Background
Glioma is a serious life-threatening disease, having the characteristics of invasiveness and a dismal prognosis. Chemotherapy and/or radiotherapy are the main approaches for glioma treatment after surgery. As
2O
3 as an antitumor agent has shown efficacy in treating glioma in laboratory tests [
1],[
2]. Additionally, As
2O
3 has potential as a mitochondrial toxin that can induce reactive oxygen species (ROS) production; these mainly originate in the intracellular mitochondrial respiratory chain and further toxic byproducts subsequently produced could lead to mitochondrial damage [
3]. In addition, excessive ROS production acts as key mediator of the apoptotic-signaling pathway. However, the detailed mechanism of As
2O
3-induced excessive ROS production remains unclear.
Among numerous possible factors, such as altering the activity of enzymes, kinases, phosphatases and transcription factors [
4], iron has been demonstrated in previous studies [
5] [
9] to play an important role during ROS production in the mitochondrion. Because of the character of its redox properties, iron can catalyze the production of ROS, which are highly toxic [
10], and increase the redox reaction [
11].
Two main forms of iron exist in the cell: non-chelatable iron and chelatable iron. Lysosomes store amounts of chelatable iron [
12], which promotes oxidative stress by catalyzing the Fenton reaction and produces highly reactive hydroxyl radicals. Excessive reactive hydroxyl radicals could damage DNA, proteins, and membranes. The concentration of cytosolic chelatable iron is low under normal conditions, but, in pathological conditions, chelatable iron released from lysosomes can increase cytosolic iron concentration [
12]. Additionally, thelysosome-damaging ability of arsenite has been demonstrated [
13] and it is also known that excessive mitochondrial ROS production requires more iron to cross the mitochondrial membrane. Therefore, we hypothesized that the mitochondrial iron transporter may participate in As
2O
3-induced excessive ROS production.
Mitochondria are considered to be the major source of intracellular ROS, which are knowingly associated with retrograde signaling and affect many cellular functions [
14] [
16]. Mitoferrin, a mitochondrial protein, has been reported to mediate ferrous iron transport across the mitochondrial inner membrane [
11],[
17],[
18]. Mitoferrin has two isoforms: mitoferrin-1 and mitoferrin-2, which are localized on the inner mitochondrial membrane and function as a requisite importer of iron for mitochondrial heme and iron-sulfur cluster (ISC) in erythroblasts [
11]
. Mitoferrin-1 is mainly distributed in erythroid cells with low levels in other tissues, whereas mitoferrin-2 is ubiquitously distributed [
19]. In non-erythroid cells, mitoferrin-2 possibly functions to maintain the levels of cellular mitochondrial iron [
11],[
19]. Previous research has reported that mitoferrin-2 transmits ferrous iron from cytoplasm to mitochondria. Additionally high mitoferrin-2-expressing cells showed higher rates of mitochondrial ferrous iron uptake compared with low mitoferrin-2-expressing cells [
20]. Therefore, the possibility that the mitoferrin-2 transporter participates in As
2O
3-induced apoptosis in glioma should be considered.
In this study, our aim is to investigate whether mitoferrin-2 participates in the cytotoxic effect of As2O3 in human glioma and mediates the production of ROS.
Methods
Source of reagents
As2O3 compound was obtained from the Department of Pharmacy, the First Affiliated Hospital of Harbin Medical University (Harbin, China), and fresh dilutions with DMEM were used in each experiment. Mitoferrin-2 siRNA was designed and purchased from GenePharma (Shanghai, China). Annexin V-FITC-PI apoptosis detection kit (Baosea Biotechnology Co., Beijing, China) was used for detection of apoptosis by flow cytometry. For detection of ROS activity, 2’,7’-dichlorofluorescin diacetate (Sigma-Aldrich, St. Louis, MO, USA) was used. Reverse transcriptase RT kit and real time PCR kit (Takara Biotechnology Co., Shiga, Japan) were used for the semiquantitation of mitoferrin-2 mRNA levels. Dimethylsulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) were used for detection of cell viability.
Cell culture
Human glioma cell lines, U87MG and T98G were cultured in DMEM(Hyclone, Logan, UT, USA) supplemented with 10% FBS(Hyclone, Logan, UT, USA) at 37 C in a humidified CO
2 incubator, and 1% penicillin-streptomycin. The cells were passaged twice weekly, and once they were nearly confluent, they were released with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) [
21].
RNA interference studies
The siRNA specific sequences of human mitoferrin-2 transporter were designed according to standard procedures, and obtained from Shanghai GenePharma (Shanghai, China). The sequences were:
5’-GGCAACAUUACUUCAUGAUTT-3’ and 5’-AUCAUGAAGUAAUGUUGCCTT-3’; and the negative control sequences were:
5’-UUCUCCGAACGUGUCACGUTT-3’ and 5’-ACGUGACACGUUCGGAGAATT-3’. Glioma cells were transfected with 40 pmol of siRNA duplex or negative groups and exposed to 5 μM As2O3 for 48 hours.
Quantitative realtimePCR (QRT-PCR)
Total RNA was isolated from glioma cells using Trizol reagent (Invitrogen, California, USA). It was then reverse-transcribed to cDNA with random primers using a reverse transcriptase RT kit (Takara Biotechnology Co., Shiga, Japan) [
22]. The mRNA levels of mitoferrin-2 expression were detected using QRT-PCR on a Light Cycler 480 (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. The primer set of mitoferrin-2 was: sense, 5’-GGAGCATTCCAGGAGACA-3’; antisense, 5’-GGGTGACCGCCTATTT-3’. Each sample was checked in triplicate, and parallel reactions were performed using primers to β-actin as an internal control. The data were analyzed using the Light Cycler 480 software.
Western blot analysis
Cell extracts were prepared in ice-cold radioimmune precipitation assay lysis buffer (150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1% Nonidet P-40, 50 mM Tris-Cl, pH 7.4) supplemented with a mixture of protease inhibitors (Roche Diagnostics, Basel, Switzerland) and centrifuged. Equivalent amounts of protein determined by Bradford assay (Bio-Rad, California, USA) in sample buffer (Invitrogen) supplemented with 10% SDS and 10% β-mercaptoethanol were resolved on NuPAGE Tris-bis 12% polyacrylamide gels (Invitrogen, California, USA). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, armstadt, Germany) and probed with anti-Mfrn-2 (1:100) (Santa Cruz Biotechnology, Shanghai, China) and anti-β-actin (1:1,000) (Santa Cruz Biotechnology). Membranes were developed by two-color infrared fluorescence imaging system (LI-COR Biosciences, Nebraska, USA).
ROS detection
Glioma cells were transfected with 40 pmol of siRNA duplex or negative groups and exposed to 5 μM As2O3 for 48 hours. Then cells were incubated with 10 μM 2’,7’-dichlorofluorescin diacetate (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes, after which they were washed. ROS generation was determined by FACS can flow cytometry (Becton-Dickinson Mountain View, CA, USA) using CellQuest software and fluorescent signals were displayed as histograms.
MTT analysis
To investigate cell viability, U87MG and T98G cells were seeded in 96-well plates at a density of 5 × 103 cells per well and stabilized for 24 hours. Glioma cells were transfected with 40 pmol of siRNA duplex or negative groups and exposed to 5 μM As2O3 for 48 hours. The cells were incubated with new culture medium containing MTT working solution (0.5mg/ml) for 4 hours at 37 C. The culture supernatant was removed from the wells, and dimethyl sulfoxide (DMSO) was added. The absorbance of each well was measured at a wavelength of 490 nm with a reader.
Apoptosis
The apoptosis-inducing effects of As2O3 in U87MG and T98G cells were determined by flow cytometery using Annexin-V/FITC and propidium iodide. About 0.5 × 106 cells were plated in each well of a 6-well plate and transfected with 40 pmol of siRNA duplex or negative groups and exposed to 5 μM As2O3 for 48 hours. Apoptosis was determined using human Annexin-V-FITC kit according to the manufacturer’s instructions and analyzed by flow cytometry. Tests were repeated in triplicate.
Statistical analysis
Statistical analysis was performed by Student’s t-test. P-values of <0.05 were considered statistically significant. All values are expressed as mean ± SEM from three different experiments.
Discussion
As
2O
3 has achieved some efficacy in treating glioma [
23] [
25] although the mechanism remains unclear. In our study, the results indicated that mitoferrin-2, by mediating ferrous transport across the mitochondrial inner membrane, plays a significant role in As
2O
3-induced glioma cell death. Our findings showed that the expression level of mitoferrin-2 was increased about 4 to 5 folds in both U87MG and T98G after pretreatment with As
2O
3 (5 μM) after 48 hours. Next, to determine whether mitoferrin-2 mediates mitochondrial ROS production or apoptosis in As
2O
3-induced glioma cell damage,
mitoferrin-2 gene expression was silenced by siRNA in glioma cells and exposed to As
2O
3 for 48 hours. Our data showed that ROS production and apoptosis in low mitoferrin-2 expression groups were reduced compared to negative control groups in As
2O
3 pretreated glioma cells. Additionally, we firstly demonstrated that down-regulation of mitoferrin-2 expression affects As
2O
3-induced cell toxicity in human glioma cell lines U87MG and T98G. As
2O
3-induced cytotoxicity was reduced after silencing mitoferrin-2. Therefore, we assume that mitoferrin-2 transporter may partly participate in As
2O
3-induced cytotoxicity through promotion of ROS production.
Human glioma treatment is especially challenging because current treatments, such as chemical therapy, drastically affect patient quality of life. As
2O
3 has been successfully used for gliomas in a number of clinical trials and experiments because of its significant anticancer property [
1],[
2]. In previous researches, As
2O
3 (3 to 10 μM) could also induce apoptosis in cultured bone marrow mesenchymal stem cells [
26],[
27]. Additionally, treatment with high concentrations of As
2O
3 (30, 60, and 90 μM) caused primary cardiomyocyte apoptosis in a dose- and time-dependent manner [
28]. Higher concentrations of As
2O
3 (4 to 32 μM) obviously reduced smooth muscle cell viability in a concentration-dependent manner [
29]. One clinical study has indicated that the plasma levels of arsenic are 5.54 to 7.30 μM in acute promyelocytic leukemia patents treated with As
2O
3 for detection of ROS activity [
30]. In the present study, we found that treatment with As
2O
3 (1 to 10 μM) in glioma cell lines significantly decreases cell viability and increases cell apoptosis.
Further, As
2O
3 is a mitochondrial toxin causing ROS generation [
31], mitochondrial transmembrane potential disappearance, and cytochrome C release into the cytoplasm. These phenomena could be the cause of apoptosis. Therefore, we presume that As
2O
3 induces apoptosis in tumor cells possibly by affecting mitochondrial function [
22],[
32] [
38]. Our data showed that highest levels of As
2O
3-induced ROS production were after 4 hours and 6 hours in U87MG and T98G cell lines respectively. Additionally, previous researches demonstrated that intracellular ROS mediates multiple cellular responses, including cell cycle progression [
39], cell differentiation [
40], and apoptotic cell death [
41]. As we know, ROS is associated with the collapse of matrix metal proteinases (MMP) and the subsequent oxidative damage to the mitochondrial membranes, leading to disruption of MMP, cytochrome release, and apoptosis [
42]. Therefore, ROS plays an important role in the antitumor effect of As
2O
3.
Previous researches have also reported that iron is associated with ROS production in some diseases [
7]. Because of its redox properties, the transient existence of ferrous in mitochondria can catalyze the production of ROS that can be highly toxic through the Fenton reaction [
10]. However, it has long been known that, when in excess, ROS are among the major determinants of toxicity in cells and organisms. Therefore, mediated mitochondrial iron accumulation may affect As
2O
3-induced ROS production in human glioma.
Mitoferrin-2-dependent mitochondrial iron uptake acts synergistically to induce photodynamic therapy (PDT)-mediated and iron-dependent mitochondrial dysfunction and subsequent cancer cell killing has been reported [
20]. Therefore, whether mitoferrin-2 can affect the antitumor effect of As
2O
3 should be further explored. In our experiment, mitoferrin-2 mRNA expression was up-regulated in glioma cells after pretreatment with As
2O
3. Thus, mitoferrin-2 may participate in As
2O
3 treatment. Additionally, previous researches have reported that up-regulated expression of mitoferrin in yeast and mouse models of Friedreich’s ataxia suggested that it is associated with the pathogenesis of diseases with mitochondrial iron accumulation [
43]. Down-regulating the expression of mitoferrin-2 or blocking the iron import channel by some approaches should alleviate the symptoms of patients by preventing iron accumulation [
44]. Therefore, we assume that mitoferrin-2 may regulate mitochondrial iron accumulation and participate in As
2O
3-induced ROS production and cell damage.
In order to further explore whether mitoferrin-2 plays a crucial role in As
2O
3-induced cytotoxicity, we measured ROS production in both down-regulated mitoferrin-2 expression groups and negative groups in glioma cells pretreated with same-concentration As
2O
3. Our data showed that ROS production in low mitoferrin-2 expression groups was reduced compared to negative groups in glioma cells pretreated with As
2O
3. This result suggests that mitoferrin-2 can regulate As
2O
3-induced ROS production in glioma cells. Additionally, mitoferrin-2 has been demonstrated to increase the iron transmitted into mitochondria and to be related to ROS production in PDT [
20]. The predominant proportion of ROS production occurs inside mitochondria, leading to mitochondrial permeability transition and cell death [
15]. Therefore, overall these findings suggest that mitoferrin-2 has anapoptotic-promotingrole in As
2O
3-induced glioma apoptosis and cell damage.
Competing interests
The authors declare that they have no competing interests.