VPA and MEL induce apoptosis by inhibiting the Nrf2-ARE signaling pathway in TMZ-resistant U251 cells
- Authors:
- Published online on: May 25, 2017 https://doi.org/10.3892/mmr.2017.6621
- Pages: 908-914
Abstract
Introduction
Glioma accounts for 30 to 40% of all intracranial tumors. Glioblastoma, which is the most lethal primary brain tumor, is the most common malignant glioma (1,2). Treatment strategies include combinations of aggressive resection, radiotherapy and temozolomide (TMZ) treatment and various novel chemotherapeutics. However, the median survival time of patients remains poor (3). Chemoresistant glioma cell subgroups are the most common cause of recurrence.
Our previous study determined that the expression of Nf-E2 related factor 2 (Nrf2), a traditional cytoprotective transcription factor, was elevated in glioma (4–8). Increased expression and continuous activation of Nrf2 in glioma may contribute to proliferation, invasion and chemoresistance of cancer cells. A previous study identified increased expression levels of Nrf2 and its target proteins in doxorubicin-resistant BEL-7402 hepatocellular carcinoma cells compared with parent cells (9). Chemoresistance was reduced in doxorubicin-resistant BEL-7402 cells following suppression of Nrf2 expression by chrysin, a potent Nrf2 inhibitor. The present study investigated the Nrf2-antioxidant response element (ARE) signaling pathway in TMZ-resistant U251. Additionally, the effects of valproic acid (VPA) and melatonin (MEL) on Nrf2 expression levels and chemoresistance in U251-TMZ cells were investigated. The present study determined that increased expression of components of the Nrf2-ARE signaling pathway in U251-TMZ cells was reversed by VPA and MEL treatment, which act as Nrf2 inhibitors. Additionally, U251-TMZ cells restored chemosensitivity to TMZ following co-treatment with VPA or MEL and exhibited increased levels of reactive oxygen species (ROS) and apoptosis.
Materials and methods
Cell culture and TMZ resistance
Human U251 glioblastoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Wisent Biotechnology Co., Ltd., Nanjing, China). The TMZ-resistant U251 cell line (termed U251-TMZ) was established by sustained low concentration TMZ (cat. no. T2577; Sigma; Merck KGaA, Darmstadt, Germany) stimulation of U251 cells, at an initial dose of 0.25 µM for 2 weeks. Then, the concentration of TMZ was doubled every 2–4 weeks. Following this stepwise increase in TMZ concentration for ~8 months, the final U251-TMZ cell line was able to survive in 100 µM TMZ. Subsequently, Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to evaluate the survival of U251-TMZ cells and the parent U251 cell line following treatment with TMZ.
Transient transfection with small interfering (si)RNA
Nrf2 siRNA (si-Nrf2) (target sequence, 5′GCA GTTCAATGAAGCTCAACT3′) and scramble siRNA (si-con; sequence, 5′GAGUACGAUCCGAGUUGAG3′) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). U251-TMZ cells (1×106/well) were seeded in 6-well plates and transfected with 2 µl Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 pmol si-Nrf2 or si-con, and 2 ml reduced serum Opti-Minimal Essential Medium (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells treated with Lipofectamine 2000 only served as the blank control group. The success of the transfection was confirmed by western blot analysis 48 h after transfection. All further experiments using U251-TMZ/si-Nrf2 cells were performed within 48 h of the transfection.
CCK-8 assay
Cells (100 µl) were seeded in 96-well culture plates at a density of 2×103 cells/well and cultured for 24 h. TMZ at 0, 12.5, 25, 50, 100, 200 and 400 µM was used to check the chemoresistance of U251-TMZ. Treatment with 200 µM TMZ + VPA at 0.5, 1, 2 and 5 mM or MEL at 0.1, 0.5, 1 and 2 mM were used to check the effect of VPA and MEL on U251-TMZ chemosensitivity. Viable cells were quantified at 0, 12, 24 and 48 h following treatment, using a CCK-8 according to the manufacturer's protocol. Briefly, 10 µl CCK-8 solution was added into every well and incubated at 37°C for 1 h. Subsequently the optical density of each well was measured at a wavelength of 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All measurements were performed six times. Data are presented as the mean ± standard deviation (SD).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated with TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) and single-stranded cDNA was synthesized from 2 ug of total RNA with the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocol. The thermocycling conditions were as follows: Denaturation for 10 min at 65°C, cDNA synthesis for 1 h at 50°C and inactivation for 5 min at 85°C. The cDNA was stored at −20°C. Quantitative analysis of Nrf2 mRNA expression levels was performed using SYBR® Green PCR Core reagents (Thermo Fisher Scientific, Inc.) and an ABI 7700 Real-time system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The primer sequences were as follows: Forward, 5′-TTCCCGGTCACATCGAGAG-3′ and reverse, 5′-TCCTGTTGCATACCGTCTAAATC-3′ for Nrf2; and forward, 5′-CATCTTCTTTTGCGTCGCCA-3′ and reverse, 5′-TTAAAAGCAGCCCTGGTGACC-3′ for GAPDH. Each amplification cycle consisted of denaturation for 15 sec at 95°C, annealing for 20 sec at 60°C and extension for 40 sec at 72°C; a total of 40 cycles were performed. A melting curve was used to distinguish specific from non-specific products and primer-dimers. Each sample was analyzed in triplicate, and expression of Nrf2 was normalized to GAPDH using the 2−∆∆Cq method (10). Alterations in Nrf2 expression were reported as fold increases (2−∆∆Cq) relative to the control group.
Protein preparation
Cytoplasmic and nuclear proteins were obtained using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, cells were treated with cytoplasmic protein extraction reagent. Lysates were subsequently incubated for 10 min on ice and centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was collected as the cytoplasmic protein. Pellets containing crude nuclei were resuspended in nuclear protein extraction regent and agitated for 2 h on ice, followed by centrifugation at 12,000 × g for 10 min at 4°C to obtain supernatants containing nuclear protein. To obtain total protein lysate, cells were harvested and homogenized using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing 1 mM phenylmethylsulfonyl fluoride, and centrifuged at 12,000 × g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein assay reagent (Pierce; Thermo Fisher Scientific, Inc.).
Western blot analysis
Protein extracts (50 mg), including cytoplasmic and nuclear protein for Nrf2 detection, nuclear protein for H3 detection and total protein for detecting all other proteins, were heat denatured in loading buffer, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. Membranes were blocked with 5% bovine serum albumin (BSA; cat. no. B2064; Sigma; Merck KGaA) for 2 h at room temperature. The following primary antibodies were used: Rabbit anti-Nrf2 (68 kDa; 1:500; cat. no. sc-722; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-histone H3 (H3; 17 kDa; 1:2,000; cat. no. 9715; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-β-actin (43 kDa; 1:10,000; cat. no. AP0060; Bioworld Technology, Inc., St. Louis Park, MN, USA), rabbit anti-HO-1 (32 kDa; 1:1,000; cat. no. ab13243; Abcam, Cambridge, UK), rabbit anti-NQO1 (31 kDa; 1:1,000; cat. no. ab34173; Abcam), rabbit anti-cleaved caspase-3 (17 kDa; 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), rabbit anti-B-cell lymphoma 2 (Bcl-2; 26 kDa; 1:1,000; cat. no. sc-492; Santa Cruz Biotechnology, Inc.), mouse anti-Bcl-2-associated X protein (Bax; 1:500; 23 kDa; cat. no. sc-20067; Santa Cruz Biotechnology, Inc.), mouse anti-phosphorylated (p)-insulin like growth factor 1 receptor (IGF-IR; 97 kDa; 1:500; cat. no. sc-81499; Santa Cruz Biotechnology, Inc.), mouse anti-IGF-IR (97 kDa; 1:500; cat. no. sc-81167; Santa Cruz Biotechnology, Inc.), rabbit anti-protein kinase B (AKT; 60 kDa; 1:1,000; cat. no. 9272; Cell Signaling Technology, Inc.), rabbit anti-p-AKT (60 kDa; 1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), rabbit anti-mammalian target of rapamycin (mTOR; 289 kDa; 1:1,000; cat. no. 2983; Cell Signaling Technology, Inc.), rabbit anti-p-mTOR (289 kDa; 1:500; cat. no. 5536; Cell Signaling Technology, Inc.) and rabbit anti-P2X purinoceptor 7 (P2RX7; 75 kDa; 1:1,000; cat. no. ab109246; Abcam). Each primary antibody was diluted appropriately in 5% BSA and incubated overnight at 4°C. The blots were washed three times in washing buffer and incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP; cat. no. BS13278; 1:10,000; Bioworld Technology, Inc.) or goat anti-mouse IgG-HRP (cat. no. BS12478; 1:10,000; Bioworld Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using an Enhanced Chemiluminescence Detection system (EMD Millipore, Billerica, MA, USA) and exposed by Tanon 5200 Chemiluminescence Image Analytical system (TanonScience and Technology Co., Ltd., Shanghai, China). β-actin served as a loading control for total or cytoplasmic protein; H3 served as a loading control for nuclear protein. Quantification of band density was performed using UN-SCAN-IT gel digitizing software version 6.1 (Silk Scientific, Inc., Orem, UT, USA) and data were normalized to β-actin or H3.
Flow cytometric analysis of apoptosis
Apoptosis was quantified using an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. The cells were washed once with incubation buffer following the aforementioned treatments, collected by centrifugation at 1,000 × g for 5 min at room temperature, and resuspended in an Annexin V-FITC/propidium iodide (PI) reactive solution. Following a 15 min incubation at room temperature, the percentage of apoptotic cells was quantified using a flow cytometer (Cytomics FC500; Beckman Coulter, Inc., Brea, CA, USA) and analyzed using CellQuest Pro software version 3.3 (Mac OS X.1; BD Biosciences, San Jose, CA, USA).
ROS detection with 2′,7′-dichlorofluorescein diacetate (DCFH-DA)
Intracellular ROS levels were quantified using the fluorescent probe DCFH-DA (cat. no. S0033; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. After 24 h treatment, cells were incubated with DCFH-DA in Opti-Minimal Essential Medium (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 37°C. Subsequently, cells were washed with PBS to remove excess dye prior to quantification using the microplate reader Fluoroscan Ascent (Thermo Fisher Scientific, Inc.) with excitation and emission wavelengths of 485and 538 nm, respectively.
Statistical analysis
Data are expressed as the mean ± SD and evaluated using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA).
Results
Establishment of the TMZ-resistant U251 cell line and protein expression levels of Nrf2-ARE signaling pathway components
The U251-TMZ cell line was established following exposure of U251 cells to continuously increasing concentrations of TMZ. CCK-8 results indicated that the survival rate was greater in in U251-TMZ cells treated with TMZ, compared with parent U251 cells (Fig. 1A).
Results from RT-qPCR (Fig. 1B) and western blotting (Fig. 1C and D) indicated that the expression levels of Nrf2 and its downstream effectors, including HO-1 and NQO1, were significantly increased in U251-TMZ cells when compared with parent U251 cells.
Silencing Nrf2 in U251-TMZ cells restores chemosensitivity to TMZ
The effect of Nrf2 silencing was determined 48 h after siRNA transfection using western blotting. si-Nrf2 successfully reduced Nrf2 protein expression levels in U251-TMZ cells (Fig. 1E and F). Protein expression levels of HO-1 and NQO1, downstream effectors of the Nrf2-ARE signaling pathway, were additionally decreased (Fig. 1E and F).
CCK-8 results indicated that the cell survival rate in the U251-TMZ si-Nrf2 group was significantly reduced when compared with the U251-TMZ si-con and blank control groups, almost to the level of parent U251 cells (Fig. 1A).
VPA or MEL treatment restores chemosensitivity of U251-TMZ cells
VPA and MEL have been identified as potent chemotherapeutic sensitizers in various tumor cell lines (11–14). The present study investigated whether these drugs may restore the chemosensitivity of U251-TMZ cells to TMZ. The results of the CCK-8 assay demonstrated that when used in combination with 200 µM TMZ, treatment with 2 or 5 mM VPA for 24 or 48 h significantly reduced the survival rate of U251-TMZ cells, when compared with TMZ treatment alone (Fig. 2A). Additionally, when U251-TMZ cells were treated with TMZ plus 1 or 2 mM MEL for 24 or 48 h, the survival rate was significantly reduced when compared with TMZ treatment alone (Fig. 2B).
VPA and MEL treatment increases ROS levels and apoptosis induced by TMZ in U251-TMZ cells
Apoptosis was investigated to determine the molecular mechanism underlying the inhibitory effect of 2 mM VPA or 1 mM MEL on 200 µM TMZ-treated U251-TMZ cells for 24 h (Fig. 3A). The rate of apoptosis in Annexin V + PI positive cells was increased in TMZ-treated U251-TMZ cells co-treated with VPA or MEL, when compared with blank control (untreated U251-TMZ cells) or TMZ only-treated cells (Fig. 3B). Following co-treatment with TMZ and VPA/MEL, the protein expression levels of cleaved caspase-3 and Bax were increased and those of Bcl-2 were reduced, as demonstrated by western blotting (Fig. 3D-F). Additionally, ROS levels were increased in U251-TMZ cells following co-treatment with TMZ and VPA or MEL, compared with the TMZ treatment alone or blank control groups (Fig. 3C).
VPA and MEL treatment may reduce Nrf2 expression via inhibition of the IGF-IR/AKT/mTOR signaling pathway
VPA and MEL are effective chemotherapeutic sensitizers in glioma (15–17). However, this effect and its association with Nrf2 have not been previously investigated in TMZ-resistant glioma cells. The present study determined that co-treatment of 2 mM VPA or 1 mM MEL with 200 µM TMZ for 24 h significantly reduced Nrf2 protein expression levels in U251-TMZ cell lines, when compared with cells treated with TMZ only (Fig. 4A, C and D). In addition, the protein expression levels of HO-1 and NQO1, downstream effectors of the Nrf2-ARE signaling pathway, which contribute to ROS clearance, were reduced following TMZ treatment in combination with VPA or MEL, when compared with cells treated with TMZ only (Fig. 4A, C and D). Inhibition of the IGF-IR/AKT/mTOR signaling pathway by VPA or MEL treatment was determined using western blotting (Fig. 4B, E and F). Furthermore, protein expression levels of P2RX7, the receptor associated with ROS generation, were increased following TMZ treatment with VPA or MEL, compared with TMZ treatment only (Fig. 4B, E and F).
Discussion
Chemoresistance reduces the chances of survival in patients with tumors, particularly in recurrent tumors. The present study investigated the possible mechanism underlying glioma chemoresistance. The findings of the current study indicated that expression levels of components of the Nrf2-ARE signaling pathway were increased in TMZ-resistant U251 cells compared with parent U251 cells. Suppressing Nrf2 expression using siRNA or the Nrf2 inhibitors VPA or MEL, restored the chemosensitivity of U251-TMZ cells to TMZ and increased the rate of apoptosis and ROS levels. Additionally, the results of the present study suggested that VPA and MEL treatment may reduce the expression levels of Nrf2-ARE signaling pathway proteins via inhibition of the IGF-IR/AKT/mTOR signaling pathway, and increase ROS levels via activation of P2RX7.
Nrf2 was initially identified as a critical transcription factor in the Kelch-like ECH-associated protein1 (Keap1)-Nrf2-ARE signaling pathway, which functions to resist oxidative stress. Under normal conditions, low constitutive quantities of Nrf2 protein are maintained by the Keap1-mediated ubiquitination and proteasomal degradation system (18). Nrf2 is activated and dissociates from Keap1 in response to oxidative stress. Following translocation from the cytoplasm to the nucleus, Nrf2 forms a heterodimer with MAF bZIP transcription factor, which binds to the ARE sequence to increase the expression of various cytoprotective genes, such as HO-1 and NQO1 (19). However, the cell protective effect of Nrf2 may additionally be present in tumor cells. Overexpression of Nrf2 accompanied by chemoresistance has been identified in non-small-cell lung cancer, breast adenocarcinoma, endometrial serous carcinoma and neuroblastoma cells (20–22). Additionally, RNA interference-mediated downregulation of Nrf2 expression in lung cancer cells induced generation of ROS and resulted in increased sensitivity to chemotherapeutic drugs in vitro and in vivo (23). The findings of the present study indicated increased Nrf2 expression in TMZ-resistant U251 cells compared with parent U251 cells. The protein expression levels of HO-1 and NQO1, Nrf2 targeted cytoprotective and antioxidant genes, were additionally significantly increased in U251-TMZ cells. HO-1 and NQO1 contribute to ROS clearance (24–26), which may be an important mechanism underlying TMZ-resistance. Silencing Nrf2 expression in U251-TMZ cells through siRNA transfection increased chemosensitivity to TMZ. The protein expression levels of HO-1 and NQO1 in U251-TMZ cells were reduced following si-Nrf2 transfection.
A recent study identified that VPA, a traditional antiepileptic drug, has chemotherapeutic effects (16). A meta-analysis revealed that patients with glioblastoma may experience prolonged survival following VPA administration. In addition, the benefit of VPA treatment was confirmed in sub-group analysis compared with non-anti-epileptic drug (AED) and other AED groups. These findings indicated the benefits of TMZ and VPA combined treatment in patients with glioblastoma. Previous studies have determined that VPA may inhibit glioma cell proliferation in vitro and in vivo by increasing apoptosis and inducing cell cycle arrest (27–29). The molecular mechanism underlying the effects of VPA in glioma treatment may in part rely on inhibition of histone deacetylase, downregulation of O-6-methylguanine-DNA methyltransferase, or activation of mitogen-activated protein kinases or reversion inducing cysteine rich protein with kazal motifs-matrix metalloproteinase pathways and redox regulation. However, the association between VPA and Nrf2 expression in glioma remains to be fully elucidated. The present study investigated the effect of VPA on Nrf2 expression in U251-TMZ cells. The findings of the current study demonstrated that VPA successfully downregulated the protein expression levels of Nrf2-ARE signaling pathway effectors in U251-TMZ cells. The suppression of the Nrf2-ARE signaling pathway may lead to an imbalanced redox equilibrium in the cell, which may result in elevation of ROS levels and induction of apoptosis. Therefore, inhibition of the Nrf2-ARE signaling pathway may contribute to the molecular mechanism underlying the effect of VPA treatment on glioma cells.
MEL, an indoleamine hormone produced by the pineal gland and a typical antioxidant, exerts antitumor activity in a wide range of neoplasms in vitro and in vivo (30). Martin et al (31) initially determined that MEL inhibited C6 glioma cell proliferation and induced cell cycle arrest in vitro and in vivo. Similar findings were published subsequently. Pharmacological concentrations of MEL (1 mM) may inhibit the ras oncogene at 85 D/protein kinase C/AKT/nuclear factor-κB signaling pathway, which may reduce local biosynthesis of estrogen and expression of the ABC transporter ATP binding cassette subfamily G member 2 (32–35). These mechanisms may participate in MEL inhibition of gliomas. However, the association between the pharmacological concentration of MEL and the Nrf2-ARE signaling pathway in glioma cells remains to be fully elucidated. A previous study suggested that MEL at low concentrations (up to 100 nM) may activate the Nrf2-ARE signaling pathway in C6 glioma cells (36). Additionally, MEL has been revealed to activate the Nrf2-ARE signaling pathway in various neurological degeneration diseases and traumatic brain injuries (37,38). However, the findings of the present study indicated that 1 mM MEL significantly reduced Nrf2 expression and that of downstream effectors, including NQO1 and HO-1, when combined with TMZ treatment in U251-TMZ cells. The differences in these findings may be due to the different concentrations and cell types used.
Expression of Nrf2 is typically regulated by the Keap1-Nrf2-ARE signaling pathway. However, previous studies identified a role for the AKT/mTOR signaling pathway in the regulation of Nrf2 expression. Phosphorylation of AKT has been demonstrated to induce activation of Nrf2 and upregulate the expression of HO-1 and NQO1. Nrf2 activators, including S-allyl cysteine, berberine, oltipraz and sulforaphane, may induce the activation of the AKT signaling pathway, thus influencing the Nrf2-ARE signaling pathway (39–42). The present study demonstrated that VPA and MEL inhibited the phosphorylation of the IGF-IR/AKT/mTOR signaling pathway and inhibited the Nrf2-ARE signaling pathway. Suppression of Nrf2-ARE may cause an imbalance of the redox equilibrium, leading to increased ROS levels and apoptosis following TMZ treatment. The inhibitory effect of VPA and MEL on Nrf2 expression partly restored chemosensitivity of U251-TMZ cells. To the best of our knowledge, the present study is the first to identify that VPA and MEL maybe potential therapeutic agents for the treatment of chemoresistant glioblastoma. Further investigations are required to confirm this effect of VPA and MEL, including in vivo experiments and preclinical studies. Additionally, potential side effects due to high doses of VPA or MEL should be considered.
Acknowledgements
The present study was supported by the National Natural Science Foundation of China (grant no. 81402072), the Natural Science Foundation of Jiangsu Province (grant no. BK20140732) and the China Postdoctoral Science Foundation (grant no. 2015M572716).
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