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Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation Geun-Hee Kwak a, Tae-Hyoung Kim b, Hwa-Young Kim a, * a b
Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu, Republic of Korea Department of Biochemistry, Chosun University School of Medicine, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2016 Accepted 18 December 2016 Available online xxx
Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. We have previously shown that depletion of MsrB3 suppresses the proliferation of normal mammalian cells by arresting cell cycle. In this study, we report the crucial role of MsrB3 in cancer cell death. Deficiency of MsrB3 induced cancer cell death, while MsrB3 overexpression stimulated cancer cell proliferation. MsrB3 depletion resulted in apoptotic cancer cell death through the activation of the intrinsic mitochondrial pathway. MsrB3 deficiency increased the levels of cellular reactive oxygen species (ROS) and led to redox imbalance, and also increased the Bax to Bcl-2 ratio and cytochrome c release, leading to caspase activation. Treatment of MsrB3-depleted cells with N-acetylcysteine, an ROS scavenger, prevented cell death, suggesting that MsrB3 deficiency-induced cell death is associated with increased ROS production. In addition, MsrB3 depletion activated poly(ADP ribose) polymerase-1 (PARP-1) and led to the translocation of apoptosis-inducing factor (AIF) to the nucleus. Taken together, our results suggest that MsrB3 plays an important role in cancer cell survival through the modulation of the intrinsic apoptosis pathway. © 2016 Elsevier Inc. All rights reserved.
Keywords: Methionine sulfoxide MsrB3 Redox imbalance Cell death Cancer
1. Introduction Reactive oxygen species (ROS) play an important role in apoptosis, a programmed cell death, by regulating the intrinsic mitochondrial apoptosis pathway [1]. An increase in ROS production activates certain pro-apoptotic Bcl-2 family members, such as Bax and Bid, which trigger permeabilization of the mitochondrial outer membrane and cytochrome c (cyt c) release, inducing apoptosis [2,3]. Furthermore, excessive levels of ROS disturb redox homeostasis, cause oxidative damage to cellular macromolecules including proteins, and induce cell death [4]. ROS can readily oxidize the sulfur-containing amino acid methionine, generating a diastereomeric mixture of methionine(R,S)-sulfoxide. To cope with methionine oxidation that induces protein damage, organisms have evolved a reductase system [5,6].
* Corresponding author. Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchung-ro, Namgu, Daegu, 42415, Republic of Korea. E-mail address: [email protected] (H.-Y. Kim).
Methionine sulfoxide reductase (Msr) is the enzyme responsible for the reduction of methionine sulfoxide to methionine [7]. In addition, cyclic oxidation and reduction of methionine is a defense mechanism that protects cells against oxidative stress [8]. Thus, Msr is thought to be an important antioxidant enzyme that scavenges cellular ROS. A number of studies have described the antioxidant role of Msr in several species ranging from bacteria to mammals [9]. There are two Msr families that differ on substrate stereospecificity. MsrA is specific for the reduction of the S-form of methionine sulfoxide, whereas MsrB acts only on the R-form. Mammalian cells contain one MsrA and three MsrB enzymes (B1eB3) [10,11]. In human cells, MsrB3 exists in two forms, MsrB3A and MsrB3B, which are generated via alternative first-exon splicing and are targeted to the endoplasmic reticulum (ER) and mitochondria, respectively [11,12]. We previously found that MsrB3 plays a critical role in the defense against oxidative stress [13,14]. In particular, overexpression of MsrB3 in fruit flies and mammalian cells increased resistance to oxidative stress [13,14], whereas its deficiency elevated susceptibility to oxidative stress [15]. We recently reported that MsrB3 is critical for cell proliferation, owing to its
http://dx.doi.org/10.1016/j.bbrc.2016.12.120 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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regulatory effect on the expression of cyclin-dependent kinase inhibitors p21 and p27 and heme oxygenase-1 (HO-1) [15,16]. Depletion of MsrB3 increased p21 and p27 levels and inhibited normal cell proliferation by arresting cell cycle at the G1/S phase [15]. Moreover, MsrB3 deficiency induced HO-1 expression, which has also an anti-proliferative effect, and led to increased cellular ROS levels [16]. A few studies to date have reported the effect of Msr enzymes on cancer cell proliferation and/or death [17]. Down-regulation of MsrA in human breast cancer cells increases cell proliferation and enhances metastatic aggressiveness [17]. Here, we investigated whether MsrB3 is also involved in the survival of cancer cells, as it is a potent regulator of normal cell proliferation. The present study demonstrates that down-regulation of MsrB3 induces apoptotic cell death in cancer cells through ROS production and activation of the intrinsic apoptosis pathway. 2. Materials and methods 2.1. Cell culture MCF-7 (human breast cancer) and A549 (human lung cancer) cells and SK-Hep1 (human liver cancer) cells were cultured in Roswell Park Memorial Institute (RPMI) medium and Dulbecco's modified Eagle's medium (DMEM), respectively, supplemented with 10% fetal bovine serum and 100 U penicillin-streptomycin antibiotics at 37 C in a 5% CO2 incubator. 2.2. siRNA-mediated knockdown of MsrB3 Cells were transfected with two different MsrB3-targeted siRNAs (siB3#1: sense, 50 -CUGGAAUUCGUAGGCUUCAdTdT-30 and antisense, 50 -UGAAGCCUACGAAUUCCAGdTdT-30 ; siB3#2: sense, 50 GGAGAGUGAUGGAAACAAAUU-30 and antisense, 50 -UUU0 GUUUCCAUCACUCUCCUU-3 ) using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. A control siRNA (sense, 50 -ACGUGACACGUUCGGAGAAUU-30 and antisense, 50 -UUCUCCGAACGUGUCACGUUU-30 ) was also used. The transfected cells were cultured for 24, 48, or 72 h. 2.3. Cell proliferation assay Cells were seeded in 24-well plates at a density of 5.0 104 cells/well. Cell proliferation was analyzed using an established colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)-based assay or by counting cells using the trypan blue exclusion method with a hemocytometer. 2.4. Colony formation assay Cells seeded in 6-well plates at a density of 1.0 105 cells/well were cultured for 48 or 72 h. The cultured cells were stained with 0.5% crystal violet in 60% methanol for 3 h at 37 C in the CO2 incubator, washed with phosphate-buffered saline (PBS), and analyzed using a LAS-4000 imaging system (Fujifilm). 2.5. Flow cytometric analysis of apoptosis Apoptosis was analyzed by flow cytometry using propidium iodide (PI) and annexin V double staining. Cells transfected with MsrB3-targeted or control siRNAs for 48 h were collected and washed twice with ice-cold PBS. The cell pellets were resuspended in 100 mL annexin V-binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2.5 mM CaCl2, pH 7.4) and transferred
to a 5-mL culture tube, containing 5 mL annexin V-FITC, to which 10 mL PI was added. The tube was gently vortexed and incubated for 15 min at room temperature in the dark. Subsequently, 300 mL of the binding buffer was added, and the cells were analyzed immediately by a FACS Canto II flow cytometer (BD Bioscience). 2.6. Western blot analysis Antibodies against MsrB3, lamin B, and glyceraldehyde 3phosphate dehydrogenase (GAPDH) were used as previously described [16,18]. Antibodies against cyt c, Bcl-2, b-tubulin, poly(ADP ribose) polymerase-1 (PARP-1), apoptosis-inducing factor (AIF), and cytochrome c oxidase IV (COX IV) were purchased from Santa Cruz Biotechnology and antibodies against Bax and caspase-7 were purchased from Cell Signaling Technology. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSePAGE) using a NuPAGE 4e12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was subsequently probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. GAPDH expression was used as a protein loading control. Quantitative analysis of blot signals was performed using the LAS-4000 imaging system and ImageJ software (NIH, USA). 2.7. Reverse transcription-PCR (RT-PCR) Total RNA was extracted from cells with the TRI-Solution (Bioscience). RT-PCR was performed using a Reverse Transcription Master Premix kit (ELPIS Biotech) containing the following oligo-dT primers: 50 -AACTGAGGAAGCGGCTAACA-30 and 50 - ACAAGGCAGCCGAATTTATG-30 for MsrB3; 50 -GGAGCCAAAAGGGTCATCAT-30 and 50 -GTGATGGCATGGACTGTGGT-30 for GAPDH. GAPDH mRNA expression was used as an internal control. 2.8. Fractionation of cytosolic, mitochondrial, and nuclear proteins Cells were harvested following detachment with trypsineEDTA, washed twice with PBS, resuspended in buffer A (0.25 M sucrose, 20 mM HEPES at pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) containing a cocktail of protease inhibitors, and homogenized using a Dounce homogenizer. The homogenate was centrifuged at 700 g for 5 min. The pellet was washed with buffer A, resuspended in RIFA lysis buffer, and used as the nuclear fraction. The supernatant was further centrifuged at 10,000 g for 15 min. The resulting pellet was washed with buffer A, resuspended in RIFA lysis buffer, and used as the mitochondrial fraction. The supernatant was further centrifuged at 10,000 g for 1 h, and the resulting supernatant was used as the cytosolic fraction. All procedures were performed at 4 C or on ice. Lamin B, COX IV, and b-tubulin were used as nuclear, mitochondrial, and cytosolic markers, respectively. 2.9. Adenovirus infection The generation of human MsrB3A-overexpressing adenovirus (Ad-MsrB3) and a control adenovirus (Ad-Con) is described elsewhere [14]. Cells were cultured for 16e24 h and following replacement of the culture medium with fresh medium, they were infected with the recombinant adenoviruses Ad-MsrB3 or Ad-Con at a multiplicity of infection (MOI) of 50, and were further incubated for 24 h. 2.10. Measurement of ROS levels Cells
were
incubated
with
50
mM
20 ,70 -
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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dichlorodihydrofluorescein diacetate (DCF-DA, SigmaeAldrich) for 20 min at 37 C or with 10 mM dihydroethidium (DHE, Invitrogen) for 10 min, washed twice with PBS, and harvested in PBS following detachment with trypsineEDTA. Fluorescence from 10,000 cells was measured and analyzed using the FACS Canto II flow cytometer. 2.11. Measurement of glutathione (GSH) contents Free and total GSH contents were measured using a GSH fluorescence detection kit (Arbor Assays) according to the manufacturer's instructions. Cell lysates were prepared in 5% sulfosalicylic acid to remove proteins. The oxidized GSH (GSSG) contents were calculated by subtracting the measured free GSH from the measured total GSH. GSH contents were expressed as nmole/ 106 cells. The GSH/GSSG ratio was used as a redox balance index. 2.12. Treatment with N-acetylcysteine Cells were treated with 5 mM N-acetylcysteine (SigmaeAldrich) for 3 h, after which they were transfected with MsrB3-targeted or control siRNAs and were cultured for an additional 48 h without medium change. 2.13. Statistical analysis Statistical analysis of the data was performed using a Student's t-test with the Prism 5 software (GraphPad). A P value of <0.05 was considered to denote statistical significance. 3. Results 3.1. MsrB3 deficiency induces apoptosis in cancer cells To investigate whether MsrB3 affects the proliferation of cancer cells, human MCF-7 breast cancer cells were transfected with two independent MsrB3-targeted siRNAs (siB3#1 and siB3#2) to deplete MsrB3. While both transfections with siB3#1 and siB3#2 decreased the expression of MsrB3, the knockdown effect was greater with siB3#2 than with siB3#1 (Fig. 1A, upper). Based on the MTT assay, a significant decrease in cell proliferation was observed in MsrB3-knockdown cells transfected with both siRNAs (Fig. 1A, lower). Moreover, the decrease in cell proliferation observed with siB3#2-transfected cells was greater than that observed with siB3#1-transfected cells, in accordance with the silencing effectiveness of the two siRNAs on MsrB3 expression. These results clearly demonstrate that down-regulation of MsrB3 leads to reduced proliferation of MCF-7 cells. Given the higher silencing effectiveness of siB3#2, this siRNA was selected to be used for the rest of the study. We also confirmed the inhibitory effect of MsrB3 depletion on proliferation of MCF-7 cells by counting cells with the trypan blue exclusion method (Fig. 1B). A significant decrease in cell number was evident in siB3-transfected cells after a 24 h transfection. We next determined whether the decreased proliferation of MCF-7 cells caused by MsrB3 down-regulation was due to cell death or inhibition of growth. Microscopic analysis revealed that at 72 h post-transfection, siB3-transfected cells were largely detached and many dead cells (cell debris) were floating in the culture medium (Fig. 1C), whereas cells transfected with a control siRNA were mostly attached on the culture plate and exhibited normal morphology. We also confirmed that cell death was induced by MsrB3 deficiency using a colony formation assay (Fig. 1D). We further investigated whether MsrB3 depletion induces cell death in other cancer cell lines. As shown in Fig. 1E, significantly increased cell death was observed in human A549 lung cancer cells
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and human SK-Hep1 hepatoma cells when MsrB3 was silenced. Thus, these data suggest that MsrB3 deficiency causes cell death across a broad range of cancer cells. We also investigated whether MsrB3 deficiency-induced cell death is apoptotic or necrotic using a flow cytometric assay with PI and annexin V double staining. We found that the PI negative and annexin V positive cell population, which undergoes early apoptosis, was significantly increased in the siB3-transfected MCF cells at 48 h post-transfection (Supplementary Fig. S1). Treatment with etoposide was used as a positive control of apoptosis. These results indicate that MsrB3 deficiency induces apoptotic cell death in cancer cells. 3.2. Overexpression of MsrB3 increases proliferation of cancer cells We investigated the effect of MsrB3 overexpression on cell proliferation in MCF-7 cells. Overexpression of MsrB3 by Ad-MsrB3 infection slightly but significantly increased proliferation of MCF7 cells (Supplementary Fig. S2A). This proliferative effect of MsrB3 overexpression was also observed in A549 lung cancer cells (Supplementary Fig. S2B). 3.3. Down-regulation of MsrB3 activates the mitochondrial apoptosis pathway To investigate how down-regulation of MsrB3 induced apoptosis in MCF-7 cells at the molecular level, we first determined the expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax molecules in MsrB3-deficient cells. In siB3-transfected cells, Bcl-2 protein levels were decreased (Fig. 2A and B), whereas Bax protein levels were significantly elevated (Fig. 2A and C). Accordingly, the Bax to Bcl-2 ratio was measured to be ~8-fold higher in MsrB3deficient cells (Fig. 2D). Since it is known that cyt c is released from mitochondria during apoptosis, we checked the levels of cyt c release to cytosol after fractionation of cytosol and mitochondria. The cytosolic levels of cyt c were significantly higher and its mitochondrial levels were lower in siB3-transfected cells than in control cells (Fig. 2E and F). We also determined whether MsrB3 deficiency induces caspase activation. To analyze caspase activation, we measured cleaved caspase-7 levels in MCF-7 cells (Fig. 2G). Notably, these cancer cells lack caspase-3 [19]. Increased levels of cleaved caspase-7 were observed in MsrB3-deficient cells, indicating activation of caspase7 by MsrB3 down-regulation. Cleavage of PARP-1 by caspases is thought to be a hallmark of apoptosis, and PAPR-1 is a substrate of caspase-7 [20,21]. We thus determined the extent of PARP-1 cleavage in MsrB3-deficient cells and compared it to that in control cells. PARP-1 cleavage was not detectable in control cells, but was clearly detected in MsrB3-deficient cells (Fig. 2G). In conclusion, down-regulation of MsrB3 resulted in increased Bax/Bcl-2 ratio, cyt c release, and activation of caspase-7 and PARP-1. Thus, our results suggest that MsrB3 deficiency activates the intrinsic mitochondrial apoptosis pathway in MCF-7 cancer cells. 3.4. MsrB3 depletion leads to AIF protein translocation to the nucleus AIF is a flavoprotein that translocates from the mitochondria to the nucleus, where it triggers caspase-independent chromatin condensation and large-scale DNA fragmentation to induce apoptosis [22]. AIF mediates PARP-1-dependent cell death and its translocation occurs quickly after PARP-1 activation [23]. Since MsrB3 knockdown induces PARP-1 activation, we investigated whether AIF protein accumulates in the nucleus of MsrB3-deficient MCF-7 cells. As shown in Fig. 3, the nuclear levels of AIF protein
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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Fig. 1. Down-regulation of MsrB3 induces cell death in cancer cells. (A) MCF-7 cells were transfected with MsrB3-targeted (siB3#1 and siB3#2) or control (siC) siRNAs for 48 h. MsrB3 mRNA levels were analyzed by RT-PCR (upper) and cell proliferation was measured using the MTT assay (lower). (BeD) MCF-7 cells were transfected with siB3#2 or siC. (B) Cell number was counted at the indicated post-transfection time points. (C) Microscopic analysis of cell morphology after 72 h transfection. In siB3#2-transfected cells, many dead cells (cell debris) were floating in the culture medium. Pictures are taken at 40 magnification. (D) Colony formation assay using crystal violet. (E) A549 and SK-Hep1 cells were transfected with siB3#2 or siC for 48 h. Cell proliferation was measured using the MTT method and the results are presented as relative cell viability. MsrB3 expression knockdown was analyzed by Western blotting (lower). All data are based on at least two independent experiments and cell proliferation (or viability) data are shown as mean ± SE of triplicates. **, P < 0.01; ***, P < 0.001, based on comparisons with the control.
were significantly elevated and its mitochondrial levels were significantly reduced in siB3-transfected cells compared to control cells. These results demonstrate that down-regulation of MsrB3 leads to translocation of AIF from the mitochondria to the nucleus that consequently triggers programmed cell death. 3.5. MsrB3 deficiency increases ROS production and disturbs redox balance Since ROS production is considered to be a factor that triggers apoptosis through the intrinsic mitochondrial pathway and MsrB3 is an antioxidant enzyme, we measured cellular ROS levels in MsrB3-deficient MCF-7 cells and compared them with those in
control cells. Cellular ROS levels detected with DCF or DHE probes were significantly higher in MsrB3-deficient cells than in control cells (Fig. 4A). Disturbance of redox homeostasis is also thought to be a trigger factor for apoptosis [4]. We determined whether MsrB3 deficiency affected cellular redox balance by measuring the ratio of free to oxidized GSH (GSH/GSSG), a representative redox balance index, together with total GSH contents in MsrB3-deficient MCF-7 and control cells. Interestingly, total GSH content was significantly lower in MsrB3-deficient cells than in control cells, suggesting that down-regulation of MsrB3 causes depletion of GSH (Fig. 4B). Free GSH levels, as well as the GSH/GSSG ratio, were significantly decreased in the MsrB3-deficient cells (Fig. 4C and D). Taken
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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Fig. 2. Down-regulation of MsrB3 activates the intrinsic mitochondrial apoptosis pathway. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Western blot analysis of Bcl-2 and Bax from cell lysates. (B) Quantitative analysis of Bcl-2 protein normalized to GAPDH (n ¼ 2; *, P < 0.05). (C) Quantitative analysis of Bax protein normalized to GAPDH (n ¼ 2; *, P < 0.05). (D) Bax to Bcl-2 ratio (n ¼ 2; *, P < 0.05). (E) Western blot analysis of cytochrome c (cyt c) from cytosolic and mitochondrial fractions. b-tubulin and COX IV were used as cytosolic and mitochondrial markers, respectively. Since the cytosolic fraction included the ER, the ER form of MsrB3 (MsrB3A) could be detected in this fraction. In the mitochondrial fraction, an MsrB3 band, which corresponds to the MsrB3B form, was also detected. (F) Quantitative analysis of cytosolic cyt c normalized to b-tubulin (n ¼ 3; **, P < 0.01). (G) Western blot analysis of caspase-7 and PARP-1 from cell lysates. Representative data are shown from at least two independent experiments.
together, these results suggest that MsrB3 depletion results in cellular redox imbalance. 3.6. N-acetylcysteine treatment increases cell viability in MsrB3depleted cells We investigated whether increased ROS production was directly related to cell death induced by MsrB3 depletion. MCF-7 cells were treated with 5 mM N-acetylcysteine as an ROS scavenger for 3 h, transfected with siRNAs, and cultured for 48 h. Under these
conditions, treatment of control cells with N-acetylcysteine resulted in a significant decrease in basal ROS levels, as well as reduced cell viability (Supplementary Fig. S3). In contrast, treatment of MsrB3-depleted cells with N-acetylcysteine significantly diminished the augmented ROS levels and increased cell viability to levels similar to those observed in control cells treated with Nacetylcysteine (Supplementary Fig. S3). These results suggest that MsrB3 deficiency-induced cell death is associated with increased ROS production.
Fig. 3. MsrB3 down-regulation leads to AIF protein accumulation in the nucleus. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Western blot analysis of AIF from nuclear and mitochondrial fractions. Lamin B and COX IV were used as nuclear and mitochondrial markers, respectively. (B) Quantitative analysis of nuclear AIF normalized to lamin B (n ¼ 3; **, P < 0.01). (C) Quantitative analysis of mitochondrial AIF normalized to COX IV (n ¼ 3; ***, P < 0.001).
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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Fig. 4. MsrB3 depletion increases cellular ROS levels and redox imbalance. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Cellular ROS levels probed with DCF and DHE. Probe treatment conditions are described in detail in Materials and Methods. (B) Total GSH content. (C) Free GSH (reduced form) content. (D) GSH (reduced) to GSSG (oxidized) ratio. All data are based on at least two independent experiments and shown as mean ± SE of triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on comparisons with the control.
4. Discussion To the best of our knowledge, we herein demonstrate for the first time a crucial role of MsrB3 in cancer cell death. MsrB3, which is targeted to the ER and mitochondria in human cells [11], plays an antioxidant role through specifically catalyzing the reduction of methionine-R-sulfoxide residues [13,15]. Recent studies by us and other groups show that MsrB3 is essential to hearing in mice and humans [24,25]. We have also recently found that MsrB3 is critical for the proliferation of normal cells through regulating the expression of p21 and HO-1 genes [15,16]. Down-regulation of MsrB3 inhibits proliferation of normal mammalian cells by arresting cell cycle at the G1/S stage [15]. Based on these findings, we investigated whether MsrB3 is associated with the regulation of proliferation and death of cancer cells. Our results demonstrate that down-regulation of MsrB3 induces apoptosis of cancer cells, whereas its up-regulation stimulates proliferation of cancer cells. The present study has revealed that MsrB3 down-regulation induces apoptotic cell death in cancer cells through activating the intrinsic mitochondrial apoptosis pathway (Supplementary Fig. S4). MsrB3 deficiency in MCF-7 cells resulted in increased ROS levels and redox imbalance, increased Bax/Bcl-2 ratio, cyt c release from mitochondria, activation of caspase-7 and PARP-1, and translocation of AIF to the nucleus. Based on these results, MsrB3 deficiency-induced apoptosis appeared to be mediated by
mechanisms both dependent on and independent of caspases. Caspase-7 activation induced by MsrB3 down-regulation represents a caspase-dependent mechanism, while AIF-mediated cell death initiated by PARP-1 activation is a caspase-independent mechanism [22,23]. Studies with inhibitors of caspases and PARP1 will help to elucidate the details of the mechanisms through which MsrB3 down-regulation induces cell death. It has previously been reported that MsrA, which is specific for the reduction of methionine S-sulfoxide, is also associated with the regulation of breast cancer cell proliferation [17]. However, in contrast to the apoptotic effect of MsrB3 down-regulation, MsrA down-regulation results in increased cell proliferation [17]. In addition, down-regulation of MsrA leads to a more aggressive cellular phenotype by increasing extracellular matrix degradation [17]. Based on the findings of the present and previous studies, it appears that MsrB3 and MsrA have opposite effects in cancer growth, suggesting that the methionine-R-sulfoxide and methionine-S-sulfoxide reduction systems are involved differently in the regulation of cancer cell survival. It will be interesting to investigate the effects of the other two MsrB enzymes, MsrB1 and MsrB2, on cancer cell survival. In conclusion, the present study has demonstrated for the first time that down-regulation of MsrB3 induces apoptotic cell death of cancer cells. One of the pathways leading to MsrB3 depletioninduced cell death is the activation of intrinsic mitochondrial
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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apoptosis, accompanied by increased ROS and redox imbalance, caspase and PARP-1 activation, and translocation of AIF to the nucleus. Conflict of interest The authors have no conflict of interest. Acknowledgement This work was supported by the 2015 Yeungnam University Research Grant. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.12.120. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.12.120. References [1] C.C. Wu, S.B. Bratton, Regulation of the intrinsic apoptosis pathway by reactive oxygen species, Antioxid. Redox Signal 19 (2013) 546e558. [2] Q. Ran, H. Liang, M. Gu, W. Qi, C.A. Walter, L.J. Roberts 2nd, B. Herman, A. Richardson, H. Van Remmen, Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis, J. Biol. Chem. 279 (2004) 55137e55146. [3] M. Torres, Mitogen-activated protein kinase pathways in redox signaling, Front. Biosci. 8 (2003) d369e391. [4] B. D'Autreaux, M.B. Toledano, ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis, Nat. Rev. Mol. Cell Biol. 8 (2007) 813e824. [5] H. Weissbach, L. Resnick, N. Brot, Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage, Biochim. Biophys. Acta 1703 (2005) 203e212. [6] J. Moskovitz, D.B. Oien, Protein carbonyl and the methionine sulfoxide reductase system, Antioxid. Redox Signal 12 (2010) 405e415. [7] H.Y. Kim, V.N. Gladyshev, Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals, Biochem. J. 407 (2007) 321e329. [8] R.L. Levine, B.S. Berlett, J. Moskovitz, L. Mosoni, E.R. Stadtman, Methionine residues may protect proteins from critical oxidative damage, Mech. Ageing Dev. 107 (1999) 323e332. [9] H.Y. Kim, The methionine sulfoxide reduction system: selenium utilization and methionine sulfoxide reductase enzymes and their functions, Antioxid.
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Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation Geun-Hee Kwak a, Tae-Hyoung Kim b, Hwa-Young Kim a, * a b
Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu, Republic of Korea Department of Biochemistry, Chosun University School of Medicine, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2016 Accepted 18 December 2016 Available online xxx
Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. We have previously shown that depletion of MsrB3 suppresses the proliferation of normal mammalian cells by arresting cell cycle. In this study, we report the crucial role of MsrB3 in cancer cell death. Deficiency of MsrB3 induced cancer cell death, while MsrB3 overexpression stimulated cancer cell proliferation. MsrB3 depletion resulted in apoptotic cancer cell death through the activation of the intrinsic mitochondrial pathway. MsrB3 deficiency increased the levels of cellular reactive oxygen species (ROS) and led to redox imbalance, and also increased the Bax to Bcl-2 ratio and cytochrome c release, leading to caspase activation. Treatment of MsrB3-depleted cells with N-acetylcysteine, an ROS scavenger, prevented cell death, suggesting that MsrB3 deficiency-induced cell death is associated with increased ROS production. In addition, MsrB3 depletion activated poly(ADP ribose) polymerase-1 (PARP-1) and led to the translocation of apoptosis-inducing factor (AIF) to the nucleus. Taken together, our results suggest that MsrB3 plays an important role in cancer cell survival through the modulation of the intrinsic apoptosis pathway. © 2016 Elsevier Inc. All rights reserved.
Keywords: Methionine sulfoxide MsrB3 Redox imbalance Cell death Cancer
1. Introduction Reactive oxygen species (ROS) play an important role in apoptosis, a programmed cell death, by regulating the intrinsic mitochondrial apoptosis pathway [1]. An increase in ROS production activates certain pro-apoptotic Bcl-2 family members, such as Bax and Bid, which trigger permeabilization of the mitochondrial outer membrane and cytochrome c (cyt c) release, inducing apoptosis [2,3]. Furthermore, excessive levels of ROS disturb redox homeostasis, cause oxidative damage to cellular macromolecules including proteins, and induce cell death [4]. ROS can readily oxidize the sulfur-containing amino acid methionine, generating a diastereomeric mixture of methionine(R,S)-sulfoxide. To cope with methionine oxidation that induces protein damage, organisms have evolved a reductase system [5,6].
* Corresponding author. Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchung-ro, Namgu, Daegu, 42415, Republic of Korea. E-mail address: [email protected] (H.-Y. Kim).
Methionine sulfoxide reductase (Msr) is the enzyme responsible for the reduction of methionine sulfoxide to methionine [7]. In addition, cyclic oxidation and reduction of methionine is a defense mechanism that protects cells against oxidative stress [8]. Thus, Msr is thought to be an important antioxidant enzyme that scavenges cellular ROS. A number of studies have described the antioxidant role of Msr in several species ranging from bacteria to mammals [9]. There are two Msr families that differ on substrate stereospecificity. MsrA is specific for the reduction of the S-form of methionine sulfoxide, whereas MsrB acts only on the R-form. Mammalian cells contain one MsrA and three MsrB enzymes (B1eB3) [10,11]. In human cells, MsrB3 exists in two forms, MsrB3A and MsrB3B, which are generated via alternative first-exon splicing and are targeted to the endoplasmic reticulum (ER) and mitochondria, respectively [11,12]. We previously found that MsrB3 plays a critical role in the defense against oxidative stress [13,14]. In particular, overexpression of MsrB3 in fruit flies and mammalian cells increased resistance to oxidative stress [13,14], whereas its deficiency elevated susceptibility to oxidative stress [15]. We recently reported that MsrB3 is critical for cell proliferation, owing to its
http://dx.doi.org/10.1016/j.bbrc.2016.12.120 0006-291X/© 2016 Elsevier Inc. All rights reserved.
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regulatory effect on the expression of cyclin-dependent kinase inhibitors p21 and p27 and heme oxygenase-1 (HO-1) [15,16]. Depletion of MsrB3 increased p21 and p27 levels and inhibited normal cell proliferation by arresting cell cycle at the G1/S phase [15]. Moreover, MsrB3 deficiency induced HO-1 expression, which has also an anti-proliferative effect, and led to increased cellular ROS levels [16]. A few studies to date have reported the effect of Msr enzymes on cancer cell proliferation and/or death [17]. Down-regulation of MsrA in human breast cancer cells increases cell proliferation and enhances metastatic aggressiveness [17]. Here, we investigated whether MsrB3 is also involved in the survival of cancer cells, as it is a potent regulator of normal cell proliferation. The present study demonstrates that down-regulation of MsrB3 induces apoptotic cell death in cancer cells through ROS production and activation of the intrinsic apoptosis pathway. 2. Materials and methods 2.1. Cell culture MCF-7 (human breast cancer) and A549 (human lung cancer) cells and SK-Hep1 (human liver cancer) cells were cultured in Roswell Park Memorial Institute (RPMI) medium and Dulbecco's modified Eagle's medium (DMEM), respectively, supplemented with 10% fetal bovine serum and 100 U penicillin-streptomycin antibiotics at 37 C in a 5% CO2 incubator. 2.2. siRNA-mediated knockdown of MsrB3 Cells were transfected with two different MsrB3-targeted siRNAs (siB3#1: sense, 50 -CUGGAAUUCGUAGGCUUCAdTdT-30 and antisense, 50 -UGAAGCCUACGAAUUCCAGdTdT-30 ; siB3#2: sense, 50 GGAGAGUGAUGGAAACAAAUU-30 and antisense, 50 -UUU0 GUUUCCAUCACUCUCCUU-3 ) using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. A control siRNA (sense, 50 -ACGUGACACGUUCGGAGAAUU-30 and antisense, 50 -UUCUCCGAACGUGUCACGUUU-30 ) was also used. The transfected cells were cultured for 24, 48, or 72 h. 2.3. Cell proliferation assay Cells were seeded in 24-well plates at a density of 5.0 104 cells/well. Cell proliferation was analyzed using an established colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)-based assay or by counting cells using the trypan blue exclusion method with a hemocytometer. 2.4. Colony formation assay Cells seeded in 6-well plates at a density of 1.0 105 cells/well were cultured for 48 or 72 h. The cultured cells were stained with 0.5% crystal violet in 60% methanol for 3 h at 37 C in the CO2 incubator, washed with phosphate-buffered saline (PBS), and analyzed using a LAS-4000 imaging system (Fujifilm). 2.5. Flow cytometric analysis of apoptosis Apoptosis was analyzed by flow cytometry using propidium iodide (PI) and annexin V double staining. Cells transfected with MsrB3-targeted or control siRNAs for 48 h were collected and washed twice with ice-cold PBS. The cell pellets were resuspended in 100 mL annexin V-binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2.5 mM CaCl2, pH 7.4) and transferred
to a 5-mL culture tube, containing 5 mL annexin V-FITC, to which 10 mL PI was added. The tube was gently vortexed and incubated for 15 min at room temperature in the dark. Subsequently, 300 mL of the binding buffer was added, and the cells were analyzed immediately by a FACS Canto II flow cytometer (BD Bioscience). 2.6. Western blot analysis Antibodies against MsrB3, lamin B, and glyceraldehyde 3phosphate dehydrogenase (GAPDH) were used as previously described [16,18]. Antibodies against cyt c, Bcl-2, b-tubulin, poly(ADP ribose) polymerase-1 (PARP-1), apoptosis-inducing factor (AIF), and cytochrome c oxidase IV (COX IV) were purchased from Santa Cruz Biotechnology and antibodies against Bax and caspase-7 were purchased from Cell Signaling Technology. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSePAGE) using a NuPAGE 4e12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was subsequently probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. GAPDH expression was used as a protein loading control. Quantitative analysis of blot signals was performed using the LAS-4000 imaging system and ImageJ software (NIH, USA). 2.7. Reverse transcription-PCR (RT-PCR) Total RNA was extracted from cells with the TRI-Solution (Bioscience). RT-PCR was performed using a Reverse Transcription Master Premix kit (ELPIS Biotech) containing the following oligo-dT primers: 50 -AACTGAGGAAGCGGCTAACA-30 and 50 - ACAAGGCAGCCGAATTTATG-30 for MsrB3; 50 -GGAGCCAAAAGGGTCATCAT-30 and 50 -GTGATGGCATGGACTGTGGT-30 for GAPDH. GAPDH mRNA expression was used as an internal control. 2.8. Fractionation of cytosolic, mitochondrial, and nuclear proteins Cells were harvested following detachment with trypsineEDTA, washed twice with PBS, resuspended in buffer A (0.25 M sucrose, 20 mM HEPES at pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) containing a cocktail of protease inhibitors, and homogenized using a Dounce homogenizer. The homogenate was centrifuged at 700 g for 5 min. The pellet was washed with buffer A, resuspended in RIFA lysis buffer, and used as the nuclear fraction. The supernatant was further centrifuged at 10,000 g for 15 min. The resulting pellet was washed with buffer A, resuspended in RIFA lysis buffer, and used as the mitochondrial fraction. The supernatant was further centrifuged at 10,000 g for 1 h, and the resulting supernatant was used as the cytosolic fraction. All procedures were performed at 4 C or on ice. Lamin B, COX IV, and b-tubulin were used as nuclear, mitochondrial, and cytosolic markers, respectively. 2.9. Adenovirus infection The generation of human MsrB3A-overexpressing adenovirus (Ad-MsrB3) and a control adenovirus (Ad-Con) is described elsewhere [14]. Cells were cultured for 16e24 h and following replacement of the culture medium with fresh medium, they were infected with the recombinant adenoviruses Ad-MsrB3 or Ad-Con at a multiplicity of infection (MOI) of 50, and were further incubated for 24 h. 2.10. Measurement of ROS levels Cells
were
incubated
with
50
mM
20 ,70 -
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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dichlorodihydrofluorescein diacetate (DCF-DA, SigmaeAldrich) for 20 min at 37 C or with 10 mM dihydroethidium (DHE, Invitrogen) for 10 min, washed twice with PBS, and harvested in PBS following detachment with trypsineEDTA. Fluorescence from 10,000 cells was measured and analyzed using the FACS Canto II flow cytometer. 2.11. Measurement of glutathione (GSH) contents Free and total GSH contents were measured using a GSH fluorescence detection kit (Arbor Assays) according to the manufacturer's instructions. Cell lysates were prepared in 5% sulfosalicylic acid to remove proteins. The oxidized GSH (GSSG) contents were calculated by subtracting the measured free GSH from the measured total GSH. GSH contents were expressed as nmole/ 106 cells. The GSH/GSSG ratio was used as a redox balance index. 2.12. Treatment with N-acetylcysteine Cells were treated with 5 mM N-acetylcysteine (SigmaeAldrich) for 3 h, after which they were transfected with MsrB3-targeted or control siRNAs and were cultured for an additional 48 h without medium change. 2.13. Statistical analysis Statistical analysis of the data was performed using a Student's t-test with the Prism 5 software (GraphPad). A P value of <0.05 was considered to denote statistical significance. 3. Results 3.1. MsrB3 deficiency induces apoptosis in cancer cells To investigate whether MsrB3 affects the proliferation of cancer cells, human MCF-7 breast cancer cells were transfected with two independent MsrB3-targeted siRNAs (siB3#1 and siB3#2) to deplete MsrB3. While both transfections with siB3#1 and siB3#2 decreased the expression of MsrB3, the knockdown effect was greater with siB3#2 than with siB3#1 (Fig. 1A, upper). Based on the MTT assay, a significant decrease in cell proliferation was observed in MsrB3-knockdown cells transfected with both siRNAs (Fig. 1A, lower). Moreover, the decrease in cell proliferation observed with siB3#2-transfected cells was greater than that observed with siB3#1-transfected cells, in accordance with the silencing effectiveness of the two siRNAs on MsrB3 expression. These results clearly demonstrate that down-regulation of MsrB3 leads to reduced proliferation of MCF-7 cells. Given the higher silencing effectiveness of siB3#2, this siRNA was selected to be used for the rest of the study. We also confirmed the inhibitory effect of MsrB3 depletion on proliferation of MCF-7 cells by counting cells with the trypan blue exclusion method (Fig. 1B). A significant decrease in cell number was evident in siB3-transfected cells after a 24 h transfection. We next determined whether the decreased proliferation of MCF-7 cells caused by MsrB3 down-regulation was due to cell death or inhibition of growth. Microscopic analysis revealed that at 72 h post-transfection, siB3-transfected cells were largely detached and many dead cells (cell debris) were floating in the culture medium (Fig. 1C), whereas cells transfected with a control siRNA were mostly attached on the culture plate and exhibited normal morphology. We also confirmed that cell death was induced by MsrB3 deficiency using a colony formation assay (Fig. 1D). We further investigated whether MsrB3 depletion induces cell death in other cancer cell lines. As shown in Fig. 1E, significantly increased cell death was observed in human A549 lung cancer cells
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and human SK-Hep1 hepatoma cells when MsrB3 was silenced. Thus, these data suggest that MsrB3 deficiency causes cell death across a broad range of cancer cells. We also investigated whether MsrB3 deficiency-induced cell death is apoptotic or necrotic using a flow cytometric assay with PI and annexin V double staining. We found that the PI negative and annexin V positive cell population, which undergoes early apoptosis, was significantly increased in the siB3-transfected MCF cells at 48 h post-transfection (Supplementary Fig. S1). Treatment with etoposide was used as a positive control of apoptosis. These results indicate that MsrB3 deficiency induces apoptotic cell death in cancer cells. 3.2. Overexpression of MsrB3 increases proliferation of cancer cells We investigated the effect of MsrB3 overexpression on cell proliferation in MCF-7 cells. Overexpression of MsrB3 by Ad-MsrB3 infection slightly but significantly increased proliferation of MCF7 cells (Supplementary Fig. S2A). This proliferative effect of MsrB3 overexpression was also observed in A549 lung cancer cells (Supplementary Fig. S2B). 3.3. Down-regulation of MsrB3 activates the mitochondrial apoptosis pathway To investigate how down-regulation of MsrB3 induced apoptosis in MCF-7 cells at the molecular level, we first determined the expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax molecules in MsrB3-deficient cells. In siB3-transfected cells, Bcl-2 protein levels were decreased (Fig. 2A and B), whereas Bax protein levels were significantly elevated (Fig. 2A and C). Accordingly, the Bax to Bcl-2 ratio was measured to be ~8-fold higher in MsrB3deficient cells (Fig. 2D). Since it is known that cyt c is released from mitochondria during apoptosis, we checked the levels of cyt c release to cytosol after fractionation of cytosol and mitochondria. The cytosolic levels of cyt c were significantly higher and its mitochondrial levels were lower in siB3-transfected cells than in control cells (Fig. 2E and F). We also determined whether MsrB3 deficiency induces caspase activation. To analyze caspase activation, we measured cleaved caspase-7 levels in MCF-7 cells (Fig. 2G). Notably, these cancer cells lack caspase-3 [19]. Increased levels of cleaved caspase-7 were observed in MsrB3-deficient cells, indicating activation of caspase7 by MsrB3 down-regulation. Cleavage of PARP-1 by caspases is thought to be a hallmark of apoptosis, and PAPR-1 is a substrate of caspase-7 [20,21]. We thus determined the extent of PARP-1 cleavage in MsrB3-deficient cells and compared it to that in control cells. PARP-1 cleavage was not detectable in control cells, but was clearly detected in MsrB3-deficient cells (Fig. 2G). In conclusion, down-regulation of MsrB3 resulted in increased Bax/Bcl-2 ratio, cyt c release, and activation of caspase-7 and PARP-1. Thus, our results suggest that MsrB3 deficiency activates the intrinsic mitochondrial apoptosis pathway in MCF-7 cancer cells. 3.4. MsrB3 depletion leads to AIF protein translocation to the nucleus AIF is a flavoprotein that translocates from the mitochondria to the nucleus, where it triggers caspase-independent chromatin condensation and large-scale DNA fragmentation to induce apoptosis [22]. AIF mediates PARP-1-dependent cell death and its translocation occurs quickly after PARP-1 activation [23]. Since MsrB3 knockdown induces PARP-1 activation, we investigated whether AIF protein accumulates in the nucleus of MsrB3-deficient MCF-7 cells. As shown in Fig. 3, the nuclear levels of AIF protein
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Fig. 1. Down-regulation of MsrB3 induces cell death in cancer cells. (A) MCF-7 cells were transfected with MsrB3-targeted (siB3#1 and siB3#2) or control (siC) siRNAs for 48 h. MsrB3 mRNA levels were analyzed by RT-PCR (upper) and cell proliferation was measured using the MTT assay (lower). (BeD) MCF-7 cells were transfected with siB3#2 or siC. (B) Cell number was counted at the indicated post-transfection time points. (C) Microscopic analysis of cell morphology after 72 h transfection. In siB3#2-transfected cells, many dead cells (cell debris) were floating in the culture medium. Pictures are taken at 40 magnification. (D) Colony formation assay using crystal violet. (E) A549 and SK-Hep1 cells were transfected with siB3#2 or siC for 48 h. Cell proliferation was measured using the MTT method and the results are presented as relative cell viability. MsrB3 expression knockdown was analyzed by Western blotting (lower). All data are based on at least two independent experiments and cell proliferation (or viability) data are shown as mean ± SE of triplicates. **, P < 0.01; ***, P < 0.001, based on comparisons with the control.
were significantly elevated and its mitochondrial levels were significantly reduced in siB3-transfected cells compared to control cells. These results demonstrate that down-regulation of MsrB3 leads to translocation of AIF from the mitochondria to the nucleus that consequently triggers programmed cell death. 3.5. MsrB3 deficiency increases ROS production and disturbs redox balance Since ROS production is considered to be a factor that triggers apoptosis through the intrinsic mitochondrial pathway and MsrB3 is an antioxidant enzyme, we measured cellular ROS levels in MsrB3-deficient MCF-7 cells and compared them with those in
control cells. Cellular ROS levels detected with DCF or DHE probes were significantly higher in MsrB3-deficient cells than in control cells (Fig. 4A). Disturbance of redox homeostasis is also thought to be a trigger factor for apoptosis [4]. We determined whether MsrB3 deficiency affected cellular redox balance by measuring the ratio of free to oxidized GSH (GSH/GSSG), a representative redox balance index, together with total GSH contents in MsrB3-deficient MCF-7 and control cells. Interestingly, total GSH content was significantly lower in MsrB3-deficient cells than in control cells, suggesting that down-regulation of MsrB3 causes depletion of GSH (Fig. 4B). Free GSH levels, as well as the GSH/GSSG ratio, were significantly decreased in the MsrB3-deficient cells (Fig. 4C and D). Taken
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Fig. 2. Down-regulation of MsrB3 activates the intrinsic mitochondrial apoptosis pathway. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Western blot analysis of Bcl-2 and Bax from cell lysates. (B) Quantitative analysis of Bcl-2 protein normalized to GAPDH (n ¼ 2; *, P < 0.05). (C) Quantitative analysis of Bax protein normalized to GAPDH (n ¼ 2; *, P < 0.05). (D) Bax to Bcl-2 ratio (n ¼ 2; *, P < 0.05). (E) Western blot analysis of cytochrome c (cyt c) from cytosolic and mitochondrial fractions. b-tubulin and COX IV were used as cytosolic and mitochondrial markers, respectively. Since the cytosolic fraction included the ER, the ER form of MsrB3 (MsrB3A) could be detected in this fraction. In the mitochondrial fraction, an MsrB3 band, which corresponds to the MsrB3B form, was also detected. (F) Quantitative analysis of cytosolic cyt c normalized to b-tubulin (n ¼ 3; **, P < 0.01). (G) Western blot analysis of caspase-7 and PARP-1 from cell lysates. Representative data are shown from at least two independent experiments.
together, these results suggest that MsrB3 depletion results in cellular redox imbalance. 3.6. N-acetylcysteine treatment increases cell viability in MsrB3depleted cells We investigated whether increased ROS production was directly related to cell death induced by MsrB3 depletion. MCF-7 cells were treated with 5 mM N-acetylcysteine as an ROS scavenger for 3 h, transfected with siRNAs, and cultured for 48 h. Under these
conditions, treatment of control cells with N-acetylcysteine resulted in a significant decrease in basal ROS levels, as well as reduced cell viability (Supplementary Fig. S3). In contrast, treatment of MsrB3-depleted cells with N-acetylcysteine significantly diminished the augmented ROS levels and increased cell viability to levels similar to those observed in control cells treated with Nacetylcysteine (Supplementary Fig. S3). These results suggest that MsrB3 deficiency-induced cell death is associated with increased ROS production.
Fig. 3. MsrB3 down-regulation leads to AIF protein accumulation in the nucleus. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Western blot analysis of AIF from nuclear and mitochondrial fractions. Lamin B and COX IV were used as nuclear and mitochondrial markers, respectively. (B) Quantitative analysis of nuclear AIF normalized to lamin B (n ¼ 3; **, P < 0.01). (C) Quantitative analysis of mitochondrial AIF normalized to COX IV (n ¼ 3; ***, P < 0.001).
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Fig. 4. MsrB3 depletion increases cellular ROS levels and redox imbalance. MCF-7 cells were transfected with MsrB3-targeted (siB3) or control (siC) siRNAs for 48 h. (A) Cellular ROS levels probed with DCF and DHE. Probe treatment conditions are described in detail in Materials and Methods. (B) Total GSH content. (C) Free GSH (reduced form) content. (D) GSH (reduced) to GSSG (oxidized) ratio. All data are based on at least two independent experiments and shown as mean ± SE of triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on comparisons with the control.
4. Discussion To the best of our knowledge, we herein demonstrate for the first time a crucial role of MsrB3 in cancer cell death. MsrB3, which is targeted to the ER and mitochondria in human cells [11], plays an antioxidant role through specifically catalyzing the reduction of methionine-R-sulfoxide residues [13,15]. Recent studies by us and other groups show that MsrB3 is essential to hearing in mice and humans [24,25]. We have also recently found that MsrB3 is critical for the proliferation of normal cells through regulating the expression of p21 and HO-1 genes [15,16]. Down-regulation of MsrB3 inhibits proliferation of normal mammalian cells by arresting cell cycle at the G1/S stage [15]. Based on these findings, we investigated whether MsrB3 is associated with the regulation of proliferation and death of cancer cells. Our results demonstrate that down-regulation of MsrB3 induces apoptosis of cancer cells, whereas its up-regulation stimulates proliferation of cancer cells. The present study has revealed that MsrB3 down-regulation induces apoptotic cell death in cancer cells through activating the intrinsic mitochondrial apoptosis pathway (Supplementary Fig. S4). MsrB3 deficiency in MCF-7 cells resulted in increased ROS levels and redox imbalance, increased Bax/Bcl-2 ratio, cyt c release from mitochondria, activation of caspase-7 and PARP-1, and translocation of AIF to the nucleus. Based on these results, MsrB3 deficiency-induced apoptosis appeared to be mediated by
mechanisms both dependent on and independent of caspases. Caspase-7 activation induced by MsrB3 down-regulation represents a caspase-dependent mechanism, while AIF-mediated cell death initiated by PARP-1 activation is a caspase-independent mechanism [22,23]. Studies with inhibitors of caspases and PARP1 will help to elucidate the details of the mechanisms through which MsrB3 down-regulation induces cell death. It has previously been reported that MsrA, which is specific for the reduction of methionine S-sulfoxide, is also associated with the regulation of breast cancer cell proliferation [17]. However, in contrast to the apoptotic effect of MsrB3 down-regulation, MsrA down-regulation results in increased cell proliferation [17]. In addition, down-regulation of MsrA leads to a more aggressive cellular phenotype by increasing extracellular matrix degradation [17]. Based on the findings of the present and previous studies, it appears that MsrB3 and MsrA have opposite effects in cancer growth, suggesting that the methionine-R-sulfoxide and methionine-S-sulfoxide reduction systems are involved differently in the regulation of cancer cell survival. It will be interesting to investigate the effects of the other two MsrB enzymes, MsrB1 and MsrB2, on cancer cell survival. In conclusion, the present study has demonstrated for the first time that down-regulation of MsrB3 induces apoptotic cell death of cancer cells. One of the pathways leading to MsrB3 depletioninduced cell death is the activation of intrinsic mitochondrial
Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120
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apoptosis, accompanied by increased ROS and redox imbalance, caspase and PARP-1 activation, and translocation of AIF to the nucleus. Conflict of interest The authors have no conflict of interest. Acknowledgement This work was supported by the 2015 Yeungnam University Research Grant. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.12.120. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.12.120. References [1] C.C. Wu, S.B. Bratton, Regulation of the intrinsic apoptosis pathway by reactive oxygen species, Antioxid. Redox Signal 19 (2013) 546e558. [2] Q. Ran, H. Liang, M. Gu, W. Qi, C.A. Walter, L.J. Roberts 2nd, B. Herman, A. Richardson, H. Van Remmen, Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis, J. Biol. Chem. 279 (2004) 55137e55146. [3] M. Torres, Mitogen-activated protein kinase pathways in redox signaling, Front. Biosci. 8 (2003) d369e391. [4] B. D'Autreaux, M.B. Toledano, ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis, Nat. Rev. Mol. Cell Biol. 8 (2007) 813e824. [5] H. Weissbach, L. Resnick, N. Brot, Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage, Biochim. Biophys. Acta 1703 (2005) 203e212. [6] J. Moskovitz, D.B. Oien, Protein carbonyl and the methionine sulfoxide reductase system, Antioxid. Redox Signal 12 (2010) 405e415. [7] H.Y. Kim, V.N. Gladyshev, Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals, Biochem. J. 407 (2007) 321e329. [8] R.L. Levine, B.S. Berlett, J. Moskovitz, L. Mosoni, E.R. Stadtman, Methionine residues may protect proteins from critical oxidative damage, Mech. Ageing Dev. 107 (1999) 323e332. [9] H.Y. Kim, The methionine sulfoxide reduction system: selenium utilization and methionine sulfoxide reductase enzymes and their functions, Antioxid.
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Please cite this article in press as: G.-H. Kwak, et al., Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/ 10.1016/j.bbrc.2016.12.120