 [14,15]. Peroxisome proliferatoractivated receptor-γ coactivator 1α (PGC1α, gene name PPARGC1A) can also coordinate the expression of multiple antioxidant genes to protect cells against oxidative stress damage. PGC1α is involved in the regulation of Nrf2 expression and activity, and studies have confirmed the proteinprotein interaction between them [16,17], but the specific mechanism is not completely clear. Therefore, the mechanism by which PGC1α and Nrf2 interact may play a synergistic role in antioxidation, but it is unclear whether their interaction is related to drug resistance.
In addition, the maintenance of mitochondrial redox homeostasis plays an important role in proteasome activity. Mitochondria are the main sources of ROS, and excessive ROS production can increase the burden on the UPS and decrease the proteasomal activity induced by increased oxidation and damaged proteasome subunits [18]. Nrf2 can inhibit mitochondrial ROS production by upregulating heme oxidase (HO-1) and the primary mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2), thereby inhibiting mitochondrial oxidative stress damage [19,20]. Mitochondrial protein stability and structural integrity are regulated through interactions with a variety of mitochondrial proteins, including PGC1α and PGC1β [21][22][23]. These results suggest that the antioxidation mediated by Nrf2 and PGC1α also regulates the homeostasis of mitochondrial function and thus maintains proteasome activity.
In this study, the "UPS-Antioxidation Axis" was taken as the entry point to explore the roles of PGC1α and Nrf2 in the regulation of antioxidant and mitochondrial functions in maintaining proteasome activity. The findings provide new ideas for reversing drug resistance in ovarian cancer.

Cell Viability
Assay. Cells (10,000 cells/well) were seeded in 96-well plates, incubated overnight, and treated with various reagents for the indicated times. Cells were then incubated with MTT reagent for 4-6 h. Absorbance values were recorded at 570 nm using an enzyme-linked immunosorbent assay reader.

Flow
Cytometry. Apoptotic cells were counted using Annexin V-FITC and propidium iodide (PI) (Annexin V Apoptosis Detection Kit, BD Pharmingen, San Jose, CA, USA) according to the manufacturer's protocol. Mitochondrial membrane potential (MMP) and ROS production were determined using JC-1 or DCFH-DA staining (Beyotime Biotechnology, Shanghai, China). Mitotracker™ Red (Sigma-Aldrich) staining was used to evaluate the alteration of mitochondrial mass. The analysis was performed using a BD Accuri C6 flow cytometer (BD Biosciences, New Jersey, USA).
2.6. Western Blotting. Whole cells were lysed in RIPA lysis buffer containing protease inhibitors. Lysates diluted with 5x SDS-PAGE loading buffer were boiled at 95°C for 2 Oxidative Medicine and Cellular Longevity 10 min, separated by SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% milk followed by incubation with primary antibodies overnight at 4°C. The next day, the membranes were incubated with HRP-conjugated secondary antibodies. The bands were incubated in ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) for chemiluminescence and visualized using Syngene Bio Imaging (Synoptics, Cambridge, UK).

2.7.
Immunoprecipitation. Cell lysates were solubilized in NP40 buffer containing a protease inhibitor mixture for 1-2 h at 4°C. After adding monoclonal antibodies, supernatants were rotated at 4°C overnight. The next day, the protein lysates were incubated with 30 μl of protein A/G Plusagarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The agarose beads were washed three times, SDS-PAGE loading buffer was added, and then, samples were analyzed via Western blotting.

Nuclear and Mitochondrial
Isolation. Cells were harvested after treatment with various reagents. Nuclear and mitochondrial fractions were extracted using a nuclear fractionation kit and mitochondrial fractionation kit, respectively (Invent Biotechnologies, MN, USA).
2.9. ATP Production. Cells were treated with 100 nM Epox for 12 h or transfected with Nrf2 or pcDNA3.1 (vector plasmid was used in the NC group). ATP production was determined using an ATP Bioluminescence Assay Kit (Beyotime Technology). An Omega luminometer was used to measure the values (BMG Labtech, Ortenberg, Germany).

Oxygen Consumption Rates (OCRs)
. Cells were seeded in 96-well plates overnight. The next day, cells were incubated with a mixture of reagents and the oxygen-sensitive probe Mito-Xpress (Luxcel Bioscience, Cork, Ireland) and examined using an Omega luminometer for 6 h.
2.13. Immunofluorescence Staining and Microscopy. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 8 min. After blocking with 5% bovine serum albumin for 30 min, cells were incubated with primary antibody overnight at 4°C. After PBS washing, the cells were incubated at room temperature for 1 h in the dark with FITC/Texas Red-conjugated secondary antibodies (Proteintech). The images were observed on an Echo-lab Revolve microscope (CA, USA).
2.14. Statistical Analysis. Data are expressed as the mean ± SD. * P < 0:05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 5 (La Jolla, CA, USA). All experiments were repeated at least three times.

Proteasome Inhibition Can
Enhance the Sensitivity of A2780 Cells to Cisplatin, but Not SKOV3 Cells. Drug resistance remains an important obstacle that limits the efficacy of platinum-based treatment in ovarian cancer [25], and targeting the ubiquitin-proteasome pathway can overcome cancer chemoresistance [26]. The MTT assay illustrated that compared with the findings in SKOV3 cells, the proteasome inhibitor Epox (a new-generation proteasome inhibitor that can noncovalently bind to the subunits of the proteasome in an irreversible manner [27]) decreased the viability of A2780 cells in a concentration-dependent manner (Figure 1(a)). As shown in Figure 1(b), the IC 50 values of cisplatin in A2780 and SKOV3 cells were 5:5 ± 0:18 and 17:89 ± 4:02 μg/ml, respectively. This demonstrated that SKOV3 cells were less sensitive to both cisplatin and proteasome inhibition. To further determine the effect of proteasomes on the sensitivity of ovarian cancer cells to cisplatin, we treated A2780 and SKOV3 cells with 100 nM Epox and different concentrations of cisplatin for 24 h and detected survival using MTT assays. The results demonstrated that Epox increased the sensitivity of A2780 cells, but not SKOV3 3 Oxidative Medicine and Cellular Longevity cells, to cisplatin. These results suggested that the proteasome plays an important role in cisplatin resistance in SKOV3 cells.

Proteasome Inhibition Promotes Nrf2
Localization to the Nucleus through the Keap1/Nrf2 Pathway. Although inhibition of the proteasome pathway has emerged as an important strategy for anticancer therapy [28], not all cancer cells are sensitive to proteasome inhibitors. Studies have identified the Nrf2 pathway as one of the mechanisms of resistance to proteasome inhibitors [29]. To investigate whether Nrf2 is involved in resistance to proteasome inhibitors, the expression of Nrf2 in the nucleus was examined. After treatment with Epox for 12 h, an increased expression of Nrf2 in the nucleus was observed in both A2780 and SKOV3 cells (Figures 2(a) and 2(b)). The immunofluorescence assay also revealed that the localization of Nrf2 in the nucleus was enhanced by Epox treatment (Figure 2(c)). As the Keap1/Nrf2 pathway plays an important role in the stabilization of Nrf2, we further detected the colocalization of Keap1 and Nrf2. The immunoprecipitation and immunofluorescence results revealed that Keap1 and Nrf2 colocalization was decreased by Epox treatment (Figures 2(d) and 2(e)). These results suggested that proteasome inhibition promotes Nrf2 localization to the nucleus through the Keap1/Nrf2 pathway.

Proteasome Inhibition Enhances Antioxidant and
Antiapoptosis Activity in SKOV3 Cells Compared with A2780 Cells. Nrf2 translocates to the nucleus to activate the transcription of ARE-containing genes [30], and thus, we determined the expression of genes in the Nrf2 pathway. To our surprise, the expression of NQO-1, GCLC, HO-1, SOD2, and G6PDH genes was all reduced after treatment with Epox for 12 h in A2780 cells, whereas the opposite results were obtained in SKOV3 cells (Figure 3(a)). To further determine the effect of proteasome inhibition on redox levels, we detected ROS levels via flow cytometry. The results revealed that Epox increased ROS levels in A2780 cells, whereas no significant changes were noted in SKOV3 cells (Figure 3(b)). In addition, Annexin V/PI staining revealed that the rate of apoptosis after exposure to Epox for 24 h was higher in A2780 cells than in SKOV3 cells (Figure 3(c)). This suggested that the increased expression  Figure 1: Proteasome inhibition can enhance the sensitivity of A2780 cells to cisplatin compared with SKOV3 cells. (a) A2780 and SKOV3 cells were treated with different concentrations of Epox for 24 h, and cell viability was determined using the MTT assay. Data are presented as the mean ± SD, n = 3, * P < 0:05, * * P < 0:01 compared with the respective controls. (b) A2780 and SKOV3 cells were treated with 100 nM Epox and different concentrations of cisplatin for 24 h, and cell viability was determined using the MTT assay. Data are presented as the mean ± SD (n = 3), * * P < 0:01, * * * P < 0:001 compared with the respective control, # P < 0:05 compared with the cisplatin-treated groups.
of Nrf2 in the nucleus did not necessarily exert antioxidant activity after proteasome inhibition; there may be regulatory factors regulating its transcriptional activity. It has been reported that PGC1α is also associated with the expression of mitochondrial ROS-detoxifying enzymes [31], and the activity of Nrf2 dose depends on PGC1α upon redox imbalance [17]. Therefore, we examined the gene expression of PGC1α and found that Epox can reduce its level in A2780 cells but increase its level in SKOV3 cells (Figure 3(d)). Notably, the expression of PGC1α was higher in SKOV3 cells than in A2780 cells (Figure 3(e)). These results indicated that compared with A2780 cells, proteasome inhibition enhances antioxidant and antiapoptosis activity in SKOV3 cells, and this may have resulted from the synergistic roles of Nrf2 and PGC1α.

PGC1α
Regulates the Antioxidant Activity of Nrf2 through GSK3β after Proteasome Inhibition in SKOV3 Cells. There is protein-protein interaction between PGC1α and Nrf2. In our experiment, proteasome inhibition increased     (Figure 4(a)). PGC1α could coactivate Nrf2 and assist the induction of antioxidant genes. So, we investigated whether PGC1α was involved in the regulation of Nrf2 antioxidant activity after proteasome inhibition in SKOV3 cells. RT-qPCR analyzed the expression of genes in the Nrf2 pathway, and the result illustrated that the increased levels of Nrf2 and antioxidant genes after Epox treatment were reversed by PGC1α silencing (Figures 4(b) and 4(c)). GSK3β interferes with Nrf2 transactivating activity, so we further investigated whether PGC1α regulated Nrf2 activity through GSK3β. Results from our Western blot analysis demonstrated that PGC1α silencing reduced the level of Ser9 phosphorylated GSK3β, whereas ZLN005 (a PGC1α transcription activator) enhanced its level (Figures 4(d) and 4(e)). When combined with GSK3β inhibitor (CHIR99021) in the presence of PGC1α silencing, the decreased levels of Nrf2 and antioxidant genes were reversed (Figure 4(f)). These results indicated that in SKOV3 cells, PGC1α regulates Nrf2 activity via GSK3β inactivation after proteasome inhibition. But whether GSK3β regulates Nrf2 directly or indirectly after Epox treatment and the specific mechanism of regulation needs to be further explored in future research.

Nrf2
Regulates PGC1α at the Transcriptional Level in SKOV3 Cells. The PGC1α promoter contains a conserved ARE sequence that can be bound by Nrf2, so we also determined whether Nrf2 directly binds to the PGC1α promoter     7 Oxidative Medicine and Cellular Longevity after proteasome inhibition in SKOV3 cells. We analyzed the PGC1α promoter sequence using the JASPAR database (http://jaspardev.genereg.net/) and predicted three potential Nrf2-binding fragments ( Figure 5(a)). To further confirm that Nrf2 regulates PGC1α promoter activity, we performed a dual-luciferase reporter assay. The PGC1α promoter fragment −2000/+1 bp was cloned in to the pGL4-basic vector and then cotransfected with a Renilla luciferase reporter plasmid into SKOV3 cells. We observed that both overexpression of Nrf2 or treatment with Epox markedly upregulated the transcriptional activity of PGC1α (Figures 5(b) and 5(c)). These data provide evidence that Nrf2 regulates PGC1α expression at the transcriptional level in SKOV3 cells.

Nrf2 Activation Enhances Mitochondrial Function in SKOV3
Cells. In addition to antioxidant properties, Nrf2 has an emerging role in mitochondrial function, and this may be through activating the transcription of PGC1α. To confirm this, we treated SKOV3 cells with Epox for 12 h or overexpressed Nrf2. JC-1 fluorescent staining was used to measure mitochondrial membrane potential (MMP, ΔΨm) (Figure 6(a)), and MitoTracker Red staining was used to measure mitochondrial mass (Figure 6(b)). The results indicated that Epox treatment or Nrf2 overexpression increased MMP and mitochondrial mass. ATP levels and mitochondrial DNA (mtDNA) copy numbers were both enhanced after treatment with Epox or Nrf2 overexpression (Figures 6(c) and 6(d)). Furthermore, we analyzed the effect of Nrf2 on the oxygen consumption rates (OCR) and found that exposure to Epox or overexpression of Nrf2 increased the OCR in SKOV3 cells (Figure 6(e)). To detect the effect of Nrf2 activation on the mitochondrial chain, we extracted mitochondria and used Western blotting to measure protein expression. As shown in Figure 6(f), Epox treatment or Nrf2 overexpression significantly increased the expression of complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (COXII), and complex V (ATP5A). However, mitochondrial-related functions were downregulated in A2780 cells (Supplementary Figure 1), further indicating that the increased nucleus expression of Nrf2 after treatment with Epox did not have activity in A2780 cells. In summary, we conclude that the activation of Nrf2 through Epox treatment or Nrf2 overexpression enhanced mitochondrial function in SKOV3 cells.

PGC1α Silencing Combined with Epox Treatment Promotes Apoptosis by Reducing Mitochondrial Function.
To further verify whether Nrf2/PGC1α signaling-mediated antioxidation and mitochondrial function play important roles in Epox resistance in SKOV3 cells, PGC1α was silenced via transient shRNA transfection. Decreased MMP is an early event of apoptosis. Flow cytometry was used to determine the effect of PGC1α silencing on MMP. The results demonstrated that PGC1α silencing reduced MMP, and the effect was further exacerbated by combined treatment with Epox (Figure 7(a)). Furthermore, Annexin V/PI staining revealed that PGC1α silencing increased the rate of apoptosis, which was further increased in cells treated with PGC1α shRNA . Relative NFE2l2 mRNA expression was measured by RT-qPCR. Data are presented as the mean ± SD, n = 3, * * P < 0:01 compared with NC group. (c) The luciferase activities of Epox-treated or Nrf2 overexpression SKOV3 cells transfected with pGL4-PGC1α and Renilla luciferase reporter (pRL-TK) plasmids were determined using dual-luciferase reporter assays. Data are presented as the mean ± SD, n = 3, * P < 0:05.

8
Oxidative Medicine and Cellular Longevity     (Figure 7(b)). In addition, we examined the expression of Bcl2 family members in SKOV3 cells after treatment with Epox and transfection with PGC1α shRNA. The Bcl2/Bax ratio was reduced by PGC1α silencing, and further decreases were observed in cells treated with both Epox and PGC1α shRNA. The expression of cleaved caspase 3 was also intensified in the combination treatment group (Figure 7(c)). What is more, the MTT assay demonstrated that PGC1α silencing increased the sensitivity of SKOV3 cells to Epox compared with the Scr-shRNA group (Figure 7(d)). These results suggested that PGC1α silencing combined with Epox treatment promotes apoptosis by reducing mitochondrial function.

Discussion
Chemotherapy resistance remains a barrier in the clinical treatment of ovarian cancer. When considering chemotherapy resistance, different aspects of drug resistance must be considered comprehensively [32]. Protein degradation and antioxidation are considered two factors of resistance to chemotherapy. Abnormally increased proteasomal activity has been observed in a variety of cancers, including breast cancer, colon cancer, and leukemia [6,7,33]. Kwak et al. demonstrated that antioxidants can induce the expression of multiple proteasome subunits in mouse fibroblasts [8]. Therefore, antioxidants are involved in the regulation of proteasomal activity, and they play important roles in tumorigenesis and development. In this study, we compared two ovarian cancer cells, A2780 and SKOV3. SKOV3 cell was chosen because it is platinum-resistant and possesses several key oncogenic characteristics, like epidermal growth factor receptor (EGFR) overexpression and p53 mutation [34,35]. In our study, compared with the findings in A2780 cells, SKOV3 cells were less sensitive to both proteasome inhibitors and cisplatin. When the proteasome was inhibited, the antioxidant capacity was decreased, and the sensitivity of A2780 cells to cisplatin was increased. The opposite results were observed in SKOV3 cells. This suggests that proteasomes and antioxidants may be involved in the differing sensitivity of A2780 and SKOV3 cells to cisplatin.
Proteasomes are closely involved in the regulation of multiple pathways within cells, and increased proteasomal activity is essential for tumor cell survival based on their ability to regulate tumor cell drug resistance through multiple pathways [7,36,37]. Inhibition of the proteasome can further     increase oxidative stress in tumor cells, thereby promoting tumor cell apoptosis. However, recent studies have found that not all tumor cells are sensitive to proteasome inhibitors. A study in multiple myeloma demonstrated that the Nrf2 signaling pathway may be involved in proteasome-mediated tumor resistance [38]. Nrf2 has seven Neh domains, and of these, Neh1 can combine with Maf protein to form a heterodimer and bind to AREs and regulate the expression of antioxidant genes such as HO-1 and NQO-1 after Nrf2 translocates to the nucleus [39,40]. In our study, although proteasome inhibition increased the nuclear expression of Nrf2 in both A2780 and SKOV3 cells, inhibiting the proteasome significantly reduced antioxidant levels and enhanced ROS production in A2780 cells. Conversely, the opposite results were obtained in SKOV3 cells. This suggested that the increased nuclear expression of Nrf2 did not necessarily exert antioxidant activity after proteasome inhibition; there may be regulatory factors regulating its transcriptional activity. Notably, PGC1α can regulate the expression of various antioxidant genes and play an important regulatory role in redox homeostasis [41]. Bruns et al. reported that PGC1α plays an important role in the clearance of ROS by regulating SOD1 and SOD2 in GBM glioblastoma cells [42]. In addition, a study using an aging disease model found that PGC1α can regulate Nrf2 expression and play a synergistic role in antioxidation [17]. Our study compared the expression of PGC1α in two cell lines and found that PGC1α expression was significantly higher in SKOV3 cells than in A2780 cells. After inhibiting proteasomes, PPARGC1A gene expression was increased in SKOV3 cells but decreased in A2780 cells, suggesting that PGC1α is also involved in proteasome inhibition-mediated antioxidant regulation. Thus, PGC1α and Nrf2 may jointly mediate the antioxidant regulation of proteasome inhibitor resistance in SKOV3 cells.
Recent studies revealed that PGC1α coactivate Nrf2, and there is a protein-protein interaction between PGC1α and Nrf2. Here, we deeply investigated the mutual regulation between PGC1α and Nrf2 in SKOV3 cells. First, we found that proteasome inhibition in SKOV3 cells can increase the colocalization of Nrf2 and PGC1α in the nucleus. Cherry et al. demonstrated that PGC1α was important for the activation of gene downstream of Nrf2 during sepsis [43]. In our study, the increased Nrf2 and antioxidant gene mRNA levels after Epox treatment were reversed by PGC1α silencing, which suggested that PGC1α was involved in the antioxidant regulation by Nrf2 during proteasome inhibition. Furthermore, Nrf2 activity is mediated by GSK3β-TrCP-dependent Cul1-based ubiquitin ligase. Choi et al. demonstrated that PGC1α can upregulate Nrf2 through the p38/GSK3β pathway to protect HK-2 cells against hydrogen peroxideinduced oxidative stress injury [44]. This is consistent with our findings that PGC1α can mediate Nrf2 activity through the inactivation of GSK3β in SKOV3 cells. Therefore, PGC1α can regulate the antioxidant activity of Nrf2 via GSK3β after proteasome inhibition. But whether GSK3β regulates Nrf2 directly or indirectly after Epox treatment and the specific mechanism of regulation needs to be further explored in future research.
Although Clark and Simon previously proposed that the PGC1α promoter region contains a conserved ARE sequence that can be bound by Nrf2, it is unclear whether a transcription factor and PGC1α can form a complex to regulate Nrf2 expression or whether Nrf2 can directly bind to the PGC1α promoter region [41,45]. After analyzing the PGC1α promoter sequence, three potential Nrf2-binding fragments were found. The dual-luciferase reporter assay found that Nrf2 upregulated the transcriptional activity of PGC1α after proteasome inhibition. The aforementioned data provided evidence that Nrf2 regulates PGC1α expression at the transcriptional level in SKOV3 cells. However, the specific molecular mechanism and promoter elements responsible for Nrf2 regulation need to be further explored. So far, we have Annexin V/PI staining was used to evaluate the apoptotic fraction, and (c) apoptosis protein expression was analyzed via Western blotting. Data are presented as the mean ± SD, n = 3, * P < 0:05, * * P < 0:01, * * * P < 0:001 compared with the control, # P < 0:05 compared with treatment with Epox+Scr-shRNA and PGC1α-shRNA. (d) SKOV3 cells were transfected with Scr-shRNA or PGC1α-shRNA plasmids and/or treated with 100 nM Epox for 24 h, and cell viability was determined using the MTT assay. Data are presented as the mean ± SD, n = 3, * P < 0:05, * * P < 0:01 compared with the control, # P < 0:05 compared with Epox+Scr-shRNA.
(e) Proposed model of the antioxidant activity regulated by Nrf2 and PGC1α after Epox treatment.