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Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS...
Park, Cheol; Choi, Sung Hyun; Jeong, Jin-Woo; Han, Min Ho; Lee, Hyesook; Hong, Su Hyun; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Choi, Yung Hyun
2020-01-02 00:00:00
ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 1, 60–68 https://doi.org/10.1080/19768354.2019.1706634 Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway a b c d e,f e,f g Cheol Park , Sung Hyun Choi , Jin-Woo Jeong , Min Ho Han , Hyesook Lee , Su Hyun Hong , Gi-Young Kim , h i e,f Sung-Kwon Moon , Wun-Jae Kim and Yung Hyun Choi a b Department of Molecular Biology, College of Natural Sciences, Dong-eui University, Busan, Republic of Korea; Department of System Management, Korea Lift College, Geochang, Republic of Korea; Freshwater Bioresources Utilization Bureau, Nakdonggang National Institute of d e Biological Resources, Sangju, Republic of Korea; National Marine Biodiversity Institute of Korea, Seocheon, Republic of Korea; Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan, Republic of Korea; Anti-Aging Research Center, Dong-eui University, g h Busan, Republic of Korea; Department of Marine Life Sciences, Jeju National University, Jeju, Republic of Korea; Department of Food and Nutrition, Chung-Ang University, Anseong, Republic of Korea; Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea ABSTRACT ARTICLE HISTORY Received 5 March 2019 Honokiol is one of the main active components of Magnolia officinalis, and has been demonstrated Revised 8 November 2019 to have multiple pharmacological activities against a variety of diseases. Recently, this phenolic Accepted 12 December 2019 compound is known to have antioxidant activity, but its mechanism of action remains unclear. The purpose of the current study was to evaluate the preventive effects of honokiol against KEYWORDS oxidative stress-induced DNA damage and apoptosis in C2C12 myoblasts. The present study Honokiol; oxidative stress; found that honokiol inhibited hydrogen peroxide (H O )-induced DNA damage and 2 2 DNA damage; apoptosis; ROS mitochondrial dysfunction, while reducing reactive oxygen species (ROS) formation. The inhibitory effect of honokiol on H O -induced apoptosis was associated with the up-regulation of 2 2 Bcl-2 and down-regulation of Bax, thus reducing the Bax/Bcl-2 ratio that in turn protected the activation of caspase-9 and -3, and inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blocking of cytochrome c release to the cytoplasm. Collectively, these results demonstrate that honokiol defends C2C12 myoblasts against H O -induced DNA damage 2 2 and apoptosis, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS. Introduction Although adequate levels of ROS activate important Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2- signaling pathways, chronic, persistent or persistence enylphenol) is a naturally occurring biphenolic com- or excessive production of ROS can cause oxidative pound derived from the medicinal plant Magnolia offici- damage to cells. Because mitochondria represent the nalis, which have been widely used in traditional major sources of ROS and the most vulnerable targets medicine (Lee et al. 2011). Many previous studies of ROS, an inadequate accumulation of ROS has been showed that honokiol had advantageous multi-pharma- recognized as one of the mechanisms leading to apop- cological effects, including antioxidant activity. For tosis associated with mitochondrial dysfunction. More- example, improvement of focal cerebral ischemia-reper- over, the accumulation of ROS could reduce fusion injury in the rat brain by honokiol was due to the mitochondrial membrane potential (MMP), resulting in inhibition of lipid peroxidation and reduction of neutro- compromise of the ATP production (Rigoulet et al. phil activation/infiltration through interference with the 2011; Sosa et al. 2013). Subsequently, the apoptogenic reactive oxygen species (ROS) production (Liou et al. factors are released into the cytoplasm from the mito- 2003). Similarly, blockade of oxidized low-density lipo- chondrial intermembrane space due to the loss of protein-induced mitochondrial mediated apoptosis by MMP, and the caspase cascade is activated, which honokiol in vascular endothelial cells was associated could eventually trigger apoptosis (Rigoulet et al. with the inhibition of ROS production (Ou et al. 2006). 2011; Sosa et al. 2013). CONTACT Wun-Jae Kim wjkim@chungbuk.ac.kr Department of Urology, College of Medicine and Institute for Tumor Research, Chungbuk National University, 776 1-Sunhwan-ro, Cheongju 28644, Republic of Korea; Yung Hyun Choi choiyh@deu.ac.kr Department of Biochemistry, Dongeui University College of Korean Medicine, 52-57, Yangjeong-ro, Busanjin, Busan 47227, Republic of Korea © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. DEVELOPMENTAL BIOLOGY ANIMAL CELLS AND SYSTEMS 61 Similar to many other types of cells, excessive ROS Aldrich Chemical Co.) for 20 min at 37°C. The relative produced by oxidative stress is participated in the devel- fluorescence intensity of the cell suspensions was opment of numerous muscle disorders and diseases measured by a flow cytometer (Becton Dickinson, San (Rodney and Pal 2016; Durgin and Straub 2018). Hydro- Jose, CA, USA). For image analysis of ROS production, gen peroxide (H O ), one of the major ROS, dissociates the cells were mounted on a microscope slide and 2 2 intracellularly to form highly reactive and destructive images were visualized using a fluorescence microscope hydroxyl radicals that contribute to DNA damage and (Carl Zeiss, Oberkochen, Germany). subsequent death in muscle cells (Vara and Pula 2014; Cobley et al. 2015). Therefore, potential antioxidants Apoptosis assay can have therapeutic as well as protective effects on Cells were fixed with 4% paraformaldehyde for 30 min at ROS-mediated damage to muscle cells. However, we room temperature (RT) and stained with 1.0 mg/ml of 4,6- still lack detailed insight into the mechanisms that diamidino-2-phenylindole (DAPI, Sigma-Aldrich Chemical control the defense efficacy of honokiol due to ROS pro- Co.) solution for 10 min at RT, and then washed with PBS. duction in muscle cells. The aim of this study was to The morphological changes in the nucleus were exam- explore the usefulness of honokiol for defensive ined using a fluorescence microscope. To determine against oxidative stress in muscle cells. Our findings the extent of apoptosis by a flow cytometer using show that honokiol effectively reduced H O -induced 2 2 Annexin V/propidium iodide (PI) double staining, the cytotoxicity through the attenuation of ROS production cells were collected and then stained with fluorescein iso- in cultured mouse C2C12 skeletal myoblasts. thiocyanate (FITC)-conjugated annexin V and PI (BD Phar- mingen, San Diego, CA, USA) at RT for 20 min. The Materials and methods fluorescence intensities of the cells were detected by a flow cytometer, and acquisition was performed using Cell culture and honokiol treatment the Cell Quest Pro software. C2C12 cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in DMEM Internucleosomal DNA fragmentation assay containing 10% fetal bovine serum and 100 U/ml penicil- lin and streptomycin (WelGENE Inc., Daegu, Republic of The cells were dissolved in lysis buffer [10 mM Tris-HCl Korea) at 37°C in humidified air with 5% CO . Honokiol, (pH 7.4), 150 mM NaCl, 5 mM Na-ethylenediaminetetraa- which was purchased from LKT Laboratories (St. Paul, cetic acid (EDTA), 0.5% Triton X-100, and 0.1 mg/ml pro- MN, USA), was dissolved in dimethyl sulfoxide (DMSO, teinase K] for 30 min at RT. DNA from the supernatant Sigma-Aldrich Chemical Co., St. Louis, MO, USA), and was extracted by chloroform/phenol/isoamyl alcohol diluted with cell culture medium to adjust the final treat- (24/25/1, v/v/v), and was precipitated by ethanol. The ment concentrations prior to use in the experiments. extracted DNA was then transferred to 1.5% agarose gel containing 0.1 µg/ml ethidium bromide (EtBr), and electrophoresis was carried out at 70 V. Cell viability assay For the cell viability study, the cells were incubated with Comet assay for DNA damage different concentrations of honokiol for 24 h, or pre-incu- After the respective treatments, cells were mixed with bated with honokiol for 1 h, before H O treatment for 2 2 0.75% low-melting agarose (LMA), and then transferred 24 h. The cells were also treated with 10 mM of N- to a microscope slide precoated with a layer of 0.75% acetyl cysteine (NAC) for 1 h in the presence or normal-melting agarose for solidification. The slides absence of H O . Subsequently, cell viability was deter- 2 2 were covered with LMA, and immersed in lysis solution mined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- [2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Triton X- trazolium bromide (MTT, Sigma-Aldrich Chemical Co.) 100, and 10% DMSO, pH 10] for 1 h at 4°C. The slides assay according to the previous study (Jeon et al. 2017). were then placed in a horizontal electrophoresis tank con- taining electrophoresis buffer (300 mM NaOH, 10 mM Na- Measurement of ROS level EDTA, pH 10) for 20 min. Thereafter, electrophoresis was To detect ROS production, cells were treated with or carried out in the same buffer for 20 min at 4°C. After elec- without honokiol and NAC for 1 h, before adding H O trophoresis, the slides were rinsed gently with the neu- 2 2 for a further 1 h. The cells were washed with cold phos- tralization buffer (0.4 M Tris-HCl, pH 7.5) for 10 min at phate buffered saline (PBS) and stained with 10 µM of 25°C. The slides were stained with 40 µg/ml EtBr and ′ ′ 2 ,7 -dichlorofluorescein diacetate (DCF-DA, Sigma- observed under a fluorescence microscope. Single cells 62 C. PARK ET AL. tail length and tail DNA percentage were analyzed by (Roche Applied Science, Indianapolis, IN, USA). Briefly, CometScore version 2.0 (TriTek, USA) software as previous the cells were lysed with the provided lysis buffer, and described (Jamialahmadi et al. 2014). the collected supernatants were mixed with an equal amount of luciferase agent, which catalyzed the light production from ATP and luciferin. The emitted light Western blot analysis was immediately measured using a microplate lumin- The cells were lysed with lysis buffer for 30 min to extract ometer, and the ATP level was calculated according to whole-cell proteins, as described in the previous study the ATP standard curve. (Kim et al. 2018). In a parallel experiment, mitochondrial and cytosolic proteins were extracted using a mitochon- Statistical analysis dria isolation kit (Active Motif, Carlsbad, CA, USA). The equal amounts of protein samples were subjected to Data were expressed as the mean ± standard deviation sodium-dodecyl sulfate-polyacrylamide gel electrophor- (SD) from at least three independent experiments. Stat- esis, and then transferred onto polyvinylidene fluoride istical significance analysis was carried out using membranes (Millipore, Bedford, MA, USA). Membranes ANOVA-Tukey’s post hoc test. A p-value of less than were probed with primary antibodies overnight at 4°C. 0.05 was considered to indicate statistical significance. The membranes were then incubated with the appropri- ate secondary antibodies conjugated with horseradish Results peroxidase for 2 h at RT. The protein bands were visual- ized by incubating the membranes in an enhanced che- Honokiol inhibits H O -induced cytotoxicity 2 2 miluminescence (ECL) reagent (Amersham Biosciences, Figure 1(A) shows that C2C12 cells treated with concen- Westborough, MA, USA). trations of 75 µM or more showed significant decrease in cell viability, but no significant change compared to ′′′′′ Determination of 8-hydroxy-2 -deoxyguanosine the control group was found until 50 µM. H O concen- 2 2 (8-OHdG) tration for inducing oxidative stress was selected to be 1 mM, which showed a survival rate of about 60%, com- The BIOXYTECH® 8-OHdG-EIA kit (OXIS Health Products pared with the control cells. To evaluate the protective Inc., Portland, OR, USA) was used for the quantitative effect of honokiol on H O -induced cytotoxicity, cells 2 2 measurement of oxidative DNA damage. Briefly, the cel- were treated with 20 and 40 mM honokiol for 1 h before lular DNA was isolated using the DNA Extraction Kit treatment with H O , and cultured for 24 h. Figure 1(B) 2 2 (iNtRON Biotechnology Inc., Sungnam, Republic of shows that pretreatment with honokiol significantly Korea), and quantified. The amount of 8-OHdG, a deoxyr- restored cell viability, as compared to H O alone. In 2 2 iboside form of 8-oxoGuanine, in the DNA was deter- addition, the H O -induced decrease in cell viability was 2 2 mined by calculation on a standard curve measured at completely suppressed to the control level in the cells pre- 450 nm absorbance using a microplate reader (Dynatech treated with NAC, a positive ROS scavenger (Figure 1(B)). Laboratories, Chantilly, VA, USA). Measurement of MMP (Δψm) Honokiol attenuates H O -induced ROS 2 2 generation To measure the loss of MMP, the cells were incubated in ′ ′ ′ ′ media containing 10 µM of 5,5 6,6 -tetrachloro-1,1 ,3,3 - Next, we investigated whether the protective effects of tetraethyl-imidacarbocyanine iodide (JC-1, Sigma- honokiol on H O -induced cytotoxicity were due to the 2 2 Aldrich Chemical Co.) at 37°C for 20 min at RT. After blockade of oxidative stress. As shown in Figure 1(C,D), washing twice with PBS to remove unbound dye, the the ROS generation was significantly increased within green (JC-1 monomers) and red (JC-1 aggregates) fluor- 1 h in the cells exposed to H O compared to the 2 2 escence ratio that monitored the proportion of mito- control; however, H O -induced accumulation of ROS in 2 2 chondrial depolarization was immediately acquired on cells pretreated with honokiol was significantly a flow cytometer. reduced. In the fluorescence microscope observation, we further confirmed that honokiol had a powerful ROS scavenging effect (Figure 1(E)). Also, the production Detection of ATP levels of ROS by H O was greatly blocked by pretreatment of 2 2 The levels of intracellular ATP were determined using a NAC, and the degree of ROS generation was not signifi- firefly-luciferase-based ATP Bioluminescence assay kit cantly changed in the honokiol alone group. ANIMAL CELLS AND SYSTEMS 63 Figure 1. The protective effects of honokiol against H O -induced ROS accumulation and cytotoxicity in C2C12 cells. (A and B) C2C12 2 2 cells were treated with various concentrations of honokiol for 24 h (A) or were treated with 1 mM H O for 24 h, after honokiol or NAC 2 2 pre-treatment (B). The cell viability was examined by the MTT assay. The data are shown as mean ± SD obtained from three indepen- dent experiments. Statistical analyses were conducted using analysis of variance (ANOVA-Tukey’s post hoc test) between groups. *p < # ## 0.05 and **p < 0.01 vs control group, p < 0.05 and p < 0.01 vs H O -treated group. (C-E) The cells were pretreated with honokiol or 2 2 10 mM NAC for 1 h, and then stimulated with or without 1 mM H O for 1 h. (C) After staining with DCF-DA, DCF fluorescence was 2 2 monitored by flow cytometer. (D) The data are shown as mean ± SD obtained from three independent experiments (**p < 0.01 and ## ### ***p < 0.001 vs control group, p < 0.01 and p < 0.001 vs H O -treated group). (E) The fluorescent images were obtained by a fluor- 2 2 escence microscope. Honokiol suppresses H O -induced apoptosis 2 2 However, the morphological changes were markedly attenuated in the cells pretreated with honokiol DAPI staining, flow cytometry, and agarose gel electro- before the treatment with H O . The results of 2 2 phoresis analysis were performed, to investigate Annexin V/PI double staining also showed that the pre- whether the cytoprotective effect of honokiol against treatment of honokiol significantly decreased the fre- H O was related to apoptosis suppression. The fluor- 2 2 quency of apoptotic cells in H O -stimulated cells 2 2 escent images in Figure 2(A) revealed that the (Figure 2(B,C)). In addition, the agarose gel electrophor- control cells had intact nuclei, while the H O -treated 2 2 esis result showed that H O -induced DNA cells showed significant chromatin condensation. 2 2 64 C. PARK ET AL. Figure 2. Attenuation of H O -induced apoptosis by honokiol. Cells were treated with honokiol for 1 h, and then stimulated with or 2 2 without 1 mM H O for 24 h. (A) The cells were collected, fixed, and stained with DAPI solution. The stained nuclei were pictured under 2 2 a fluorescence microscopye. (B and C) The cells cultured under the same conditions were collected, and stained with FITC-conjugated Annexin V and PI for flow cytometry analysis. (B) The percentages of apoptotic cells were determined by counting the percentage of Annexin V-positive cells. (C) Data were expressed as the mean ± SD of three independent experiments. Statistical analyses were con- ducted using analysis of variance (ANOVA-Tukey’s post hoc test) between groups. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control ## ## group, p < 0.01 and p < 0.001 vs H O -treated group. (D) DNA fragmentation was analyzed by extracting genomic DNA, electrophor- 2 2 esis in 1.5% agarose gel, and then visualizing by EtBr staining. fragmentation was completely attenuated by the pre- honokiol on DNA damage by assessing the level of 8- treatment of honokiol (Figure 2(D)). OHdG, a specific marker of DNA oxidative damage. Figure 3(D) shows that H O treatment significantly 2 2 increased the production of 8-OHdG adduct compared Honokiol reduces H O -induced DNA damage 2 2 to the control group, but pretreatment of honokiol mark- edly reduced the production of 8-OHdG by H O . 2 2 To determine whether honokiol prevents DNA damage, the comet assay was performed. Figure 3(A,B) shows that similar to the control cells, the smeared pattern of Honokiol diminishes H O -induced mitochondrial 2 2 nuclear DNA was not observed in cells treated with hon- dysfunction okiol alone. However, there was a clear increase in the length of tail in H O -treated cells. On the other hand, To examine the protective effect of honokiol on mito- 2 2 in honokiol-pretreated cells, tail length and tail DNA chondrial dysfunction by H O , MMP and intracellular 2 2 were obviously shortened. Additionally, the immuno- ATP levels were evaluated. As shown in Figure 4(A,B), blotting results showed a marked increase in γH2AX changes in ratio of the polarized and depolarized cell phosphorylation (at serine 139, p-γH2AX) in H O -stimu- populations were observed in cells treated with H O , 2 2 2 2 lated cells, compared to the untreated control cells. and the increase in depolarized mitochondrial mem- However, the increased levels of p–γH2AX by H O brane was about 8 times higher than in the control 2 2 were inhibited in the presence of honokiol (Figure 3 group. Along with the results, the concentration of ATP (C)). We also investigated the protective effect of in cells exposed to H O was significantly decreased; 2 2 ANIMAL CELLS AND SYSTEMS 65 Figure 3. Protection of H O -induced DNA damage by honokiol. Cells were pretreated with honokiol for 1 h, and then stimulated with 2 2 or without 1 mM H O for 24 h. (A) The comet assay was performed, and representative images of comet assay were taken. (B) The 2 2 statistical analysis of tail length and tail DNA percentage. Statistical analyses were conducted using analysis of variance (ANOVA- ## ### Tukey’s post hoc test) between groups. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control group, p < 0.01 and p < 0.001 vs H O - 2 2 treated group. (C) The cellular proteins were prepared, and p-γH2AX and γH2AX protein levels were assayed by Western blot analysis. (D) The amount of 8-OHdG in DNA was determined using an 8-OHdG-EIA kit. The measurements were made in triplicate, and values are expressed as the mean ± SD. Statistical analyses were conducted using analysis of variance (ANOVA-Tukey’s post hoc test) between # ## groups. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control group, p < 0.05 and p < 0.01 vs H O -treated group. 2 2 however, honokiol could significantly block these Discussion changes (Figure 4(C)). As seen in many previous studies, the accumulation of ROS due to the imbalance of ROS production and Honokiol restores H O -induced alteration of the 2 2 defense of antioxidant systems of skeletal muscle cells apoptosis regulatory genes can oxidize important components of the cells ultimately leading to DNA damage and apoptosis (Terrill et al. 2013; To further investigate the mechanisms of the anti-apopto- Pohjoismäki and Goffart 2017). Such skeletal muscle cell tic effect of honokiol, we examined the effect of honokiol death plays a crucial role in the development of muscle on H O -induced changes of apoptosis-regulated gene 2 2 atrophy, and the elevation of ROS levels is related to expression. The immunoblotting results show that the the degree of skeletal muscle cell damage in atrophic anti-apoptotic Bcl-2 protein was significantly down-regu- conditions (Zuo and Pannell 2015; Gao et al. 2018). lated in H O -treated cells, while the pro-apoptotic Bax 2 2 Recently, H O -induced oxidative stress has been protein was up-regulated. Additionally, the expression of 2 2 shown to induce functional impairment of mitochondria, pro-caspase-9 and −3 was markedly reduced in H O - 2 2 which can lead to myoblast damage (Wang et al. 2016; treated cells, and the expression of cleaved poly (ADP- Kim and Yi 2018; Ábrigo et al. 2018). Thus, these obser- ribose) polymerase (PARP) was increased (Figure 4(D)). vations suggest that the prevention of myoblast death Further, the expression of cytochrome c in H O -stimu- 2 2 by oxidative stress may be a promising strategy to lated cells was increased in the cytoplasmic fraction, indi- prevent skeletal muscle wasting. Current results have cating that cytochrome c was released from the shown that H O activated DNA damage and induced mitochondria into the cytoplasm (Figure 4(E)). However, 2 2 apoptosis by inducing ROS production in C2C12 myo- these changes by H O treatment were relatively con- 2 2 blasts; however, honokiol significantly prevented H O - served in the honokiol-pretreated cells. 2 2 66 C. PARK ET AL. Figure 4. Attenuation of H O -induced mitochondrial dysfunction and changes of apoptosis regulatory proteins by honokiol. Cells were 2 2 pretreated with honokiol for 1 h, and then stimulated with or without 1 mM H O for 24 h. (A) The cells were incubated with 10 µM JC- 2 2 1, and the values of MMP were evaluated by a flow cytometer. (B) The data are shown as mean ± SD obtained from three independent experiments. (C) ATP production was monitored using a luminometer. The results are the mean ± SD obtained from three independent experiments. Statistical analyses were conducted using analysis of variance (ANOVA-Tukey’s post hoc test) between groups. *p < 0.05, # ## **p < 0.01 and ***p < 0.001 vs control group, p < 0.05 and p < 0.01 vs H O -treated group. (D) The cellular proteins were prepared, 2 2 and the protein levels were assayed by Western blot analysis. (E) The mitochondrial and cytosolic proteins isolated from cells were separated by SDS polyacrylamide gel electrophoresis, and transferred to the membranes. The membranes were probed with anti-cyto- chrome c antibody. Equal protein loading was confirmed by the analysis of cytochrome c oxidase subunit IV (COX IV) and actin in each protein extract (M.F., mitochdrial fraction; C.F., cytosolic fraction). induced cytotoxicity, by inhibiting DNA damage through respiratory chain’s electron transport pathways, ulti- reduction of ROS accumulation. The results indicate that mately interfering with the production of intracellular H O -mediated cytotoxicity in C2C12 myoblasts were ATP (Valero 2014; Tian et al. 2017). According to 2 2 achieved by inducing ROS-dependent DNA damage. current studies, pre-treatment with honokiol signifi- The present finding is summarized in Figure 5. cantly reversed H O -induced loss of MMP and ATP 2 2 In inducing excessive ROS-mediated apoptosis, ROS contents. These results are in good agreement with overload causes free radical attack of the membrane previous studies that show that the protective effects phospholipid, which in turn leads to mitochondrial of apoptosis against oxidative stress in various muscle membrane depolarization leading to the loss of cell models are related to the maintenance of ATP pro- MMP, which is considered to be the onset of the intrin- duction by the preservation of mitochondrial function sic apoptosis pathway (Rigoulet et al. 2011; Sosa et al. (Valero 2014; Tian et al. 2017). Therefore, we consider 2013). At the same time, mitochondrial dysfunction that the conservation of ATP production due to the promotes abnormalities in the mitochondrial retention of mitochondrial membrane function is one ANIMAL CELLS AND SYSTEMS 67 ATP (Gustafsson and Gottlieb 2007; Kiraz et al. 2016). On the other hand, pro-apoptotic proteins, including Bax, antagonize anti-apoptotic proteins, or translocate to mitochondrial membranes to induce mitochondrial pore formation leading to the loss of MMP, resulting in the cytosolic release of apoptotic factors (Imahashi et al. 2004; Kulikov et al. 2012). Therefore, the balance of apoptotic Bax family proteins to the anti- apoptotic Bcl-2 family proteins serves as a determinant for activating or inhibiting the intrinsic apoptosis pathway. Many previous studies have shown that the induction of apoptosis by H O in C2C12 cells was 2 2 associated with a decrease in the Bcl-2/Bax ratio and/ or activation of caspases (Siu et al. 2009; Haramizu et al. 2017). Consistent with previous findings, our results also showed that the cytosolic release of cyto- chrome c, and the decreased expression of Bcl-2 and increased expression of Bax observed in H O -treated 2 2 cells abolished in the presence of honokiol. In addition, H O -induced activation of caspase-9 and -3, and the 2 2 degradation of PARP, were also significantly blocked by honokiol. In this respect, it is suggested that hono- Figure 5. Schematic pathways for the inhibitory effect of hono- kiol inhibits H O -induced apoptosis by decreasing the 2 2 kiol on oxidative stress-mediated DNA damage and apoptosis in C2C12 cells. increase of the Bax/Bcl-2 expression ratio, which means that the reduction reduces cytochrome c release from the mitochondria to the cytoplasm. This, in turn, pro- possible mechanism of honokiol to preserve the cell tects the activation of caspase cascade signaling survival pathway from oxidative stress. pathway. The signalings for apoptosis initiation vary depend- In summary, the present study demonstrates that ing on the stimulus, but are largely divided into the honokiol could effectively prevent H O -induced oxi- 2 2 death receptor (DR)-mediated extrinsic and mitochon- dative stress, mitochondrial dysfunction, DNA damage dria-mediated intrinsic pathways. In the extrinsic and apoptosis, through its antioxidant action in C2C12 pathway, the interaction between the death ligands myoblasts. Honokiol was also able to alter the reduced and the corresponding DRs ultimately activates ratio of Bcl-2/Bax and activation of caspases, which caspase-8. On the other hand, the initiation of the may contribute to the protective effect of honokiol on intrinsic pathway requires activation of caspase-9 by H O exposure-induced apoptosis, as shown in the com- 2 2 the release of apoptogenic factors, including cyto- posite scheme in Figure 5. Although the results of this chrome c from the mitochondria to the cytoplasm study were performed using a cell line and in vitro (Rigoulet et al. 2011; Tummers and Green 2017). Acti- assays, it may provide important information about sig- vation of the initiating caspases, including caspase-9 naling molecules protecting muscle cells under oxidative and −8, ultimately activates downstream effector cas- damage. pases, including caspase-3/-7, eventually leading to apoptosis. This process is accompanied by degradation of the substrate proteins of effector caspases, such as Disclosure statement PARP, as evidence that caspase-dependent apoptosis No potential conflict of interest was reported by the authors. is induced (Kiraz et al. 2016; Tummers and Green 2017). The activity of the caspase cascade for the induction of apoptosis is regulated by various proteins, Funding including Bcl-2 family members. Among the Bcl-2 This research was a part of the Basic Science Research Program family members, anti-apoptotic proteins, such as Bcl- through the National Research Foundation of Korea grant 2, are located on the outer mitochondrial membrane (2017R1D1A1B03032689 and 2018R1A2B2005705), and Inter- to prevent the release of apoptogenic factors, and national Science and Business Belt Program through the Minis- provide protection by inhibiting the consumption of try of Science (2017K000490) funded by the Korea government. 68 C. PARK ET AL. 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