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mTOR signaling pathway by acting on mitochondria
SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR signaling pathway by acting on mitochondria Peng Zhao, Yannong Dou, Li Chen, Linhu Li, Zhifeng Wei, Juntao Yu, Xin Wu, Yue Dai, Yufeng Xia PII: DOI: Reference:
S0367-326X(15)00109-4 doi: 10.1016/j.fitote.2015.05.002 FITOTE 3176
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
3 April 2015 28 April 2015 3 May 2015
Please cite this article as: Peng Zhao, Yannong Dou, Li Chen, Linhu Li, Zhifeng Wei, Juntao Yu, Xin Wu, Yue Dai, Yufeng Xia, SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR signaling pathway by acting on mitochondria, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.05.002
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ACCEPTED MANUSCRIPT SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR
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signaling pathway by acting on mitochondria
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Peng Zhaoa, 1, Yannong Doua, 1, Li Chen2, Linhu Li2, Zhifeng Wei1, Juntao Yu1, Xin
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Wu1, Yue Dai1, Yufeng Xia3,*
Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of
Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24
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2
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Jia Xiang, Nanjing 210009, China.
Tong Jia Xiang, Nanjing 210009, China. Department of Chinese Materia Medica Analysis, China Pharmaceutical University,
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a
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24 Tong Jia Xiang, Nanjing 210009, China.
These authors equally contribute to this paper.
*Corresponding author. Tel.: + 862583271400; fax: + 862585301528. E-mail addresses: [email protected] (Y.-f. Xia)
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ACCEPTED MANUSCRIPT Abstract (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)
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acrylamide (SC-III3), a newly synthesized derivative of scopoletin, was previously shown to reduce the viability of HepG2 cells and tumor growth of HepG2 xenograft
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mouse model. It induces the death of HepG2 cells by a way irrelvant to apoptosis and necrosis. To shed light on the cytotoxic mechanisms of SC-III3, the present study
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address whether and how it can induce autophagic cell death. When HepG2 cells were incubated with various concentrations of SC-III3, autophagic vacuoles could be observed by transmission electron microscopy and monodansylcadaverine staining.
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Increased expressions of LC3-II to LC3-I and Beclin-1, required for autophagosome
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formation, were accompanied. These characteristics integrally indicated that SC-III3 could initiate autophagy in HepG2 cells. N-acetyl-l-cysteine (NAC), a ROS scavenger,
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could reverse SC-III3-caused ROS accumulation, but it did not affect SC-III3-induced
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autophagy, suggesting that ROS was not involved in SC-III3-mediated autophagy in HepG2 cells. SC-III3 significantly depressed mitochondrial function, as evidenced by disruption of mitochondrial transmembrane potential and loss of the mitochondrial cristae structure, as well as decrease of Cox-I, Cox-III, Cox-IV, and ATP levels. The autophagy and activation of AMPK-TSC2-mTOR-p70s6k pathways induced by SC-III3 in HepG2 cells could be efficiently blocked by pretreatments of compound C (an inhibitor of AMPK). Moreover, addition of extracellular ATP to the cell culture media could reverse SC-III3-caused activation of AMPK-TSC2-mTOR-p70s6k pathway, autophagy and cell viability decrease in HepG2 cells. Collectively, SC-III3 2
ACCEPTED MANUSCRIPT leads to autophagy through inducing mitochondrial dysfunction, depleting ATP, and activating AMPK-mTOR pathway, which thus reflects the cytotoxic effect of SC-III3
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in HepG2 cells.
Keywords: SC-III3, HepG2 cell, Autophagy, Mitochondria, ATP, AMPK, mTOR
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Chemical compounds studied in the article: Scopoletin (PubChem CID: 5280460); N-acetyl-l-cysteine (PubChem CID: 12035); Monodansylcadaverine (PubChem CID:
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4247); Doxorubicin (PubChem CID: 31703); Adenosine triphosphate (PubChem CID: 5957); Lactate: (PubChem CID: 612); Compound C (PubChem CID: 11524144); Glutaraldehyde (PubChem CID: 3485); Dimethyl sulfoxide (PubChem CID: 679);
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Phenylmethylsulfonyl fluoride (PubChem CID: 4784)
Introduction
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Hepatocellular carcinoma (HCC), one of the most common lethal malignancies,
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severely impacts the health and even the lives of patients [1]. Surgical resection, liver transplantation, and chemotherapy constitute the main modalities of HCC therapy [2]. However, HCC is usually diagnosed at an advanced stage or with progression after locoregional therapy and has a dismal prognosis, owing to the underlying liver disease and the lack of effective therapeutic options [3, 4]. Therefore, novel therapeutic drugs are urgently needed to improve the efficacy of HCC therapy. Scopoletin (6-methoxy-7-hydroxycoumarin), a coumarin compound present in many plants, has been proven to possess a wide range of pharmacological properties, such as anti-angiogenic, anti-inflammatory and hypouricemic activities [5-9]. In 3
ACCEPTED MANUSCRIPT recent years, the antitumor effects of scopoletin have also been reported. For instance, it was shown to inhibit human prostate tumor cells and leukemia cells through
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inducing cell cycle arrest and triggering apoptosis [10, 11]. However, either in vitro or in vivo antitumor effect of scopoletin is far less profound, and a high elimination rate
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leads its in vivo effect to maintain only a few minutes. Some derivatives of scopoletin have been reported to exhibit better antitumor effects in vitro and in vivo than
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scopoletin [12]. We recently synthesized a series of new scopoletin derivatives with different substituents and founded that SC-III3, one of the novel scopoletin derivatives, showed potent antitumor activity at lower concentrations against most of
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the tested human tumor cell lines in vitro, and significant inhibition of the
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transplanted mouse Lewis lung carcinomas in vivo [13]. Of note, SC-III3 showed well inhibition of the growth of human hepatoma cells (HepG2 cells) in vitro and in vivo
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by inducing the generation of intracellular ROS [14].
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Further mechanistic studies in HepG2 cells demonstrated that SC-III3 induced neither cell necrosis nor apoptosis (type I programmed cell death), the primary pathway of anticancer drugs-induced cell death [15, 16]. We therefore speculate that SC-III3 might function through inducing autophagic cell death (type II programmed cell death), an alternative pattern of cell death different from apoptosis. The present study was performed to investigate the effect of SC-III3 on the autophagy of HepG2 cells, and explore its underlying mechanisms in view of mitochondrial dysfunction.
Materials and Methods Materials 4
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(E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-
3-yl) acrylamide, was prepared by Dr. Chen Li (China Pharmaceutical University).
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The structure was identified by IR, 1H NMR, and HRMS. The purity was 99.5% determined with HPLC. It was applied in DMSO (Sigma-Aldrich) to 10 mM and
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stored at -20°C. The concentrations used here were 0.03, 0.1, 0.3 and 1 µM for cellular treatment in vitro and freshly diluted with DMEM to final concentration.
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Cells in control groups were treated with the same amount of DMSO (0.01%) as used in the corresponding experiments. Doxorubicin was obtained from Shenzhen Main Luck Pharmaceuticals, Inc. and dissolved in phosphate buffered saline (PBS) to give a
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stock solution of 10 mM. The purity was 99.2% determined with HPLC. The solution
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was stored at -20°C, and kept away from light. ATP, N-acetyl-l-cysteine (NAC), and compound C were obtained from Sigma-Aldrich and applied in PBS. Antibodies
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against p-AMPK (Thr172), AMPK, p-TSC2 (Ser1387), TSC2, p-mTOR (Ser2448),
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mTOR, p-p70s6k (Thr389), p70s6k were purchased from Cell Signaling Technology. Light Chain 3B (LC3B). Beclin-1 monoclonal antibody was purchased from Beyotime Institute of Biotechnology. GAPDH monoclonal antibody was purchased from Kangchen Bio-tech; Cox-I, Cox-III and Cox-IV monoclonal antibodies were purchased from Bioworld Technology. Cell culture The human hepatoma HepG2 cell line was obtained from American Type Culture Collection. Cells were grown in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 units/mL penicillin and 60 µg/mL 5
ACCEPTED MANUSCRIPT streptomycin (Gibco) at 37°C in a humidified atmosphere comprised of 95% O2 and 5% CO2.
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Transmission electron microscopy
Cells were treated and then harvested by trypsinization, washed twice with PBS,
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fixed with a buffer containing 2.5% glutaraldehyde for 24 h and refixed in 1% osmium tetroxide for 30 min. Then, they were dehydrated in graded ethanol, washed
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with propylene oxide, embedded in Epong, and finally sectioned on a Reichert-Jung ultramicrotome at 90 nm thickness. Before being viewed with a Philips electron microscope CM-120, sections were stained with methylene buffer ArumeII according
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to the previous report [16].
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Measurement of autophagic vacuoles HepG2 cells were cultured in 96-well plates (1 × 104 cells/well), and treated with
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SC-III3. Autophagic vacuoles were detected with monodansylcadaverine (MDC) by
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incubating cells with MDC (50 μM) in PBS at 37°C for 10 min. Then, cells were washed three times with PBS and immediately analyzed by fluorescence microscopy using an inverted microscope (Nikon Eclipse TE 300). Assay of mitochondrial depolarization HepG2 cells were treated with SC-III3. Then, cells were harvested by trypsinization and washed with PBS. Cells were stained with JC-1 dye according to manufacturer's instruction (Beyotime Biotech), and then analyzed on a FACSCanto II flow cytometer (FACSCanto I, Becton Dickinson). Data acquisition and analysis were performed with a Becton-Dickinson FACSCalibur flow cytometer using Cell Quest 6
ACCEPTED MANUSCRIPT software™. Measurement of the amount of ATP in the cell
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HepG2 cells were treated and collected. Intracellular ATP levels were measured with a commercially available Colorimetric ATP Assay Kit (Jiancheng Biotech)
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according to the manufacturer’s instructions. ATP levels were normalized to the protein levels.
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Measurement of lactate generation
The Lactic Acid Assay Kit was used to quantify extracellular L-lactate and was conducted according to the manufacturer’s instructions (Jiancheng Biotech). In brief,
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cells were treated with SC-III3 for 6, 12 and 24 h, respectively. Following incubation,
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the culture media supernatant was collected for the detection of lactate generation. Absorbance values at 490 nm were obtained by Varioskan multimode microplate
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spectrophotometer (Thermo). The amount of lactate generation was calculated as
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follows: lactate generation (mM) = 3 × (ODsample − ODblank)/(ODstandard − ODblank). Apoptosis detection HepG2 cells were pretreated with/without compound C for 2 h and then exposed to SC-III3 for 24 h. Cells were harvested by trypsinization and washed with PBS. Then, cells were stained with Annexin V-FITC and PI, and loaded on a flow cytometer, set for FL1 (AnnexinV) and FL2 (PI) bivariate analysis. Data acquisition and analysis were performed with a Becton-Dickinson FACSCalibur flow cytometer using Cell Quest software. Cell viability assay 7
ACCEPTED MANUSCRIPT HepG2 cells were initiated in 96-well microplate at a density of 5×103 cells per well in 100 μL culture medium. The cells were incubated overnight and exposed to 1
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μM of SC-III3 for 24 h with or without ATP (5 mM). Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well. Plate was incubated at 37°C in a 5% CO2
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atmosphere for 4 h, the supernatants were removed and 150 μL/well DMSO was added to dissolve formazan crystals. Plate was placed on an orbital shaker for 5 min,
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and the absorbance per well was recorded at 570 nm with a Model 1500 Multiskan spectrum microplate Reader (Thermo). Data were analyzed from three independent experiments and then normalized to the absorbance of the wells containing media
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Western blotting assay
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only (0%) and untreated cells (100%).
HepG2 cells were washed twice with PBS buffer (pH 7.2) and lysed with lysis
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buffer (15 mM Tris [tris (hydro xymethyl) aminomethane] - HCl, 50 mM NaCl, 5 mM
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EGTA [ethyleneglycoltetraacetic acid], 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS [sodium dodecyl sulfate], 1 mM NaF, 1 mM PMSF [phenylmethylsulfonyl fluoride], 1 mM Na3VO4, 10 µg/mL aprotinin, and 10 µg/mL leupeptin, pH 7.4) and centrifuged. The concentrations of total proteins were measured by Bradford assay with a Varioskan spectrofluorometer and spectrophotometer (Thermo) at 595 nm. Samples were fractionated on a 10% SDS-PAGE, stacked at 80 V for 30 min and separated at 120 V for 1 h and transferred to PVDF membranes at 15 V. Membranes were blocked for 2 h at room temperature with 10% nonfat-milk and then incubated with different primary antibodies overnight at 4°C. Then, they were incubated with 8
ACCEPTED MANUSCRIPT secondary antibodies for 1 h at 37°C. The bands were visualized using film exposure with ECL reagent.
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Statistical analysis
For analysis of data, the values were presented as mean ± SEM for three
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independent experiments. The differences between the groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by a
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post hoc Tukey’s test with Prism 5 software. Results
Previously, we dmonstrated that SC-III3 was able to efficiently inhibit the growth
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of hepatocellular carcinoma through inducing the generation of intracellular ROS and
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cell death, but lack significant effect on apoptosis and necrosis of HepG2 cells. Given that autophagy is well recognized to be closely implicated in the cell death process
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[17-20], we evaluated the effect of SC-III3 on cell autophagy in HepG2 cells.
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Structural analysis via transmission electron microscopy (TEM) showed that cells treated with SC-III3 (0.03, 0.1, 0.3 and 1 μM) developed many autophagic vacuoles (autophagosomes) in the cytoplasm (Fig. 1A). Because monodansylcadaverine (MDC) accumulates in mature autophagic vacuoles such as autophago-lysosomes, MDC staining can reflect autophagic vacuoles [21]. Fig. 1B showed that SC-III3 treatment sharply increased fluorescence intensity of MDC, indicating that it increased the formation of autophagosome in HepG2 cells. We also examined the effect of SC-III3 on the expressions autophagy-related proteins. As shown in Fig. 1C, treatment of HepG2 cells with SC-III3 for 24 h increased the conversion of the microtubule 9
ACCEPTED MANUSCRIPT associated protein 1 light chain 3 (LC3-I) to the autophagosome-associated form (LC3-II), and up-regulated the protein level of Beclin-1, a key factor in the autophagy
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process. Taken together, these results indicated that SC-III3 could significantly induce
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autophagy in HepG2 cells.
Previous study demonstrated that SC-III3 could induce a significant accumulation
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of ROS in HepG2 cells, which contributes to its inhibition of cell growth [14]. Then, we tried to gain insight into the relative contribution of ROS accumulation in SC-III3-mediated autophagy. As shown in Fig. 2, NAC, a ROS scavenger, did not
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markedly reduce the fluorescence intensity of MDC and the up-regulated expressions
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of LC3-II and Beclin-1 induced by SC-III3. The data suggested that SC-III3-induced
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autophagy was not primarily dependent on ROS.
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Mitochondria, the energy factory of cells, plays an important role in cell autophagy. Reducing mitochondrial function and altering content of different intracellular parameters (e.g. ATP/AMP, ROS, Ca2+) can induce autophagy [22]. We therefore investigated whether SC-III3-mediated autophagy response is secondary to the mitochondrial dysfunction. Firstly, we examined the effect of SC-III3 treatment on mitochondrial membrane potential (MMP). The results indicated that SC-III3 (0.03, 0.1, 0.3 and 1 µM) treatment for 12 h clearly increased the population of cells with depolarized mitochondria (Fig. 3A). To further determine changes in mitochondria, we examined the ultra-structure of mitochondria by TEM. As shown in Fig. 3B, the 10
ACCEPTED MANUSCRIPT untreated HepG2 cells exhibited a normal complement of healthy-looking mitochondria having an intact cristae structure. The mitochondria in SC-III3-treated
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cells seemed to have an intact outer membrane but disrupted cristae structure. Secondly, we asked whether SC-III3 could inhibit the specific complexes of the
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electron transport chain, the ultimate end point of cellular metabolism [23]. Evaluation of the components of the electron transport chain such as Cox-I, Cox-III, and Cox-IV
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showed a concentration-dependent decrease in the levels of Cox-I Cox-III, and Cox-IV after SC-III3 treatment (Fig. 3C). The results indicated that SC-III3 could destruct the electron transport chain, the major source of cellular ATP [24]. Therefore,
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we measured the ATP content in HepG2 cells treated with SC-III3. As depicted in Fig.
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4A and B, treatment with SC-III3 resulted in a time- and concentration-dependent reduction of cellular ATP levels in HepG2 cells. After 3, 6, 12, 24 and 48 h of
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treatment, SC-III3 (1 μM) obviously reduced the ATP levels, and the inhibition rates
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were 16.7, 26.2, 41.5, 70.1 and 85.3%, respectively. Meanwhile, after 24 h of treatment, the inhibition rates caused by SC-III3 (0.03, 0.1, 0.3 and 1 μM) were 17.2, 43.9, 52.5 and 72.1%, respectively. Then, we sought to determine whether SC-III3 impairs glycolysis, another mode of energy generation for maintenance of cellular ATP levels, in HepG2 cells by measuring the production of extracellular L-lactate, the end product of glycolysis [25]. Compared with the control group, SC-III3 had no effect on the production of L-lactate, which suggested that glycolysis was not inhibited by SC-III3 (Fig. 4C, D). These observations showed that SC-III3 could destruct the function and structure 11
ACCEPTED MANUSCRIPT of mitochondria, inhibit the mitochondrial ATP production and subsequently promote
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a state of cellular starvation in HepG2 cells.
Cellular energy homeostasis is regulated by AMPK, which is activated by a
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decrease of ATP that associates with high level of AMP. Activated AMPK induces the inactivation of mTOR and its downstream effector p70s6k by phosphorylation of
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TSC2 [26-28]. In this study, the changes of the effector proteins of AMPK signaling, including AMPK, TSC2, mTOR and p70s6k, in response to SC-III3 treatment were investigated. As shown in Fig. 5A, exposing HepG2 cells to SC-III3 (0.03, 0.1, 0.3
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and 1 µM) for 24 h clearly increased the phosphorylation levels of AMPK at Thr172
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residue and TSC2 at Ser1387 residue, and dephosphorylation levels of mTOR at Ser2448 residue and p70s6k at Thr389 residue, while did not affect the protein levels
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of AMPK, TSC2, mTOR and p70s6k.
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Time-course for the activation of AMPK pathway in HepG2 cells showed that SC-III3 (1 µM) increased the phosphorylation level of AMPK (Thr172) at 3 h, which persisted up to 48 h. The phosphorylation of TSC2 (Ser1387) was also increased by SC-III3 treatment for 6 h, and maintained the elevated levels up to 48 h. SC-III3 decreased the phosphorylation level of mTOR (Ser2448) and p70s6k (Thr389) at 6 h and 12 h, respectively (Fig. 5B). These results suggested that the activation of AMPK-mTOR pathway is involved in the effect of SC-III3.
To gain insight into the relative contribution of AMPK pathway in 12
ACCEPTED MANUSCRIPT SC-III3-induced autophagy, compound C (an inhibitor of AMPK) was adopted. Pre-treatment with compound C significantly decreased the fluorescence intensity of
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MDC induced by SC-III3 (Fig. 6A). Meanwhile, the up-regulated expressions of LC3-II and Beclin-1 by SC-III3 were also reduced by compound C (Fig. 6B).
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Furthermore, compound C pretreatment markedly inhibited the ability of SC-III3 to promote the phosphorylations of AMPK and TSC2, and dephosphorylations of mTOR
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and p70s6k (Fig. 6C). It has been reported that autophagy may inhibit apoptosis of cancer cell in adverse microenvironment, and inhibition of autophagy may trigger increased induction of apoptosis in cells [29, 30]. We, therefore, explored whether
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inhibition of autophagy could increase apoptosis of HepG2 cells under SC-III3
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treatment. As shown in Fig. 6D, E, combination with compound C increased the proportion of apoptotic cells when compared with those with SC-III3 alone.
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Together, these data demonstrated that AMPK-mTOR pathway played critical
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roles in SC-III3-induced autophagy in HepG2 cells, and the autophagy might protect cells from apoptotic cell death.
Our findings mentioned above showed that SC-III3 could depress mitochondrial function, reduce ATP production, activate the AMPK-mTOR pathway, and induce autophagy in HepG2 cells. However, whether ATP depletion contributed to the autophagy by SC-III3 was unclear. By compensating for reduced ATP levels in SC-III3-treated cells through adding extracellular ATP (5 mM) to the cell culture media, it was shown that the ATP loss caused by SC-III3 was well reversed by 13
ACCEPTED MANUSCRIPT addition of extracellular ATP (Fig. 7A), and the abrogation of ATP loss led to a decrease of MDC fluorescence intensity (Fig. 7B) and up-regulated expressions of
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LC3-II and Beclin-1 induced by SC-III3 (Fig. 7C). Meanwhile, SC-III3-induced phosphorylations of AMPK and TSC2, and dephosphorylations of mTOR and p70s6k
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were down-regulated by the addition of ATP (Fig. 7D). More importantly,
extracellular ATP (Fig. 7E).
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SC-III3-mediated cell viability inhibition was dramatically reversed by addition of
These results indicated that SC-III3 evoked autophagy of HepG2 cells by inducing ATP loss and subsequently activating the AMPK-mTOR pathway, which
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participated in SC-III3-induced inhibition of cell viability.
Discussion
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In this study, SC-III3 was shown to induce mitochondrial dysfunction and
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resultant decrease of ATP production in HepG2 cells. The decreased ATP levels created a depleted energy status that led to inhibition of the mTOR pathway through AMPK, and consequently elicited autophagic cell death of the hepatoma carcinoma cells. Cell death can be executed by different mechanisms, including apoptosis, autophagy, necrosis, or combinations of these processes [29]. Autophagy, a different programmed cell death from apoptosis, is reported to play an important role in the treatment of cancer. Exposure to therapeutic agents subjects cancer cells to stress, which can elicit an autophagic response in cancer cells. Moreover, if the stress is too 14
ACCEPTED MANUSCRIPT severe or too prolonged, autophagy can become cytotoxic and lead to cell death [17, 31-35].Our previous studies showed that SC-III3 could induce cell death of HepG2
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cells, but had limited effects on cell apoptosis and necrosis [14]. Then, we explored the effects of SC-III3 on autophagic cell death. It was found that SC-III3 induced
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autophagy of HepG2 cells, which was strongly supported by the following evidence. Firstly, the presence of autophago-lysosomes in cells was confirmed by transmission
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electron microscopy, which suggested that SC-III3 induced late autophagy where the fusion between auto-phagosomes and acidic lysosomes occurred. We also determined the presence of autophagic vacuoles by staining with MDC. The fluorescence
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intensity in MDC-stained cells increased significantly with the treatment of SC-III3.
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Furthermore, we confirmed that SC-III3 could increase the specific hallmarks such as the expression levels of LC3-II and Beclin-1.
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Accumulated data point to an essential role of ROS in the activation of autophagy.
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Moreover, connections between ROS and autophagy can be observed in diverse pathological conditions [22, 25]. Previously, we showed that SC-III3-mediated cell viability inhibition was associated with the accumulation of ROS [14]. Therefore, we examined the relative contributions of ROS to SC-III3-induced autophagy. Unexpectedly, the results demonstrated that ROS was not involved in SC-III3-induced autophagy, which indicated that the autophagy by SC-III3 was executed by other mechanisms. Mitochondria are crucial regulators of the intrinsic pathway of autophagy in cancer cells [36, 37]. Mitochondrial dysfunction can be initiated by a serial of triggers, 15
ACCEPTED MANUSCRIPT including starvation, radiation, cell stress and chemicals, which can affect components of the mitochondrial respiratory chain result in inefficient ATP production [26].
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Moreover, it is well established that autophagy is induced by different types of mitochondrial stress, such as decrease of ATP production [38-40]. Our results
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indicated that SC-III3 was able to significantly inhibit mitochondrial function in HepG2 cells. For example, SC-III3 induced loss of mitochondrial membrane potential,
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destruction of mitochondrial membrane structure, and degradation of the mitochondrial electron transport chain, such as Cox-I, III and IV, in HepG2 cells. Moreover, SC-III3 could significantly decrease ATP level in a time- and
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concentration-dependent manner. Furthermore, glycolysis is likely stimulated to
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compensate for the loss of energy production by oxidative phosphorylation [25]. However, our results showed that SC-III3 had no influence on the glycolysis of
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HepG2 cells. Taken together, the mitochondrial dysfunction and consequent ATP
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depletion were involved in the effect of SC-III3. AMPK, the highly conserved energy sensing protein kinase, plays an important role in monitoring the change of cell energy and promotes cell autophagy, growth arrest, or apoptosis under stress conditions. Under conditions of energy depletion, AMPK is activated and phosphorylates TSC2, leading to turn off the mTOR pathway [27, 28, 41]. Our current findings demonstrated that the SC-III3-induced decrease of ATP level was able to create a depleted energy status, which activated AMPK and led to the inhibition of mTOR. Additionally, compound C, an AMPK inhibitor, significantly suppressed the autophagy and AMPK-TSC2-mTOR-p70s6k activation 16
ACCEPTED MANUSCRIPT triggered by SC-III3, demonstrating that AMPK played a key role in SC-III3-stimulated autophagy in HepG2 cell. However, addition of compound C also
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significantly increased the proportion of apoptotic cells when compared with those with SC-III3 alone. It was suggested that autophagy may facilitate cell survival in
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adverse microenvironment, and inhibition of autophagy may trigger increased induction of apoptosis in cells. While, excessive autophagy can also cause the death
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of cancer cells.
ATP depletion could promote a state of cellular starvation that activated the autophagy pathway through the inhibition of AMPK-TSC2-mTOR-p70s6k pathway
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[27, 28]. In this study, we found that addition of extracellular ATP to the cell culture
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media significantly increased intracellular ATP levels in SC-III3-treated cells. Moreover, addition of extracellular ATP almost completely blocked SC-III3-induced
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autophagy, AMPK-mTOR response pathways, and cell viability decrease. Collectively,
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these data demonstrated that SC-III3 induced AMPK-mTOR response pathways, subsequent autophagy and cell viability inhibition through inducing intracellular ATP-depletion.
In conclusion, we found that autophagy activation occurred in HepG2 cells treated with SC-III3, and that the change was induced by mitochondrial dysfunction and subsequent ATP depletion, which in turn activated AMPK-TSC2-mTOR-p70s6k pathway, leading to the autophagy activation. Our study provided a new insight into the anti-cancer mechanisms of SC-III3 and proposed that SC-III3 would be a potential compound for treating hepatocellular carcinoma. 17
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Acknowledgements
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This work was supported by the Fundamental Research Funds for the Central Universities (JKZ2011016), Qing Lan Project of Jiangsu Province and the Priority
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Academic Program Development of Jiangsu Higher Education Institutions, Colleges and Universities in Jiangsu Province plans to graduate research and innovation
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(CXZZ12_0326), and was partially funded by Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193). No additional external funding received for this study. The funders had no role in study design, data
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collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
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The authors declare no competing interests.
Reference
[1] El-Serag HB. Hepatocellular carcinoma, N Engl J Med 2011; 365: 1118-1127. [2] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics 2002. CA Cancer J Clin 2005; 55: 74-8. [3] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362: 1907-17. [4] Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, Christensen E, Pagliaro L, Colombo M, Rodés J. Clinical management of 18
ACCEPTED MANUSCRIPT hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: 421-30.
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T
[5] Booth NL, Nikolic D, van Breemen RB, Geller SE, Banuvar S, Shulman LP, Farnsworth NR. Confusion regarding anticoagulant coumarins in dietary supplements.
SC
Clin Pharmacol Ther 2004; 76: 511-6.
[6] Ojewole JA, Adesina SK. Cardiovascular and neuromuscular actions of scopoletin
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from Tetrapleura tetraptera. Planta Med 1983a; 49: 99-102.
[7] Pan R, Gao XH, Li Y, Xia YF, Dai Y. Anti-arthritic effect of scopoletin, a coumarin compound occurring in Erycibe obtusifolia Benth stems, is associated with
ED
decreased angiogenesis in synovium. Fundam Clin Pharmacol 2010; 24: 477-90.
factor-induced
PT
[8] Pan R, Dai Y, Gao XH, Lu D, Xia YF. Inhibition of vascular endothelial growth angiogenesis
by
scopoletin
through
interrupting
the
CE
autophosphorylation of VEGF receptor 2 and its downstream signaling pathways.
AC
Vascul Pharmacol 2011; 54: 18-28. [9] Ding ZQ, Dai Y, Wang ZT. Hypouricemic acion of scopoletin arising from xanthine oxidase inhibition and uricosuric activity. Planta Med 2005; 71: 183-5. [10] Kim EK, Kwon KB, Shin BC, Seo EA, Lee YR, Kim JS, Park JW, Park BH, Ryu DG. Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor kappaB and caspase-3. Life Sci 2005; 77: 824-36. [11] Liu XL, Zhang L, Fu XL, Chen K, Qian BC. Effect of scopoletin on PC3 cell proliferation and apoptosis. Acta Pharmacol Sin 2001; 22: 929-33.
19
ACCEPTED MANUSCRIPT [12] Bhattacharyya SS, Paul S, De A, Das D, Samadder A, Boujedaini N, Khuda-Bukhsh AR. Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic
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T
coumarin: cytotoxicity and bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target. Toxicol Appl Pharmacol 2011; 253: 270-81.
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[13] Li L, Zhao P, Hu J, Liu J, Liu Y, Wang Z, Xia Y, Dai Y, Chen L. Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid hybrids. Eur J Med
MA NU
Chem 2015; 93: 300-7.
[14] Zhao P, Chen L, Li LH, Wei ZF, Tong B, Jia YG, Kong LY, Xia YF, Yue D. SC-III3, a novel scopoletin derivative, targeted the mitochondria to induce autophagy
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cancer 2014; 14: 987.
ED
through AMPK/mTOR signaling pathway in human hepatoma HepG2 cells. BMC
[15] Vyas VK, Chintha C, Pandya MR. Biology and medicinal chemistry approaches
CE
towards various apoptosis inducers. Anticancer Agents Med Chem 2013; 13: 433-55.
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[16] Wen M, Wu J, Luo H, Zhang H. Galangin induces autophagy through upregulation of p53 in HepG2 Cells. Pharmacology 2012; 89: 247-55. [17] Levy JM, Thorburn A. Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacol Ther 2011; 131: 130-41. [18] Liu YL, Yang PM, Shun CT, Wu MS, Weng JR, Chen CC. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy 2010; 6: 1057-65. [19] Chen N, Karantza-Wadsworth V. Role and regulation of autophagy in cancer. Biochim Biophys Acta 2009; 1793: 1516-23. 20
ACCEPTED MANUSCRIPT [20] Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005; 5: 726-34.
RI P
T
[21] Balduini W, Carloni S, Buonocore G. Autophagy in hypoxia-ischemia induced brain injury. J Matern Fetal Neonatal Med 2012; 25: 30-34.
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[22] Schiavi A, Ventura N. The interplay between mitochondria and autophagy and its role in the aging process. Exp Gerontol 2014; 56: 147-53.
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[23] Scherz-Shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011; 36: 30-38.
[24] Lee I, Hüttemann M. Energy crisis: The role of oxidative phosphorylation in
ED
acute inflammation and sepsis. Biochim Biophys Acta 2014; 1842: 1579-86.
PT
[25] Venkatasubramanian R, Henson MA, Forbes NS. Incorporating energy
242: 440-53.
CE
metabolism into a growth model of multicellular tumor spheroids. J Theor Biol 2006;
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[26] Ryter SW, Mizumura K, Choi AM. The impact of autophagy on cell death modalities. Int J Cell Biol 2014; 2014: 502676. [27] Hardie DG. AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function. Genes and Development 2011; 25: 1895-908. [28] Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk B E, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 2008; 30: 214-26. [29] Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discov. 2010; 9: 447-64. 21
ACCEPTED MANUSCRIPT [30] Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated
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T
protein kinase. Cell Death Differ 2007; 14: 1948-57.
[31] White E, Karp C, Strohecker AM, Guo Y, Mathew R. Role of autophagy in
SC
suppression of inflammation and cancer. Curr Opin Cell Biol 2010; 22: 212-17. [32] Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol
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Ther 2011; 11: 157-68.
[33] O’Donovan TR, O’Sullivan GC, McKenna SL. Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following
ED
treatment with chemotherapeutics. Autophagy 2011; 7: 509-24.
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[34] Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 2004; 304: 1500-2.
AC
1773-85.
CE
[35] Yin XM, Ding WX, Gao W. Autophagy in the liver. Hepatology 2008; 47:
[36] Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 2007; 87: 99163. [37] Galluzzi L, Joza N, Tasdemir E, Maiuri M C, Hengartner M, Abrams JM, Tavernarakis N, Penninger J, Madeo F, Kroemer G. No death without life: vital functions of apoptotic effectors. Cell Death Differ. 2008; 15: 1113-23. [38] Lemasters JJ, Qian T, He L, Kim JS, Elmore SP. Cascio W. E., Role of mitochondria l inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid. Redox Signal 2002; 4: 769-81. 22
ACCEPTED MANUSCRIPT [39] Skulachev VP. Bioenergetic aspects of apoptosis, necrosis and mitoptosis. Apoptosis 2006; 11: 473-85.
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[40] Song WH, Ballard JW, Yi YJ. Sutovsky P., Regulation of Mitochondrial Genome Inheritance by Autophagy and Ubiquitin-Proteasome System: Implications for Health,
SC
Fitness, and Fertility. Biomed Res Int 2014; 2014: 9818-67.
[41] Alers S, Löffler AS, Wesselborg S, Stork B. Role of AMPK-mTOR-Ulk1/2 in the
MA NU
regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 2012; 32:
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2-11.
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Figure legends
Figure 1 SC-III3 induces evident autophagy in HepG2 cells. (A) HepG2 cells were treated with different concentrations of SC-III3 for 24 h. The images were taken by
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transmission electron microscope, and were partially enlarged to show details. The images showed numerous double-membraned cytoplasmic vacuolation (black arrows).
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(B) After treated with SC-III3 for 24 h, HepG2 cells were stained with MDC. Then,
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morphology of HepG2 cells were taken by fluorescence microscopy. (C) HepG2 cells were treated with SC-III3 for 24 h. The protein levels of LC3-II and Beclin-1 were
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analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
Figure 2 Role of ROS in SC-III3-mediated autophagy. (A) HepG2 cells were pretreated with NAC for 2 h and then exposed to SC-III3 for 24 h. The cells were stained with MDC, and morphology was taken by fluorescence microscopy. (B) HepG2 cells were pretreated with NAC for 2 h and then exposed to SC-III3 for 24 h. The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
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ACCEPTED MANUSCRIPT Figure 3 SC-III3 depresses mitochondrial function in HepG2 cells. (A) HepG2 cells were treated with SC-III3 for 24 h. Then, the cells were stained with a potential
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sensitive dye JC-1, and mitochondrial membrane potential was determined by flow cytometry. (B) HepG2 cells were treated with SC-III3 for 24 h. Then, images were
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taken by transmission electron microscope. The images showed numerous mitochondria (arrows). (C) HepG2 cells were treated with SC-III3 for 24 h. The
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protein levels of Cox-I, Cox-III, and Cox-IV were analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
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Figure 4 The effect of SC-III3 on cellular metabolism. (A) HepG2 cells were
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treated with SC-III3 (1 μM) for different times as indicated. Then, cellular ATP was measured. (B) HepG2 cells were treated with different concentrations of SC-III3 for
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24 h. Then, cellular ATP was measured. (C) HepG2 cells were treated with 1 μM
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SC-III3 for the indicated time periods and the production of extracellular lactate was measured. (D) HepG2 cells were treated with different concentrations of SC-III3 for 24 h. Then, the production of extracellular lactate was measured. The data were presented as mean ± SEM, n = 3. The difference were significant at *p < 0.05 and **p < 0.01 compared with control.
Figure 5 Effect of SC-III3 on AMPK-mTOR pathways. (A) HepG2 cells were treated with SC-III3 for 24 h. Western blot analyses of the AMPK-mTOR signaling-related proteins in HepG2 cells were performed. (B) HepG2 25
ACCEPTED MANUSCRIPT cells were treated with 1 μM of SC-III3 for different times as indicated. Western blot analyses of the AMPK-mTOR signaling-related proteins were performed in HepG2
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cells. The data were presented as mean ± SEM, n = 3.
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Figure 6 Role of AMPK-mTOR in SC-III3-mediated autophagy and apoptosis. (A) HepG2 cells were pre-incubated with 10 μM of compound C for 2 h, followed by
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incubation with 1 μM SC-III3 for 24 h. Then, cell was stained with MDC, and morphology was taken by fluorescence microscopy. (B) The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. (C) Western blot analyses of
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p-AMPK, p-TSC2, p-mTOR, and p-p70s6k in HepG2 cells were performed. (D)
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Annexin V/PI double-staining assay of HepG2 was analyzed by flow cytometry. (E) The percentages of cells in apoptosis were represented. The data were presented as
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mean ± SEM, n = 3. The difference were significant at **p < 0.01 vs. control.
Figure 7 Role of ATP in SC-III3-mediated autophagy and activation of AMPK-mTOR pathway. (A) HepG2 cells were exposed to 1 μM of SC-III3 for 24 h with or without addition of 5 mM ATP. Then, cellular ATP was measured. (B) The cell was stained with MDC, and morphology was taken by fluorescence microscopy. (C) The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. (D) Western blot analyses of p-AMPK, p-TSC2, p-mTOR, and p-p70s6k in HepG2 cells were performed. (E) The cell viability was analyzed by MTT assay. The data were presented as mean ± SEM, n = 3. **p < 0.01 compared with SC-III3 (0 μM) and ##p < 26
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Graphical abstract
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S0367-326X(15)00109-4 doi: 10.1016/j.fitote.2015.05.002 FITOTE 3176
To appear in:
Fitoterapia
Received date: Revised date: Accepted date:
3 April 2015 28 April 2015 3 May 2015
Please cite this article as: Peng Zhao, Yannong Dou, Li Chen, Linhu Li, Zhifeng Wei, Juntao Yu, Xin Wu, Yue Dai, Yufeng Xia, SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR signaling pathway by acting on mitochondria, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.05.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT SC-III3, a novel scopoletin derivative, induces autophagy of human hepatoma HepG2 cells through AMPK/mTOR
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signaling pathway by acting on mitochondria
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Peng Zhaoa, 1, Yannong Doua, 1, Li Chen2, Linhu Li2, Zhifeng Wei1, Juntao Yu1, Xin
1
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Wu1, Yue Dai1, Yufeng Xia3,*
Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of
Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24
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2
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Jia Xiang, Nanjing 210009, China.
Tong Jia Xiang, Nanjing 210009, China. Department of Chinese Materia Medica Analysis, China Pharmaceutical University,
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3
a
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24 Tong Jia Xiang, Nanjing 210009, China.
These authors equally contribute to this paper.
*Corresponding author. Tel.: + 862583271400; fax: + 862585301528. E-mail addresses: [email protected] (Y.-f. Xia)
1
ACCEPTED MANUSCRIPT Abstract (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)
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acrylamide (SC-III3), a newly synthesized derivative of scopoletin, was previously shown to reduce the viability of HepG2 cells and tumor growth of HepG2 xenograft
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mouse model. It induces the death of HepG2 cells by a way irrelvant to apoptosis and necrosis. To shed light on the cytotoxic mechanisms of SC-III3, the present study
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address whether and how it can induce autophagic cell death. When HepG2 cells were incubated with various concentrations of SC-III3, autophagic vacuoles could be observed by transmission electron microscopy and monodansylcadaverine staining.
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Increased expressions of LC3-II to LC3-I and Beclin-1, required for autophagosome
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formation, were accompanied. These characteristics integrally indicated that SC-III3 could initiate autophagy in HepG2 cells. N-acetyl-l-cysteine (NAC), a ROS scavenger,
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could reverse SC-III3-caused ROS accumulation, but it did not affect SC-III3-induced
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autophagy, suggesting that ROS was not involved in SC-III3-mediated autophagy in HepG2 cells. SC-III3 significantly depressed mitochondrial function, as evidenced by disruption of mitochondrial transmembrane potential and loss of the mitochondrial cristae structure, as well as decrease of Cox-I, Cox-III, Cox-IV, and ATP levels. The autophagy and activation of AMPK-TSC2-mTOR-p70s6k pathways induced by SC-III3 in HepG2 cells could be efficiently blocked by pretreatments of compound C (an inhibitor of AMPK). Moreover, addition of extracellular ATP to the cell culture media could reverse SC-III3-caused activation of AMPK-TSC2-mTOR-p70s6k pathway, autophagy and cell viability decrease in HepG2 cells. Collectively, SC-III3 2
ACCEPTED MANUSCRIPT leads to autophagy through inducing mitochondrial dysfunction, depleting ATP, and activating AMPK-mTOR pathway, which thus reflects the cytotoxic effect of SC-III3
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in HepG2 cells.
Keywords: SC-III3, HepG2 cell, Autophagy, Mitochondria, ATP, AMPK, mTOR
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Chemical compounds studied in the article: Scopoletin (PubChem CID: 5280460); N-acetyl-l-cysteine (PubChem CID: 12035); Monodansylcadaverine (PubChem CID:
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4247); Doxorubicin (PubChem CID: 31703); Adenosine triphosphate (PubChem CID: 5957); Lactate: (PubChem CID: 612); Compound C (PubChem CID: 11524144); Glutaraldehyde (PubChem CID: 3485); Dimethyl sulfoxide (PubChem CID: 679);
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Phenylmethylsulfonyl fluoride (PubChem CID: 4784)
Introduction
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Hepatocellular carcinoma (HCC), one of the most common lethal malignancies,
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severely impacts the health and even the lives of patients [1]. Surgical resection, liver transplantation, and chemotherapy constitute the main modalities of HCC therapy [2]. However, HCC is usually diagnosed at an advanced stage or with progression after locoregional therapy and has a dismal prognosis, owing to the underlying liver disease and the lack of effective therapeutic options [3, 4]. Therefore, novel therapeutic drugs are urgently needed to improve the efficacy of HCC therapy. Scopoletin (6-methoxy-7-hydroxycoumarin), a coumarin compound present in many plants, has been proven to possess a wide range of pharmacological properties, such as anti-angiogenic, anti-inflammatory and hypouricemic activities [5-9]. In 3
ACCEPTED MANUSCRIPT recent years, the antitumor effects of scopoletin have also been reported. For instance, it was shown to inhibit human prostate tumor cells and leukemia cells through
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inducing cell cycle arrest and triggering apoptosis [10, 11]. However, either in vitro or in vivo antitumor effect of scopoletin is far less profound, and a high elimination rate
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leads its in vivo effect to maintain only a few minutes. Some derivatives of scopoletin have been reported to exhibit better antitumor effects in vitro and in vivo than
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scopoletin [12]. We recently synthesized a series of new scopoletin derivatives with different substituents and founded that SC-III3, one of the novel scopoletin derivatives, showed potent antitumor activity at lower concentrations against most of
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the tested human tumor cell lines in vitro, and significant inhibition of the
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transplanted mouse Lewis lung carcinomas in vivo [13]. Of note, SC-III3 showed well inhibition of the growth of human hepatoma cells (HepG2 cells) in vitro and in vivo
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by inducing the generation of intracellular ROS [14].
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Further mechanistic studies in HepG2 cells demonstrated that SC-III3 induced neither cell necrosis nor apoptosis (type I programmed cell death), the primary pathway of anticancer drugs-induced cell death [15, 16]. We therefore speculate that SC-III3 might function through inducing autophagic cell death (type II programmed cell death), an alternative pattern of cell death different from apoptosis. The present study was performed to investigate the effect of SC-III3 on the autophagy of HepG2 cells, and explore its underlying mechanisms in view of mitochondrial dysfunction.
Materials and Methods Materials 4
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(E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-
3-yl) acrylamide, was prepared by Dr. Chen Li (China Pharmaceutical University).
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The structure was identified by IR, 1H NMR, and HRMS. The purity was 99.5% determined with HPLC. It was applied in DMSO (Sigma-Aldrich) to 10 mM and
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stored at -20°C. The concentrations used here were 0.03, 0.1, 0.3 and 1 µM for cellular treatment in vitro and freshly diluted with DMEM to final concentration.
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Cells in control groups were treated with the same amount of DMSO (0.01%) as used in the corresponding experiments. Doxorubicin was obtained from Shenzhen Main Luck Pharmaceuticals, Inc. and dissolved in phosphate buffered saline (PBS) to give a
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stock solution of 10 mM. The purity was 99.2% determined with HPLC. The solution
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was stored at -20°C, and kept away from light. ATP, N-acetyl-l-cysteine (NAC), and compound C were obtained from Sigma-Aldrich and applied in PBS. Antibodies
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against p-AMPK (Thr172), AMPK, p-TSC2 (Ser1387), TSC2, p-mTOR (Ser2448),
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mTOR, p-p70s6k (Thr389), p70s6k were purchased from Cell Signaling Technology. Light Chain 3B (LC3B). Beclin-1 monoclonal antibody was purchased from Beyotime Institute of Biotechnology. GAPDH monoclonal antibody was purchased from Kangchen Bio-tech; Cox-I, Cox-III and Cox-IV monoclonal antibodies were purchased from Bioworld Technology. Cell culture The human hepatoma HepG2 cell line was obtained from American Type Culture Collection. Cells were grown in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 units/mL penicillin and 60 µg/mL 5
ACCEPTED MANUSCRIPT streptomycin (Gibco) at 37°C in a humidified atmosphere comprised of 95% O2 and 5% CO2.
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Transmission electron microscopy
Cells were treated and then harvested by trypsinization, washed twice with PBS,
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fixed with a buffer containing 2.5% glutaraldehyde for 24 h and refixed in 1% osmium tetroxide for 30 min. Then, they were dehydrated in graded ethanol, washed
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with propylene oxide, embedded in Epong, and finally sectioned on a Reichert-Jung ultramicrotome at 90 nm thickness. Before being viewed with a Philips electron microscope CM-120, sections were stained with methylene buffer ArumeII according
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to the previous report [16].
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Measurement of autophagic vacuoles HepG2 cells were cultured in 96-well plates (1 × 104 cells/well), and treated with
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SC-III3. Autophagic vacuoles were detected with monodansylcadaverine (MDC) by
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incubating cells with MDC (50 μM) in PBS at 37°C for 10 min. Then, cells were washed three times with PBS and immediately analyzed by fluorescence microscopy using an inverted microscope (Nikon Eclipse TE 300). Assay of mitochondrial depolarization HepG2 cells were treated with SC-III3. Then, cells were harvested by trypsinization and washed with PBS. Cells were stained with JC-1 dye according to manufacturer's instruction (Beyotime Biotech), and then analyzed on a FACSCanto II flow cytometer (FACSCanto I, Becton Dickinson). Data acquisition and analysis were performed with a Becton-Dickinson FACSCalibur flow cytometer using Cell Quest 6
ACCEPTED MANUSCRIPT software™. Measurement of the amount of ATP in the cell
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HepG2 cells were treated and collected. Intracellular ATP levels were measured with a commercially available Colorimetric ATP Assay Kit (Jiancheng Biotech)
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according to the manufacturer’s instructions. ATP levels were normalized to the protein levels.
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Measurement of lactate generation
The Lactic Acid Assay Kit was used to quantify extracellular L-lactate and was conducted according to the manufacturer’s instructions (Jiancheng Biotech). In brief,
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cells were treated with SC-III3 for 6, 12 and 24 h, respectively. Following incubation,
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the culture media supernatant was collected for the detection of lactate generation. Absorbance values at 490 nm were obtained by Varioskan multimode microplate
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spectrophotometer (Thermo). The amount of lactate generation was calculated as
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follows: lactate generation (mM) = 3 × (ODsample − ODblank)/(ODstandard − ODblank). Apoptosis detection HepG2 cells were pretreated with/without compound C for 2 h and then exposed to SC-III3 for 24 h. Cells were harvested by trypsinization and washed with PBS. Then, cells were stained with Annexin V-FITC and PI, and loaded on a flow cytometer, set for FL1 (AnnexinV) and FL2 (PI) bivariate analysis. Data acquisition and analysis were performed with a Becton-Dickinson FACSCalibur flow cytometer using Cell Quest software. Cell viability assay 7
ACCEPTED MANUSCRIPT HepG2 cells were initiated in 96-well microplate at a density of 5×103 cells per well in 100 μL culture medium. The cells were incubated overnight and exposed to 1
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μM of SC-III3 for 24 h with or without ATP (5 mM). Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well. Plate was incubated at 37°C in a 5% CO2
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atmosphere for 4 h, the supernatants were removed and 150 μL/well DMSO was added to dissolve formazan crystals. Plate was placed on an orbital shaker for 5 min,
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and the absorbance per well was recorded at 570 nm with a Model 1500 Multiskan spectrum microplate Reader (Thermo). Data were analyzed from three independent experiments and then normalized to the absorbance of the wells containing media
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Western blotting assay
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only (0%) and untreated cells (100%).
HepG2 cells were washed twice with PBS buffer (pH 7.2) and lysed with lysis
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buffer (15 mM Tris [tris (hydro xymethyl) aminomethane] - HCl, 50 mM NaCl, 5 mM
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EGTA [ethyleneglycoltetraacetic acid], 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS [sodium dodecyl sulfate], 1 mM NaF, 1 mM PMSF [phenylmethylsulfonyl fluoride], 1 mM Na3VO4, 10 µg/mL aprotinin, and 10 µg/mL leupeptin, pH 7.4) and centrifuged. The concentrations of total proteins were measured by Bradford assay with a Varioskan spectrofluorometer and spectrophotometer (Thermo) at 595 nm. Samples were fractionated on a 10% SDS-PAGE, stacked at 80 V for 30 min and separated at 120 V for 1 h and transferred to PVDF membranes at 15 V. Membranes were blocked for 2 h at room temperature with 10% nonfat-milk and then incubated with different primary antibodies overnight at 4°C. Then, they were incubated with 8
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Statistical analysis
For analysis of data, the values were presented as mean ± SEM for three
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independent experiments. The differences between the groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by a
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post hoc Tukey’s test with Prism 5 software. Results
Previously, we dmonstrated that SC-III3 was able to efficiently inhibit the growth
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of hepatocellular carcinoma through inducing the generation of intracellular ROS and
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cell death, but lack significant effect on apoptosis and necrosis of HepG2 cells. Given that autophagy is well recognized to be closely implicated in the cell death process
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[17-20], we evaluated the effect of SC-III3 on cell autophagy in HepG2 cells.
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Structural analysis via transmission electron microscopy (TEM) showed that cells treated with SC-III3 (0.03, 0.1, 0.3 and 1 μM) developed many autophagic vacuoles (autophagosomes) in the cytoplasm (Fig. 1A). Because monodansylcadaverine (MDC) accumulates in mature autophagic vacuoles such as autophago-lysosomes, MDC staining can reflect autophagic vacuoles [21]. Fig. 1B showed that SC-III3 treatment sharply increased fluorescence intensity of MDC, indicating that it increased the formation of autophagosome in HepG2 cells. We also examined the effect of SC-III3 on the expressions autophagy-related proteins. As shown in Fig. 1C, treatment of HepG2 cells with SC-III3 for 24 h increased the conversion of the microtubule 9
ACCEPTED MANUSCRIPT associated protein 1 light chain 3 (LC3-I) to the autophagosome-associated form (LC3-II), and up-regulated the protein level of Beclin-1, a key factor in the autophagy
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process. Taken together, these results indicated that SC-III3 could significantly induce
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autophagy in HepG2 cells.
Previous study demonstrated that SC-III3 could induce a significant accumulation
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of ROS in HepG2 cells, which contributes to its inhibition of cell growth [14]. Then, we tried to gain insight into the relative contribution of ROS accumulation in SC-III3-mediated autophagy. As shown in Fig. 2, NAC, a ROS scavenger, did not
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markedly reduce the fluorescence intensity of MDC and the up-regulated expressions
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of LC3-II and Beclin-1 induced by SC-III3. The data suggested that SC-III3-induced
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autophagy was not primarily dependent on ROS.
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Mitochondria, the energy factory of cells, plays an important role in cell autophagy. Reducing mitochondrial function and altering content of different intracellular parameters (e.g. ATP/AMP, ROS, Ca2+) can induce autophagy [22]. We therefore investigated whether SC-III3-mediated autophagy response is secondary to the mitochondrial dysfunction. Firstly, we examined the effect of SC-III3 treatment on mitochondrial membrane potential (MMP). The results indicated that SC-III3 (0.03, 0.1, 0.3 and 1 µM) treatment for 12 h clearly increased the population of cells with depolarized mitochondria (Fig. 3A). To further determine changes in mitochondria, we examined the ultra-structure of mitochondria by TEM. As shown in Fig. 3B, the 10
ACCEPTED MANUSCRIPT untreated HepG2 cells exhibited a normal complement of healthy-looking mitochondria having an intact cristae structure. The mitochondria in SC-III3-treated
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cells seemed to have an intact outer membrane but disrupted cristae structure. Secondly, we asked whether SC-III3 could inhibit the specific complexes of the
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electron transport chain, the ultimate end point of cellular metabolism [23]. Evaluation of the components of the electron transport chain such as Cox-I, Cox-III, and Cox-IV
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showed a concentration-dependent decrease in the levels of Cox-I Cox-III, and Cox-IV after SC-III3 treatment (Fig. 3C). The results indicated that SC-III3 could destruct the electron transport chain, the major source of cellular ATP [24]. Therefore,
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we measured the ATP content in HepG2 cells treated with SC-III3. As depicted in Fig.
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4A and B, treatment with SC-III3 resulted in a time- and concentration-dependent reduction of cellular ATP levels in HepG2 cells. After 3, 6, 12, 24 and 48 h of
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treatment, SC-III3 (1 μM) obviously reduced the ATP levels, and the inhibition rates
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were 16.7, 26.2, 41.5, 70.1 and 85.3%, respectively. Meanwhile, after 24 h of treatment, the inhibition rates caused by SC-III3 (0.03, 0.1, 0.3 and 1 μM) were 17.2, 43.9, 52.5 and 72.1%, respectively. Then, we sought to determine whether SC-III3 impairs glycolysis, another mode of energy generation for maintenance of cellular ATP levels, in HepG2 cells by measuring the production of extracellular L-lactate, the end product of glycolysis [25]. Compared with the control group, SC-III3 had no effect on the production of L-lactate, which suggested that glycolysis was not inhibited by SC-III3 (Fig. 4C, D). These observations showed that SC-III3 could destruct the function and structure 11
ACCEPTED MANUSCRIPT of mitochondria, inhibit the mitochondrial ATP production and subsequently promote
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a state of cellular starvation in HepG2 cells.
Cellular energy homeostasis is regulated by AMPK, which is activated by a
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decrease of ATP that associates with high level of AMP. Activated AMPK induces the inactivation of mTOR and its downstream effector p70s6k by phosphorylation of
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TSC2 [26-28]. In this study, the changes of the effector proteins of AMPK signaling, including AMPK, TSC2, mTOR and p70s6k, in response to SC-III3 treatment were investigated. As shown in Fig. 5A, exposing HepG2 cells to SC-III3 (0.03, 0.1, 0.3
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and 1 µM) for 24 h clearly increased the phosphorylation levels of AMPK at Thr172
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residue and TSC2 at Ser1387 residue, and dephosphorylation levels of mTOR at Ser2448 residue and p70s6k at Thr389 residue, while did not affect the protein levels
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of AMPK, TSC2, mTOR and p70s6k.
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Time-course for the activation of AMPK pathway in HepG2 cells showed that SC-III3 (1 µM) increased the phosphorylation level of AMPK (Thr172) at 3 h, which persisted up to 48 h. The phosphorylation of TSC2 (Ser1387) was also increased by SC-III3 treatment for 6 h, and maintained the elevated levels up to 48 h. SC-III3 decreased the phosphorylation level of mTOR (Ser2448) and p70s6k (Thr389) at 6 h and 12 h, respectively (Fig. 5B). These results suggested that the activation of AMPK-mTOR pathway is involved in the effect of SC-III3.
To gain insight into the relative contribution of AMPK pathway in 12
ACCEPTED MANUSCRIPT SC-III3-induced autophagy, compound C (an inhibitor of AMPK) was adopted. Pre-treatment with compound C significantly decreased the fluorescence intensity of
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MDC induced by SC-III3 (Fig. 6A). Meanwhile, the up-regulated expressions of LC3-II and Beclin-1 by SC-III3 were also reduced by compound C (Fig. 6B).
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Furthermore, compound C pretreatment markedly inhibited the ability of SC-III3 to promote the phosphorylations of AMPK and TSC2, and dephosphorylations of mTOR
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and p70s6k (Fig. 6C). It has been reported that autophagy may inhibit apoptosis of cancer cell in adverse microenvironment, and inhibition of autophagy may trigger increased induction of apoptosis in cells [29, 30]. We, therefore, explored whether
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inhibition of autophagy could increase apoptosis of HepG2 cells under SC-III3
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treatment. As shown in Fig. 6D, E, combination with compound C increased the proportion of apoptotic cells when compared with those with SC-III3 alone.
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Together, these data demonstrated that AMPK-mTOR pathway played critical
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roles in SC-III3-induced autophagy in HepG2 cells, and the autophagy might protect cells from apoptotic cell death.
Our findings mentioned above showed that SC-III3 could depress mitochondrial function, reduce ATP production, activate the AMPK-mTOR pathway, and induce autophagy in HepG2 cells. However, whether ATP depletion contributed to the autophagy by SC-III3 was unclear. By compensating for reduced ATP levels in SC-III3-treated cells through adding extracellular ATP (5 mM) to the cell culture media, it was shown that the ATP loss caused by SC-III3 was well reversed by 13
ACCEPTED MANUSCRIPT addition of extracellular ATP (Fig. 7A), and the abrogation of ATP loss led to a decrease of MDC fluorescence intensity (Fig. 7B) and up-regulated expressions of
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LC3-II and Beclin-1 induced by SC-III3 (Fig. 7C). Meanwhile, SC-III3-induced phosphorylations of AMPK and TSC2, and dephosphorylations of mTOR and p70s6k
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were down-regulated by the addition of ATP (Fig. 7D). More importantly,
extracellular ATP (Fig. 7E).
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SC-III3-mediated cell viability inhibition was dramatically reversed by addition of
These results indicated that SC-III3 evoked autophagy of HepG2 cells by inducing ATP loss and subsequently activating the AMPK-mTOR pathway, which
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participated in SC-III3-induced inhibition of cell viability.
Discussion
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In this study, SC-III3 was shown to induce mitochondrial dysfunction and
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resultant decrease of ATP production in HepG2 cells. The decreased ATP levels created a depleted energy status that led to inhibition of the mTOR pathway through AMPK, and consequently elicited autophagic cell death of the hepatoma carcinoma cells. Cell death can be executed by different mechanisms, including apoptosis, autophagy, necrosis, or combinations of these processes [29]. Autophagy, a different programmed cell death from apoptosis, is reported to play an important role in the treatment of cancer. Exposure to therapeutic agents subjects cancer cells to stress, which can elicit an autophagic response in cancer cells. Moreover, if the stress is too 14
ACCEPTED MANUSCRIPT severe or too prolonged, autophagy can become cytotoxic and lead to cell death [17, 31-35].Our previous studies showed that SC-III3 could induce cell death of HepG2
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cells, but had limited effects on cell apoptosis and necrosis [14]. Then, we explored the effects of SC-III3 on autophagic cell death. It was found that SC-III3 induced
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autophagy of HepG2 cells, which was strongly supported by the following evidence. Firstly, the presence of autophago-lysosomes in cells was confirmed by transmission
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electron microscopy, which suggested that SC-III3 induced late autophagy where the fusion between auto-phagosomes and acidic lysosomes occurred. We also determined the presence of autophagic vacuoles by staining with MDC. The fluorescence
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intensity in MDC-stained cells increased significantly with the treatment of SC-III3.
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Furthermore, we confirmed that SC-III3 could increase the specific hallmarks such as the expression levels of LC3-II and Beclin-1.
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Accumulated data point to an essential role of ROS in the activation of autophagy.
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Moreover, connections between ROS and autophagy can be observed in diverse pathological conditions [22, 25]. Previously, we showed that SC-III3-mediated cell viability inhibition was associated with the accumulation of ROS [14]. Therefore, we examined the relative contributions of ROS to SC-III3-induced autophagy. Unexpectedly, the results demonstrated that ROS was not involved in SC-III3-induced autophagy, which indicated that the autophagy by SC-III3 was executed by other mechanisms. Mitochondria are crucial regulators of the intrinsic pathway of autophagy in cancer cells [36, 37]. Mitochondrial dysfunction can be initiated by a serial of triggers, 15
ACCEPTED MANUSCRIPT including starvation, radiation, cell stress and chemicals, which can affect components of the mitochondrial respiratory chain result in inefficient ATP production [26].
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Moreover, it is well established that autophagy is induced by different types of mitochondrial stress, such as decrease of ATP production [38-40]. Our results
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indicated that SC-III3 was able to significantly inhibit mitochondrial function in HepG2 cells. For example, SC-III3 induced loss of mitochondrial membrane potential,
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destruction of mitochondrial membrane structure, and degradation of the mitochondrial electron transport chain, such as Cox-I, III and IV, in HepG2 cells. Moreover, SC-III3 could significantly decrease ATP level in a time- and
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concentration-dependent manner. Furthermore, glycolysis is likely stimulated to
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compensate for the loss of energy production by oxidative phosphorylation [25]. However, our results showed that SC-III3 had no influence on the glycolysis of
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HepG2 cells. Taken together, the mitochondrial dysfunction and consequent ATP
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depletion were involved in the effect of SC-III3. AMPK, the highly conserved energy sensing protein kinase, plays an important role in monitoring the change of cell energy and promotes cell autophagy, growth arrest, or apoptosis under stress conditions. Under conditions of energy depletion, AMPK is activated and phosphorylates TSC2, leading to turn off the mTOR pathway [27, 28, 41]. Our current findings demonstrated that the SC-III3-induced decrease of ATP level was able to create a depleted energy status, which activated AMPK and led to the inhibition of mTOR. Additionally, compound C, an AMPK inhibitor, significantly suppressed the autophagy and AMPK-TSC2-mTOR-p70s6k activation 16
ACCEPTED MANUSCRIPT triggered by SC-III3, demonstrating that AMPK played a key role in SC-III3-stimulated autophagy in HepG2 cell. However, addition of compound C also
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significantly increased the proportion of apoptotic cells when compared with those with SC-III3 alone. It was suggested that autophagy may facilitate cell survival in
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adverse microenvironment, and inhibition of autophagy may trigger increased induction of apoptosis in cells. While, excessive autophagy can also cause the death
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of cancer cells.
ATP depletion could promote a state of cellular starvation that activated the autophagy pathway through the inhibition of AMPK-TSC2-mTOR-p70s6k pathway
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[27, 28]. In this study, we found that addition of extracellular ATP to the cell culture
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media significantly increased intracellular ATP levels in SC-III3-treated cells. Moreover, addition of extracellular ATP almost completely blocked SC-III3-induced
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autophagy, AMPK-mTOR response pathways, and cell viability decrease. Collectively,
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these data demonstrated that SC-III3 induced AMPK-mTOR response pathways, subsequent autophagy and cell viability inhibition through inducing intracellular ATP-depletion.
In conclusion, we found that autophagy activation occurred in HepG2 cells treated with SC-III3, and that the change was induced by mitochondrial dysfunction and subsequent ATP depletion, which in turn activated AMPK-TSC2-mTOR-p70s6k pathway, leading to the autophagy activation. Our study provided a new insight into the anti-cancer mechanisms of SC-III3 and proposed that SC-III3 would be a potential compound for treating hepatocellular carcinoma. 17
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Acknowledgements
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This work was supported by the Fundamental Research Funds for the Central Universities (JKZ2011016), Qing Lan Project of Jiangsu Province and the Priority
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Academic Program Development of Jiangsu Higher Education Institutions, Colleges and Universities in Jiangsu Province plans to graduate research and innovation
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(CXZZ12_0326), and was partially funded by Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193). No additional external funding received for this study. The funders had no role in study design, data
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collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
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The authors declare no competing interests.
Reference
[1] El-Serag HB. Hepatocellular carcinoma, N Engl J Med 2011; 365: 1118-1127. [2] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics 2002. CA Cancer J Clin 2005; 55: 74-8. [3] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362: 1907-17. [4] Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, Christensen E, Pagliaro L, Colombo M, Rodés J. Clinical management of 18
ACCEPTED MANUSCRIPT hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: 421-30.
RI P
T
[5] Booth NL, Nikolic D, van Breemen RB, Geller SE, Banuvar S, Shulman LP, Farnsworth NR. Confusion regarding anticoagulant coumarins in dietary supplements.
SC
Clin Pharmacol Ther 2004; 76: 511-6.
[6] Ojewole JA, Adesina SK. Cardiovascular and neuromuscular actions of scopoletin
MA NU
from Tetrapleura tetraptera. Planta Med 1983a; 49: 99-102.
[7] Pan R, Gao XH, Li Y, Xia YF, Dai Y. Anti-arthritic effect of scopoletin, a coumarin compound occurring in Erycibe obtusifolia Benth stems, is associated with
ED
decreased angiogenesis in synovium. Fundam Clin Pharmacol 2010; 24: 477-90.
factor-induced
PT
[8] Pan R, Dai Y, Gao XH, Lu D, Xia YF. Inhibition of vascular endothelial growth angiogenesis
by
scopoletin
through
interrupting
the
CE
autophosphorylation of VEGF receptor 2 and its downstream signaling pathways.
AC
Vascul Pharmacol 2011; 54: 18-28. [9] Ding ZQ, Dai Y, Wang ZT. Hypouricemic acion of scopoletin arising from xanthine oxidase inhibition and uricosuric activity. Planta Med 2005; 71: 183-5. [10] Kim EK, Kwon KB, Shin BC, Seo EA, Lee YR, Kim JS, Park JW, Park BH, Ryu DG. Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor kappaB and caspase-3. Life Sci 2005; 77: 824-36. [11] Liu XL, Zhang L, Fu XL, Chen K, Qian BC. Effect of scopoletin on PC3 cell proliferation and apoptosis. Acta Pharmacol Sin 2001; 22: 929-33.
19
ACCEPTED MANUSCRIPT [12] Bhattacharyya SS, Paul S, De A, Das D, Samadder A, Boujedaini N, Khuda-Bukhsh AR. Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic
RI P
T
coumarin: cytotoxicity and bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target. Toxicol Appl Pharmacol 2011; 253: 270-81.
SC
[13] Li L, Zhao P, Hu J, Liu J, Liu Y, Wang Z, Xia Y, Dai Y, Chen L. Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid hybrids. Eur J Med
MA NU
Chem 2015; 93: 300-7.
[14] Zhao P, Chen L, Li LH, Wei ZF, Tong B, Jia YG, Kong LY, Xia YF, Yue D. SC-III3, a novel scopoletin derivative, targeted the mitochondria to induce autophagy
PT
cancer 2014; 14: 987.
ED
through AMPK/mTOR signaling pathway in human hepatoma HepG2 cells. BMC
[15] Vyas VK, Chintha C, Pandya MR. Biology and medicinal chemistry approaches
CE
towards various apoptosis inducers. Anticancer Agents Med Chem 2013; 13: 433-55.
AC
[16] Wen M, Wu J, Luo H, Zhang H. Galangin induces autophagy through upregulation of p53 in HepG2 Cells. Pharmacology 2012; 89: 247-55. [17] Levy JM, Thorburn A. Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacol Ther 2011; 131: 130-41. [18] Liu YL, Yang PM, Shun CT, Wu MS, Weng JR, Chen CC. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy 2010; 6: 1057-65. [19] Chen N, Karantza-Wadsworth V. Role and regulation of autophagy in cancer. Biochim Biophys Acta 2009; 1793: 1516-23. 20
ACCEPTED MANUSCRIPT [20] Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005; 5: 726-34.
RI P
T
[21] Balduini W, Carloni S, Buonocore G. Autophagy in hypoxia-ischemia induced brain injury. J Matern Fetal Neonatal Med 2012; 25: 30-34.
SC
[22] Schiavi A, Ventura N. The interplay between mitochondria and autophagy and its role in the aging process. Exp Gerontol 2014; 56: 147-53.
MA NU
[23] Scherz-Shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011; 36: 30-38.
[24] Lee I, Hüttemann M. Energy crisis: The role of oxidative phosphorylation in
ED
acute inflammation and sepsis. Biochim Biophys Acta 2014; 1842: 1579-86.
PT
[25] Venkatasubramanian R, Henson MA, Forbes NS. Incorporating energy
242: 440-53.
CE
metabolism into a growth model of multicellular tumor spheroids. J Theor Biol 2006;
AC
[26] Ryter SW, Mizumura K, Choi AM. The impact of autophagy on cell death modalities. Int J Cell Biol 2014; 2014: 502676. [27] Hardie DG. AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function. Genes and Development 2011; 25: 1895-908. [28] Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk B E, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 2008; 30: 214-26. [29] Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discov. 2010; 9: 447-64. 21
ACCEPTED MANUSCRIPT [30] Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated
RI P
T
protein kinase. Cell Death Differ 2007; 14: 1948-57.
[31] White E, Karp C, Strohecker AM, Guo Y, Mathew R. Role of autophagy in
SC
suppression of inflammation and cancer. Curr Opin Cell Biol 2010; 22: 212-17. [32] Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol
MA NU
Ther 2011; 11: 157-68.
[33] O’Donovan TR, O’Sullivan GC, McKenna SL. Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following
ED
treatment with chemotherapeutics. Autophagy 2011; 7: 509-24.
PT
[34] Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 2004; 304: 1500-2.
AC
1773-85.
CE
[35] Yin XM, Ding WX, Gao W. Autophagy in the liver. Hepatology 2008; 47:
[36] Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 2007; 87: 99163. [37] Galluzzi L, Joza N, Tasdemir E, Maiuri M C, Hengartner M, Abrams JM, Tavernarakis N, Penninger J, Madeo F, Kroemer G. No death without life: vital functions of apoptotic effectors. Cell Death Differ. 2008; 15: 1113-23. [38] Lemasters JJ, Qian T, He L, Kim JS, Elmore SP. Cascio W. E., Role of mitochondria l inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid. Redox Signal 2002; 4: 769-81. 22
ACCEPTED MANUSCRIPT [39] Skulachev VP. Bioenergetic aspects of apoptosis, necrosis and mitoptosis. Apoptosis 2006; 11: 473-85.
RI P
T
[40] Song WH, Ballard JW, Yi YJ. Sutovsky P., Regulation of Mitochondrial Genome Inheritance by Autophagy and Ubiquitin-Proteasome System: Implications for Health,
SC
Fitness, and Fertility. Biomed Res Int 2014; 2014: 9818-67.
[41] Alers S, Löffler AS, Wesselborg S, Stork B. Role of AMPK-mTOR-Ulk1/2 in the
MA NU
regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 2012; 32:
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CE
PT
ED
2-11.
23
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ACCEPTED MANUSCRIPT
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Figure legends
Figure 1 SC-III3 induces evident autophagy in HepG2 cells. (A) HepG2 cells were treated with different concentrations of SC-III3 for 24 h. The images were taken by
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transmission electron microscope, and were partially enlarged to show details. The images showed numerous double-membraned cytoplasmic vacuolation (black arrows).
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(B) After treated with SC-III3 for 24 h, HepG2 cells were stained with MDC. Then,
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morphology of HepG2 cells were taken by fluorescence microscopy. (C) HepG2 cells were treated with SC-III3 for 24 h. The protein levels of LC3-II and Beclin-1 were
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analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
Figure 2 Role of ROS in SC-III3-mediated autophagy. (A) HepG2 cells were pretreated with NAC for 2 h and then exposed to SC-III3 for 24 h. The cells were stained with MDC, and morphology was taken by fluorescence microscopy. (B) HepG2 cells were pretreated with NAC for 2 h and then exposed to SC-III3 for 24 h. The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
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ACCEPTED MANUSCRIPT Figure 3 SC-III3 depresses mitochondrial function in HepG2 cells. (A) HepG2 cells were treated with SC-III3 for 24 h. Then, the cells were stained with a potential
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sensitive dye JC-1, and mitochondrial membrane potential was determined by flow cytometry. (B) HepG2 cells were treated with SC-III3 for 24 h. Then, images were
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taken by transmission electron microscope. The images showed numerous mitochondria (arrows). (C) HepG2 cells were treated with SC-III3 for 24 h. The
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protein levels of Cox-I, Cox-III, and Cox-IV were analyzed with western blotting. The data were presented as mean ± SEM, n = 3.
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Figure 4 The effect of SC-III3 on cellular metabolism. (A) HepG2 cells were
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treated with SC-III3 (1 μM) for different times as indicated. Then, cellular ATP was measured. (B) HepG2 cells were treated with different concentrations of SC-III3 for
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24 h. Then, cellular ATP was measured. (C) HepG2 cells were treated with 1 μM
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SC-III3 for the indicated time periods and the production of extracellular lactate was measured. (D) HepG2 cells were treated with different concentrations of SC-III3 for 24 h. Then, the production of extracellular lactate was measured. The data were presented as mean ± SEM, n = 3. The difference were significant at *p < 0.05 and **p < 0.01 compared with control.
Figure 5 Effect of SC-III3 on AMPK-mTOR pathways. (A) HepG2 cells were treated with SC-III3 for 24 h. Western blot analyses of the AMPK-mTOR signaling-related proteins in HepG2 cells were performed. (B) HepG2 25
ACCEPTED MANUSCRIPT cells were treated with 1 μM of SC-III3 for different times as indicated. Western blot analyses of the AMPK-mTOR signaling-related proteins were performed in HepG2
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cells. The data were presented as mean ± SEM, n = 3.
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Figure 6 Role of AMPK-mTOR in SC-III3-mediated autophagy and apoptosis. (A) HepG2 cells were pre-incubated with 10 μM of compound C for 2 h, followed by
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incubation with 1 μM SC-III3 for 24 h. Then, cell was stained with MDC, and morphology was taken by fluorescence microscopy. (B) The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. (C) Western blot analyses of
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p-AMPK, p-TSC2, p-mTOR, and p-p70s6k in HepG2 cells were performed. (D)
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Annexin V/PI double-staining assay of HepG2 was analyzed by flow cytometry. (E) The percentages of cells in apoptosis were represented. The data were presented as
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mean ± SEM, n = 3. The difference were significant at **p < 0.01 vs. control.
Figure 7 Role of ATP in SC-III3-mediated autophagy and activation of AMPK-mTOR pathway. (A) HepG2 cells were exposed to 1 μM of SC-III3 for 24 h with or without addition of 5 mM ATP. Then, cellular ATP was measured. (B) The cell was stained with MDC, and morphology was taken by fluorescence microscopy. (C) The protein levels of LC3-II and Beclin-1 were analyzed with western blotting. (D) Western blot analyses of p-AMPK, p-TSC2, p-mTOR, and p-p70s6k in HepG2 cells were performed. (E) The cell viability was analyzed by MTT assay. The data were presented as mean ± SEM, n = 3. **p < 0.01 compared with SC-III3 (0 μM) and ##p < 26
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