Silencing of NRF2 Reduces the Expression of ALDH1A1 and ALDH3A1 and Sensitizes to 5-FU in Pancreatic Cancer Cells

Pancreatic cancer remains an intractable cancer with a poor five-year survival rate, which requires new therapeutic modalities based on the biology of pancreatic oncogenesis. Nuclear factor E2 related factor-2 (NRF2), a key cytoprotective nuclear transcription factor, regulates antioxidant production, reduction, detoxification and drug efflux proteins. It also plays an essential role in cell homeostasis, cell proliferation and resistance to chemotherapy. We aimed to evaluate the possibility that modulation of NRF2 expression could be effective in the treatment of pancreatic cancer cells. We investigated whether the depletion of NRF2 by using small interfering RNAs (siRNAs) is effective in the expression of biomarkers of pancreatic cancer stemness such as aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1). NRF2 knockdown markedly reduced the expression of NRF2 and glutamate-cysteine ligase catalytic subunit (GCLC) in cell lines established from pancreatic cancers. NRF2 silencing also decreased the ALDH1A1 and ALDH3A1 expression. Furthermore, this NRF2 depletion enhanced the antiproliferative effects of the chemotherapeutic agent, 5-fluorouracil (5-FU) in pancreatic cancer cells.


Introduction
Pancreatic cancer is one of the most lethal solid malignancies and remains the fourth most common cause of cancer-associated mortality worldwide, with a 5-year survival rate of less than 5% [1,2]. The disease is highly aggressive, often discovered late and typically develops resistance to conventional treatment like chemo-and radiotherapy [3]. Therefore, a better understanding of the pathogenic mechanisms involved in the occurrence and progression of pancreatic cancer is required to devise more effective therapeutic strategies.
Cancer stem-like cells have been identified and characterized in various solid tumors, including pancreatic cancer [4][5][6]. Pancreatic cancer stem-like cells are defined as having high tumorigenicity, self-renewal and differentiation capacities [7,8], as well as involvement in chemoresistance along with aggressive behavior [9,10]. The aldehyde dehydrogenase (ALDH) activity is a hallmark of

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay
Pancreatic cancer cells (2000 cells per well) were plated into 96-well flat bottom plates and treated with two different concentrations (50 µM and 100 µM) of 5-FU for 72 h. After then, 25 µL of 1mg/mL MTT (Sigma, St. Louis, MO, USA) dissolved in PBS was added to each well followed by incubation for 4 h. Finally 150 µL of DMSO (Sigma, St. Louis, MO, USA) was added to each well to dissolve the formazan crystals. The absorbance (560 nm) was measured, and the data was analyzed using an ELx808 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The mean value and standard deviation (SD) were then determined.

Determination of Synergism
For the determination of synergism, classification index (CI) was calculated with the equation of (%A × %B)/(%AB × 100), where %A and %B are the percent cell viability of individual 5-FU treated or NRF2 siRNA transfected cell and %AB is the combination of them. Supra-additivity was defined as CI > 1; additivity was defines as CI = 1; and subadditivity was defined as CI < 1 [32,33].

Statistical Analysis
All data are expressed as mean ± SD of triplicate experiments. Statistical comparison was carried out using two-tailed student's t-test. The results were regarded statistically significant when the p value was lower than 0.05. In all experiments, * represents p < 0.05, ** represents p < 0.01 and *** represents p < 0.001.

NRF2 Knockdown Reduces the Expression of NRF2 and GCLC
To evaluate the important role of NRF2 in regulation of the expression of ALDH1A1 and ALDH3A1 in pancreatic cancer cells, we first knocked down the expression of NRF2 in pancreatic cancer AsPC-1, COLO-357 and PANC-1 cells using siRNA. The RT-PCR results at 48 h and 72 h post-transfection revealed that the NRF2 mRNA levels were reduced in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared with those in control-siRNA transfected cells ( Figure 1).

NRF2 Knockdown Reduces the Expression of NRF2 and GCLC
To evaluate the important role of NRF2 in regulation of the expression of ALDH1A1 and ALDH3A1 in pancreatic cancer cells, we first knocked down the expression of NRF2 in pancreatic cancer AsPC-1, COLO-357 and PANC-1 cells using siRNA. The RT-PCR results at 48 h and 72 h posttransfection revealed that the NRF2 mRNA levels were reduced in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared with those in control-siRNA transfected cells (Figure 1). Western blot analysis also showed that the expression of NRF2 was significantly reduced in NRF2-siRNA transfected AsPC-1 and COLO-357 cells, compared to those in control cells ( Figure 2). To further evaluate whether decreased levels of NRF2 downregulated the expression of genes in the NRF2 signaling pathway, we measured the mRNA levels of glutamate-cysteine ligase catalytic subunit (GCLC), one of NRF2 downstream target genes. RT-PCR results showed that the mRNA expression of GCLC were distinctly decreased in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared to those in control cells ( Figure 1). Western blot analysis also showed that the protein expression of GCLC was also significantly decreased in NRF2-siRNA transfected AsPC-1 and COLO-357 cells compared to those in control cells ( Figure 2). Western blot analysis also showed that the expression of NRF2 was significantly reduced in NRF2-siRNA transfected AsPC-1 and COLO-357 cells, compared to those in control cells ( Figure 2). Antioxidants 2017, 6, 52 4 of 10

NRF2 Knockdown Reduces the Expression of NRF2 and GCLC
To evaluate the important role of NRF2 in regulation of the expression of ALDH1A1 and ALDH3A1 in pancreatic cancer cells, we first knocked down the expression of NRF2 in pancreatic cancer AsPC-1, COLO-357 and PANC-1 cells using siRNA. The RT-PCR results at 48 h and 72 h posttransfection revealed that the NRF2 mRNA levels were reduced in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared with those in control-siRNA transfected cells ( Figure 1). Western blot analysis also showed that the expression of NRF2 was significantly reduced in NRF2-siRNA transfected AsPC-1 and COLO-357 cells, compared to those in control cells ( Figure 2). To further evaluate whether decreased levels of NRF2 downregulated the expression of genes in the NRF2 signaling pathway, we measured the mRNA levels of glutamate-cysteine ligase catalytic subunit (GCLC), one of NRF2 downstream target genes. RT-PCR results showed that the mRNA expression of GCLC were distinctly decreased in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared to those in control cells ( Figure 1). Western blot analysis also showed that the protein expression of GCLC was also significantly decreased in NRF2-siRNA transfected AsPC-1 and COLO-357 cells compared to those in control cells ( Figure 2). To further evaluate whether decreased levels of NRF2 downregulated the expression of genes in the NRF2 signaling pathway, we measured the mRNA levels of glutamate-cysteine ligase catalytic subunit (GCLC), one of NRF2 downstream target genes. RT-PCR results showed that the mRNA expression of GCLC were distinctly decreased in NRF2-siRNA transfected AsPC-1, COLO-357 and PANC-1 cells compared to those in control cells (Figure 1). Western blot analysis also showed that the protein expression of GCLC was also significantly decreased in NRF2-siRNA transfected AsPC-1 and COLO-357 cells compared to those in control cells (Figure 2).

NRF2 Knockdown Reduces the Expression of ALDH1A1 and ALDH3A1
To investigate whether silencing NRF2 reduced the expression of ALDH1A1 and ALDH3A1, we performed RT-PCR to determine the effect of NRF2 inhibition by siRNA on ALDH1A1 and ALDH3A1 expression in AsPC-1, COLO-357 and PANC-1 cells. The RT-PCR results demonstrated that the mRNA of ALDH1A1 and ALDH3A1 were significantly decreased in NRF2-siRNA transfected pancreatic cancer cells compared to those in control-siRNA transfected cells (Figure 3).

NRF2 Knockdown Reduces the Expression of ALDH1A1 and ALDH3A1
To investigate whether silencing NRF2 reduced the expression of ALDH1A1 and ALDH3A1, we performed RT-PCR to determine the effect of NRF2 inhibition by siRNA on ALDH1A1 and ALDH3A1 expression in AsPC-1, COLO-357 and PANC-1 cells. The RT-PCR results demonstrated that the mRNA of ALDH1A1 and ALDH3A1 were significantly decreased in NRF2-siRNA transfected pancreatic cancer cells compared to those in control-siRNA transfected cells (Figure 3). Western blot analysis also confirmed the effect of NRF2 knockdown on reducing the expression of ALDH1A1 and ALDH3A1. Compared to cells transfected with control-siRNA, cells transfected with NRF2-siRNA showed the distinct decrease in the protein expression of ALDH1A1 and ALDH3A1 in AsPC-1 cells (Figure 4), suggesting that NRF2 knockdown suppresses the expression of ALDH1A1 and ALDH3A1. The NRF2 dependent expression of ALDH1A1 and ALDH3A1 was in accord with our previous experiment of cDNA microarray study after knockdown or induction of NRF2 in AsPC-1 cell line [34].  Western blot analysis also confirmed the effect of NRF2 knockdown on reducing the expression of ALDH1A1 and ALDH3A1. Compared to cells transfected with control-siRNA, cells transfected with NRF2-siRNA showed the distinct decrease in the protein expression of ALDH1A1 and ALDH3A1 in AsPC-1 cells (Figure 4), suggesting that NRF2 knockdown suppresses the expression of ALDH1A1 and ALDH3A1. The NRF2 dependent expression of ALDH1A1 and ALDH3A1 was in accord with our previous experiment of cDNA microarray study after knockdown or induction of NRF2 in AsPC-1 cell line [34].

NRF2 Knockdown Reduces the Expression of ALDH1A1 and ALDH3A1
To investigate whether silencing NRF2 reduced the expression of ALDH1A1 and ALDH3A1, we performed RT-PCR to determine the effect of NRF2 inhibition by siRNA on ALDH1A1 and ALDH3A1 expression in AsPC-1, COLO-357 and PANC-1 cells. The RT-PCR results demonstrated that the mRNA of ALDH1A1 and ALDH3A1 were significantly decreased in NRF2-siRNA transfected pancreatic cancer cells compared to those in control-siRNA transfected cells (Figure 3). Western blot analysis also confirmed the effect of NRF2 knockdown on reducing the expression of ALDH1A1 and ALDH3A1. Compared to cells transfected with control-siRNA, cells transfected with NRF2-siRNA showed the distinct decrease in the protein expression of ALDH1A1 and ALDH3A1 in AsPC-1 cells (Figure 4), suggesting that NRF2 knockdown suppresses the expression of ALDH1A1 and ALDH3A1. The NRF2 dependent expression of ALDH1A1 and ALDH3A1 was in accord with our previous experiment of cDNA microarray study after knockdown or induction of NRF2 in AsPC-1 cell line [34]. Since the highly expressed NRF2 levels potentiated the resistance to chemotherapeutic agents in pancreatic cancer cells, we then investigated the role of NRF2 in determination of the sensitivity of AsPC-1, COLO-357 and PANC-1 cells to the chemotherapeutic agents 5-fluorouracil (5-FU). NRF2depleted or control cells at 48 h post-transfection were treated with two different concentrations of 5-FU (0, 50 or 100 µM) for 72 h. The results of MTT assay revealed that the depletion of NRF2 by siRNA significantly enhanced the sensitivity of pancreatic cancer cells to 5-FU ( Figure 5). Due to the limited numbers of combination the calculation of classification index (CI) was chosen to assess synergistic effect of combination rather than combination index [32,33]. The calculated CI of 5-FU and NRF2 knockdown combination revealed that AsPC-1 and COLO-357 cell lines showed supra-additivity with mean CI values 1.35, 1.23 (50 µM, 100 µM 5-FU each in AsPC-1 cell line); 1.49, 1.55 (50 µM, 100 Since the highly expressed NRF2 levels potentiated the resistance to chemotherapeutic agents in pancreatic cancer cells, we then investigated the role of NRF2 in determination of the sensitivity of AsPC-1, COLO-357 and PANC-1 cells to the chemotherapeutic agents 5-fluorouracil (5-FU). NRF2-depleted or control cells at 48 h post-transfection were treated with two different concentrations of 5-FU (0, 50 or 100 µM) for 72 h. The results of MTT assay revealed that the depletion of NRF2 by siRNA significantly enhanced the sensitivity of pancreatic cancer cells to 5-FU ( Figure 5). Due to the limited numbers of combination the calculation of classification index (CI) was chosen to assess synergistic effect of combination rather than combination index [32,33]. The calculated CI of 5-FU and NRF2 knockdown combination revealed that AsPC-1 and COLO-357 cell lines showed supra-additivity with mean CI values 1. 35  All together, these results suggest that inhibition of NRF2 is related with the enhancement of the sensitivity of these cells to chemotherapeutic agents and the expression of cancer cell stemness biomarker as ALDH1A1 and ALDH3A1.

Discussion
Over the last decade, numerous studies have shaped the hierarchical theory to explain the intratumoral heterogeneity observed in many tumors including pancreatic cancer [4,[7][8][9]. This theory argues that a tumor comprises cells of not just a single clone, but multiple subclones with distinct cell morphologies, gene expression profiles, cell growth kinetics and cell surface markers. Among these heterogeneous subclones, a minor one generally comprising less than 5% of a tumor is termed the cancer stem cell. It has a potential for unlimited self-renewal with the capacity to provide committed progeny constituting the volume of a tumor through asymmetric cell division. This feature naturally renders cells of this specific subclone responsible for cancer initiation, progression, invasion, metastasis and resistance to various modes of therapy [7][8][9][10]. In pancreatic cancer, investigation of cancer stem cell lineages enabled the identification of cell membrane markers such as CD44, CD24, epithelial specific antigen (ESA) and CD133 and the expression pattern of ALDH1A1 as an important biomarker of pancreatic cancer stem cells [8,35]. Considering the poor outcomes from conventional treatments currently available for pancreatic cancer, more effective therapy needs immediate development, and pancreatic cancer stem cells can be a highly desirable target worthy of investigation. Related to this study, our recent study has demonstrated that dasatinib enhances gemcitabine-induced decreased ALDH1A1 expression and increased cell death of MIA PaCa-2 pancreatic cancer cells with acquired resistance to gemcitabine [17].
The biochemical role of NRF2 and its representative downstream proteins are: (1) NADPH production for reduction and monooxygenation: glucose-6 phosphate dehydrogenase (G6PD), a ratelimiting step enzyme of NADPH production, 6-phosphogluconate dehydrogenase (6PGD) and malic enzyme (ME); (2) glutathione dependent superoxide removal system: GCLC, several kinds of superoxide dismutases (SODs), glutathione reductase (GSR) and the family of thioredoxin reductases (TXNRDs); (3) drug conjugating or oxidizing system: several kinds of glutathione s-transferases (GSTs), cytochrome p450 family enzymes (CYPs), NQO-1 an oxidized mitochondrial quinone detoxifying enzyme, aldo-keto reductase family enzymes (AKRs) and ALDHs; and (4) drug efflux system: the family of multidrug resistant proteins (MDRs) and ABC transporters [19,36,37]. In addition to catalyzing the endogenous substrate, the substrate cross-reactivities of these proteins enable them to turn over xenobiotics to eliminate them from the body, or to produce metabolic products with unidentified function. All together, these results suggest that inhibition of NRF2 is related with the enhancement of the sensitivity of these cells to chemotherapeutic agents and the expression of cancer cell stemness biomarker as ALDH1A1 and ALDH3A1.

Discussion
Over the last decade, numerous studies have shaped the hierarchical theory to explain the intra-tumoral heterogeneity observed in many tumors including pancreatic cancer [4,[7][8][9]. This theory argues that a tumor comprises cells of not just a single clone, but multiple subclones with distinct cell morphologies, gene expression profiles, cell growth kinetics and cell surface markers. Among these heterogeneous subclones, a minor one generally comprising less than 5% of a tumor is termed the cancer stem cell. It has a potential for unlimited self-renewal with the capacity to provide committed progeny constituting the volume of a tumor through asymmetric cell division. This feature naturally renders cells of this specific subclone responsible for cancer initiation, progression, invasion, metastasis and resistance to various modes of therapy [7][8][9][10]. In pancreatic cancer, investigation of cancer stem cell lineages enabled the identification of cell membrane markers such as CD44, CD24, epithelial specific antigen (ESA) and CD133 and the expression pattern of ALDH1A1 as an important biomarker of pancreatic cancer stem cells [8,35]. Considering the poor outcomes from conventional treatments currently available for pancreatic cancer, more effective therapy needs immediate development, and pancreatic cancer stem cells can be a highly desirable target worthy of investigation. Related to this study, our recent study has demonstrated that dasatinib enhances gemcitabine-induced decreased ALDH1A1 expression and increased cell death of MIA PaCa-2 pancreatic cancer cells with acquired resistance to gemcitabine [17].
The biochemical role of NRF2 and its representative downstream proteins are: (1) NADPH production for reduction and monooxygenation: glucose-6 phosphate dehydrogenase (G6PD), a rate-limiting step enzyme of NADPH production, 6-phosphogluconate dehydrogenase (6PGD) and malic enzyme (ME); (2) glutathione dependent superoxide removal system: GCLC, several kinds of superoxide dismutases (SODs), glutathione reductase (GSR) and the family of thioredoxin reductases (TXNRDs); (3) drug conjugating or oxidizing system: several kinds of glutathione s-transferases (GSTs), cytochrome p450 family enzymes (CYPs), NQO-1 an oxidized mitochondrial quinone detoxifying enzyme, aldo-keto reductase family enzymes (AKRs) and ALDHs; and (4) drug efflux system: the family of multidrug resistant proteins (MDRs) and ABC transporters [19,36,37]. In addition to catalyzing the endogenous substrate, the substrate cross-reactivities of these proteins enable them to turn over xenobiotics to eliminate them from the body, or to produce metabolic products with unidentified function.
NRF2 protects cells from oxidative stress by upregulating the expression of various kinds of genes involved in cytoprotection and detoxification [18,19]. The protective role of NRF2 from oxidants and electrophiles is essential in various malignant cancers, where high expression of NRF2 can promote cancer cell viability, growth and resistance to conventional chemotherapy and radiotherapy [23,24,26,27]. NRF2 has also been shown to be an essential factor of differentiation and self-renewal capability of glioma stem cells [28]. Silencing NRF2 decreases the self-renewal capacity of glioma stem cells by critically reducing the expression of B lymphoma Mo-MLV insertion region 1 homolog (Bmi1, a transcription regulation factor for stem cells), SRY-box 2 (Sox2, a regulator of growth factors) and Cyclin E [28]. NRF2 also regulates hematopoietic stem cell survival [38,39]. In contrast, inhibition of NRF2-dependent antioxidant response by siRNA in various cancers, including pancreatic cancer, can lead to increased sensitivity to gemcitabine, 5-FU, cisplatin, camptothecin, etoposide and doxorubicin [23,24,31]. Consistent with previous studies, this study also revealed that NRF2 inhibition by siRNA enhanced the sensitivity of pancreatic cancer AsPC-1, COLO-357 and PANC-1 cells to 5-FU. These observations support that decreasing NRF2-dependent protective response can promote the sensitivity of cancer cells including pancreatic cancer to anticancer therapeutic agents. The degradation of 5-FU is initiated by the reduction to dihydrofluorouracil by dihydropyrimidine dehydrogenase, a rate-liming NADPH dependent enzyme [40]. The 5-FU treatment increases intracellular reactive oxygen species (ROS) and the removal of them by antioxidants rescues cancer cells from apoptotic death [41]. We also previously reported that the synergistic effect of chk2 inhibitor with gemcitabine revealed increased production of ROS and the ROS scavenger treatment reversed apoptotic cell death [42]. This protective effect of NRF2 arises from the effectiveness of removal of ROS under the treatment of cytotoxic drugs and possibly the increased viability of cells due to the NRF2 related stemness property [43].
Although the mechanism or direct temporal cause-result relationship between chemoresistance and the expression of ALDH1A1 and ALDH3A1 was not addressed in this experiment, the role of NRF2 in pancreatic cancer cell survival is evident by modulating the expression of these drug metabolizing enzymes including possibly ALDH1A1, ALDH3A1 and other potent downstream target of NRF2 [37,44]. The pharmacological inhibition of ALDH1A1 resulted in decreased formation of sphere-like colonies along with down regulation of cancer stem cell markers in head and neck squamous cell carcinoma cell lines [45]. In addition to ALDH1A1 and ALDH3A1, ALDH7A1 is also known as an ALDH family member of NRF2 downstream targets. ALDH7A1 is functionally involved with stemness and metastatic activity of prostate cancer and recurrence of non-small cell lung cancer [46,47]. Beyond to the simple enzymatic function of detoxification as aldehyde turn over to carboxylic acid, ALDHs have numerous non-enzymatic functions. The production of retinoic acids, γ-amino butyric acid and other unidentified carboxylic acid products may participate various cellular processes including cell proliferation, differentiation and survival [12]. Interestingly, in our previous study, siRNA-mediated ALDH1A1 knockdown inhibited cell proliferation in pancreatic cancer MIA PaCa-2 cells [10]. Moreover, the treatment of ALDH1A1-siRNA perturbed gemcitabine resistance, resulting in decreased cell viability, increased apoptotic cell death and the accumulation of cells at S-phase [10].
The effective modulation of specific NRF2 downstream effectors could be a therapeutic target for the development of novel therapy for pancreatic cancer. Further detailed study, such as the functional assay of sphere formation under the knockdown of ALDH1A1 or ALDH3A1, is needed to define the molecular mechanisms involved in the function of NRF2 and its effectors, including members of ALDHs in pancreatic cancer cells in relevance with drug resistance and cancer cell stemness.