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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition Q4
Feng-Ze Wang a, b, Zheng-Yao Chang a, Hong-Rong Fei c, Ming-Feng Yang b, Xiao-Yi Yang b, Bao-Liang Sun b, d, * a
Q1
School of Biological Science, Taishan Medical University, Taian 271016, PR China Key Lab of Cerebral Microcirculation in Universities of Shandong, Taishan Medical University, Taian 271000, PR China School of Pharmacology, Taishan Medical University, Taian 271016, PR China d Department of Neurology, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2014 Accepted 22 April 2014 Available online xxx
PI3K/Akt/mTOR pathway plays an important role in tumor progression and anti-cancer drug resistance. The aim of the present study is to determine the antitumor effect of CCT128930, a novel small molecule inhibitor of Akt, in the HepG2 hepatoma cancer cells. Our results showed that at low concentrations, CCT128930 increased, but not inhibited, the phosphorylation of Akt in HepG2 and A549 cells. CCT128930 inhibited cell proliferation by inducing cell cycle arrest in G1 phase through downregulation of cyclinD1 and Cdc25A, and upregulation of p21, p27 and p53. A higher dose (20 mM) of CCT128930 triggered cell apoptosis with activation of caspase-3, caspase-9, and PARP. Treatment with CCT128930 increased phosphorylation of ERK and JNK in HepG2 cells. CCT128930 activated DNA damage response of HepG2 cell characterized by phosphorylation of H2AX, ATM (ataxia-telangiectasia mutated), Chk1 and Chk2. Upon exposure to CCT128930 at a higher concentration, HepG2 cells exhibited autophagy was accompanied by an increase the levels of LC3-II and Beclin-1. Blocking autophagy using chloroquine magnified CCT128930-induced apoptotic cell death and the phosphorylation of H2AX. The results in this study have advanced our current understandings of the anti-cancer mechanisms of CCT128930 in cancer cells. Ó 2014 Published by Elsevier Masson SAS.
Keywords: CCT128930 Akt Cell cycle DNA damage Autophagy
1. Introduction It is widely accepted that PI3K/Akt/mTOR pathway plays a key role in controlling cell growth, apoptosis and cell differentiation. Mutations or genetic alterations of many components of the PI3K/ Akt pathway frequently occur in human cancers [1]. Signaling through PI3K drives a number of cell functions by the activation of the Akt protein (also known as protein kinase B, PKB). The downstream effector of PI3K, Akt, increases the cell cycle progression by blocking FOXO-mediated transcription of cell cycle inhibitors that lead to the accumulation and activation of the mTOR-raptor kinase complex, which in turn mediates protein kinase p70S6K1 activation to regulate protein synthesis and cell proliferation [2]. mTOR functions as a central element in a signaling pathway involved in the control of cell proliferation, apoptosis and autophagy, an ubiquitous and evolutionarily conserved process that degrades
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* Corresponding author. Department of Neurology, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, PR China. E-mail addresses: [email protected], [email protected] (B.-L. Sun).
cytosolic components via the lysosomes and allows cells to survive various forms of stress [3]. Many studies have shown that DNA damage or replication errors results in the inhibition of Cdks activity via activation of the cell cycle checkpoint, which is essential for preventing entry into mitosis in the presence of genomic defects [4,5]. The key regulators of the checkpoint pathways in eukaryotic cell DNA damage response are ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases, which are activated in response to DNA damaging agents such as ionizing radiation, ultraviolet light and DNA replication inhibitors [6]. The downstream target kinases of ATR/ATM in response to DNA damage are Chk1 and Chk2. ATR/ATM activate Chk1 and Chk2 protein kinases responsible for the inhibition via phosphorylation of the Cdc25 phosphatase. Although Chk1 or Chk2 are known to trigger G2/M phase cell cycle arrest, these kinases are also known to mediate ATM-dependent G1 checkpoint due to activating p53 tumor suppressor protein by phosphorylation [7,8]. Autophagy is a conservative process of protein degradation through autophagosome and lysosome system, which generally
http://dx.doi.org/10.1016/j.biochi.2014.04.008 0300-9084/Ó 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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functions as a homeostasis mechanism to maintain a balance between the synthesis and degradation of cellular products. The role of autophagy in cancer and cell death is apparently quite complex and poorly understood. Evidence indicates that the predominant function of autophagy in cancer cells is to confer stress tolerance, which contributes to tumor cell survival during cancer progression. On the other hand, induction of autophagic cell death has been proposed as a mechanism of cell death, given that characteristics of autophagy have been discovered in dying cells [9]. Similar to its potential to either promote cell survival or induce cell death, a large body of research shows a paradoxical role of autophagy during chemotherapy [10]. There are increasing evidences indicates that PI3K/Akt/mTOR inhibitors initiate autophagy as a survival program that may interfere with their antitumor activity [11]. Therefore, autophagy may be a potential resistance mechanism and its abrogation may be increase the cytotoxicity of mTOR inhibition. The prevalence of PI3K/Akt/mTOR signaling abnormalities in cancer cells has suggested the potential use of this pathway as a novel therapeutic target. Inhibitors of this signaling pathway are being actively developed and tested for tumor therapy [12e14]. In this study, we investigate a potential antitumor activity of CCT128930 in vitro models. We demonstrate that CCT128930 inhibits the cell proliferation by inducing G1 cell cycle arrest. At high concentrations, CCT128930 causes apoptosis. Activation of g-H2AX, ATM, Chk1 and Chk2 may be responsible for the DNA damage in CCT128930-treated HepG2 cells. CCT128930 also triggers the autophagy, as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by chloroquine (CQ) further increases the cytotoxicity of CCT128930. Our results indicate that CCT128930 might represent a promising drug for the treatment of cancer.
medium was then removed, and cells were treated with various concentrations of CCT128930. An amount of 20 mL MTT (5 mg/mL) was added for 4 h. After removing supernatant, 150 mL DMSO was added to resolve formazan crystals, and the absorbance was detected at 490 nm. For cell colony formation assay, HepG2 cells were seeded at 1500 cells per well (6-well cell culture plate) and treated with different concentrations of CCT128930 for 2 weeks. Cells were fixed in 1% glutaraldehyde and stained with 0.5% crystal violet. Colonies with >30 cells were counted under an inverted microscope. 2.4. Cell cycle analysis Cells were treated with CCT128930 for 24 h. At the end of the treatment, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS), and fixed overnight in cold 70% ethanol at 4 C. After washing with PBS, the cells was digested with RNase A and stained with PI. Samples were analyzed for their DNA content by using a FACSCalibur flow cytometry with CellQuest software (Becton Dickinson, USA). 2.5. Annexin V-FITC/PI staining Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. To assess the extent of apoptosis induction, cells were harvested (adherent and suspended cells) and stained according to the manufacturer’s instructions. Samples were analyzed with the FACScan flow cytometry and CellQuest analysis software (Becton Dickinson). 2.6. Western blot analysis
2. Materials and methods 2.1. Reagent CCT128930 was purchased from Sata Cruz Biotechnology (Shanghai, China), and dissolved with DMSO. Crystal violet and 3[4,5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) and chloroquine were obtained from SigmaeAldrich (St. Louis, USA). PD98059 was purchased from Promega (Madison, USA). SP600129 was obtained from Calbiochem (CA, USA). Annexin V-FITC apoptosis detection kit was obtained from KeyGEN Biotech (Nanjing, China). Western Lightning Plus ECL was purchased from perkinelmer (MA, USA). Antibodies specific to b-actin, Beclin-1 and LC3-II were purchased from SigmaeAldrich. Rabbit anti-Akt, p-Akt, p70SK6, caspase-9, caspase-3, PARP, ATM, p-ATM, ATR, p-ATR, p-Chk2, Chk1, p-Chk1, g-H2AX, p-p53, p-p38, JNK and Cdc2 polyclonal antibodies were obtained from Cell Signaling Technology (Shanghai, China). Mouse anti-Chk2, p21, p27 and p-JNK were purchased from BD Biosciences. Anti-cyclinD1, Cdc25A, Cdc25B, Cdc25C, cyclinB1, p53, ERK and p-ERK were purchased from Santa cruz Biotechnology.
After cells were lysed with ice-cold lysis buffer (150 mM NaCl, 20 mM TriseHCl, 1% NP-40, 0.5% Na-DOC, 0.1% SDS, 0.2 mmol/L PMSF, and protease inhibitor cocktails) for 20 min on ice. Cell supernatants were obtained by centrifugation and the protein concentration was determined. 30 mg protein was used for western blotting analysis. Detection of the target proteins on the membranes was performed using the ECL Western Blotting Detection Reagents. 2.7. Immunoflourescence assay Cultured cells were fixed with cold 100% methanol at 4 C for 10 min and permeabilized with PBS-0.1%Triton X-100 for 10 min. After blocked with 3% BSA, the cells were incubated with anti-gH2AX antibody. After washing, cells were incubated with FITCconjugated secondary antibodies (Santa cruz, 1:200 dilution), and DAPI was used to stain the nuclei. Fluorescence images were obtained using a fluorescence microscope with appropriate filter sets. 2.8. Statistical analysis
2.2. Cells and cell culture HepG2 cell and A549 cell were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and was cultured in RMPI 1640 medium containing 10% fetal calf serum (Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified atmosphere (37 C; 5% CO2).
All data were expressed as mean SD. Statistical analysis was performed by the student’s t-test. P < 0.05 was indicated to be statistical significant. 3. Results
2.3. Cell viability assay and cell colony formation assay
3.1. CCT128930 inhibits cell proliferation in a concentrationdependent manner
MTT assay was used to detect cell viability, as reported elsewhere [15]. Briefly, cells were plated in 96-well plates for 24 h. The
CCT128930 was reported to be a PI3K/mTOR inhibitor [16], and then we detect the effects of CCT128930 on the phosphorylation of
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 1. The effect of CCT128930 on the phosphorylation state of Akt and p70S6K in HepG2 and A549 cells. Cells were incubated with CCT128930 at the indicated concentrations for 24 h and then processed for western blotting analysis. b-actin was used as the loading control.
Akt, an activator of mTOR, and its downstream effector p70 ribosomal protein S6 kinase (p70S6K) in HepG2 and A549 cells. As shown in Fig. 1, CCT128930 treatment did not inhibit the phosphorylation of Akt (Ser473) and p70S6K (Thr389) at indicated concentrations. Much to our surprise, when the cells were treated with lower concentration of CCT128930, an increase in phosphorylation of Akt was observed. To investigate the effects of CCT128930 on the growth of cancer cells, MTT assay was used to evaluate the HepG2 and A549 cell viability. The results showed that CCT128930 decreased the viability of cancer cells in a dose-dependent manner (IC50 ¼ 30.03 mM in HepG2 cell and IC50 ¼ 32.94 mM in A549 cell) (Fig. 2A). Colony formation assay revealed that treatment with 10 mM CCT128930 totally blocked the formation of colonies,
confirming the anti-cancer activity of CCT128930 against cancer cells (Fig. 2B). Light microscopy observation of CCT128930-treated HepG2 cells revealed morphological changes accompanying shrinkage of the cell size and constriction of cell shape, which may be an early indicator of responses to cell growth inhibition or apoptotic stimuli (Fig. 2C). 3.2. CCT128930 induces G1 phase arrest in HepG2 and A549 cells For finding whether the growth inhibitory function of CCT128930 was associated with its effect on cell cycle progression, we investigated changes of cell cycle distribution in CCT128930treated cancer cells by flow cytometric analysis. As shown in Fig. 3A, CCT128930 treatment significantly increased G1 phase cells
Fig. 2. CCT128930 inhibited the growth of cancer cells. (A) CCT128930 reduced the viability of HepG2 and A549 cells. Cells were seeded at a density of 1 106 cells/mL in 96-well polystyrene culture plates at 37 C with 5% (v/v) CO2 for one day. After 24 h of incubation, cells were incubated with CCT128930 for 24 h and then processed for MTT assay. (B) CCT128930 inhibits the colony formation ability of HepG2 cell. The histogram represents the mean colony number of 3 independent experiments (mean SD). *P < 0.05, **P < 0.01 compared with the control. (C) Morphological changes in CCT128930-treated HepG2 cells. Morphological changes observed in the HepG2 cells after 24 h of treatment with various concentrations of CCT128930.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 3. CCT128930 induced G1 phase arrest in HepG2 and A549 cells. (A) CCT128930 was observed to induce G1 phase arrest. Upon exposure to CCT128930 at indicated concentrations for 24 h, both HepG2 cells and A549 cells all exhibited G1 phase arrest. (B) Expression of the cell cycle-associated proteins. HepG2 cells were treated with CCT128930 for the indicated concentrations. Cell lysates were fractionated on SDS-polyacrylamide gels and analyzed by western blotting with antibodies against cell cycle associated proteins. bactin was shown as loading control.
in HepG2 and A549 cells. Investigation of the pivotal proteins involved in cell cycle transition by CCT128930 showed that expression of p21, p27 and p53 was increased by CCT128930 treatment (Fig. 3B). 3.3. High doses of CCT128930-induced HepG2 cell apoptosis Next, we performed experiments to determine whether this inhibitory effect of CCT128930 on cell viability resulted from apoptotic cell death. Apoptotic or necrotic cells were detected after incubation with increasing concentrations of CCT128930 for 24 h.
Annexin V/PI flow cytometry results revealed that apoptotic rate in 20 mM CCT128930 treatment HepG2 cells increased from 5.33% to 12.62% (Fig. 4A), these results indicated that high dose of CCT128930 induced the cell death in HepG2 cells. To elucidate whether CCT128930-treated hepG2 cells express typical apoptotic markers, we used western blotting analysis to determine the cleavage of apical pro-caspase-3, pro-caspase-9, and PARP in HepG2 cells. As shown in Fig. 4B, HepG2 cells exhibited significantly increased the cleavage of caspase-3, caspase-9 and PARP after exposure to 20 mM CCT128930, suggesting enhanced apoptotic activity in HepG2 cells.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 4. High dose of CCT128930 induced cell apoptosis in HepG2 cells. (A) Annexin V/FITC-PI FACS assay of HepG2 cells exposed to various doses of CCT128930 (0e20 mM) for 24 h. The percentage of early apoptotic cells with exposed CCT128930 is located in the lower right quadrant and the percentage of necrotic/apoptosis in terminal stages is located in the upper right quadrant. Data are representative of one of three repeats. (B) Western blotting analysis of apoptosis-related proteins. HepG2 cells were exposed to CCT128930 for 24 h, and western blot analysis was carried out to detect the cleavage of caspases-9, caspase-3 and PARP.
3.4. ERK and JNK are required for CCT128930-induced cytotoxicity in HepG2 cells
3.5. CCT128930 activates DNA damage sensor kinases and associates cellular checkpoints in HepG2 cells
Mitogen-activated protein kinase (MAPK) signaling pathway activation contributes to decrease cell proliferation and inducing apoptosis in response to a variety of extra cellular stresses [17], we then determined whether the activity of ERK, JNK and p38 MAPK were upregulated in HepG2 cells after treatment with CCT128930. Using western blotting analysis, we found that phosphorylation of ERK and JNK protein was increased in HepG2 cells after exposure to CCT128930 for 24 h (Fig. 5A). However, no significant changes of phosphorylated p38 MAPK were observed from the western blotting results. To assess whether CCT128930-induced cell viability inhibition was dependent on ERK and JNK activation, HepG2 cells were cultured with CCT128930 (20 mM) either alone or in combination with inhibitors of ERK (PD98059) and JNK1/2 (SP600129) for 24 h, and MTT assays were carried out. As shown in Fig. 5B, inhibition of cell viability by CCT128930 was partially alleviated in the presence of PD98059 or SP600125, suggesting a dependence on ERK and JNK signaling for the CCT128930 mediates the cell growth and survival.
To further elucidate the possible mechanism of CCT128930induced cell cycle arrest and apoptosis, we detected the DNA damage after CCT128930 exposure by examined the expression of phosphorylation of H2AX (g-H2AX), an indicator of the presence of DNA damage. We observed an increased phosphorylation of H2AX at Ser-139 in CCT128930-treated HepG2 cells (Fig. 6A). Immunofluorescence staining also showed an increased formation of gH2AX foci in CCT128930-treated HepG2 cells (Fig. 6B). Both ATM and ATR play a central role in coordinating the DNA damage response, including cell cycle checkpoint control and apoptosis [18]. We therefore measured the effect of CCT128930 on the key signaling proteins of this pathway. Treatment of HepG2 cells with indicated concentrations of CCT128930 increased the phosphorylation of ATM at Ser-1981 without any change in the total levels of ATM. However, we did not observe any changes in the phosphorylation of ATR (Ser428) in CCT128930-treated cells (Fig. 6C). Since phosphorylation of Chk1 and Chk2 also serves as an indicator of checkpoint activation in response to DNA damage, we determined whether the phosphorylation of Chk1 and Chk2 were affected in CCT128930-
Fig. 5. Pretreatment ERK and JNK inhibitor partially reduced CCT128930-inhbited cell viability. (A) Cells were treated with different doses of CCT128930 for 24 h and protein expression was determined by western blotting analysis. (B) To investigate the effect of the ERK and JNK inhibitor on CCT128930-inhibited cell viability, HepG2 cells were pretreated as before with 20 mM PD98059 (PD) and 20 mM SP600125 (SP), respectively. After 24 h incubation with CCT128930 (20 mM), cell viability was analyzed using MTT assay. *Indicates that P < 0.05 versus control cells.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 6. Effect of CCT128930 on DNA damage responses. (A) DNA double-strand breaks, as measured by g-H2AX, were induced by CCT128930 treatment. Whole cell lysates were prepared and subjected to western blotting using antibodies against indicated proteins. b-actin antibody was used as an internal control to show equal protein loading. (B) Detection of g-H2AX by immunofluorescence. Cells were treated with the indicated concentrations of CCT128930 for 24 h, stained with rabbit anti-g-H2AX (Ser139) antibody and goat antirabbit FITC-conjugated secondary antibody (green), and then counterstained with DAPI (blue). (C) CCT128930 regulates the phosphorylation of ATM, Chk1, and Chk2 in HepG2 cells. Cells were treated with different doses of CCT128930 for 24 h, and then western blotting analysis was performed with antibodies against the indicated proteins. b-actin antibody was used as a loading control.
treated cells. As shown in Fig. 6C, exposure to CCT128930 significantly increased the phosphorylation of Chk1 and Chk2 in HepG2 cells. 3.6. Inhibition of autophagy exacerbates the cytotoxic activity of CCT128930
we found that the inhibition of autophagy with CQ may be potentiating CCT128930-evoked DNA damage response, because the inhibition of autophagy significantly enhanced CCT128930induced phosphorylation of H2AX in HepG2 cells (Fig. 8). 4. Discussion
Autophagy is the basic catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes; we then determined whether CCT128930 induced autophagy in HepG2 cells. We detected the levels of LC3-II for monitoring autophagy because it has been widely used for estimating the abundance of the autophagosome or autophagy [19]. HepG2 cells were transfected with RFP-GFP-LC3 for 24 h; the cells were then treated with CCT128930 for 24 h. As presented in Fig. 7A, CCT128930 induced the redistribution of GFP-LC3 from a diffuse pattern to punctate structures. Moreover, an increase in LC3-II expression was observed after treatment of CCT128930 in HepG2 cells. Beclin1 (Atg6), a wellknown key regulator of autophagy, was also upregulated in CCT128930-treated HepG2 cells (Fig. 7B). To determine whether induction of autophagy by CCT128930 is a survival or death mechanism, we detected the CCT128930induced apoptosis in the presence of CQ, a lysosomal protease inhibitor. As shown in Fig. 7C, the presence of 20 mM CQ markedly enhanced the HepG2 cells apoptosis, indicating that the inhibition of autophagy enhanced CCT128930-induced apoptosis of HepG2 cells. In agreement, the combination of CCT128930 with CQ was also more potent than CCT128930 alone in inducing apoptosis evidenced by increased cleavage of caspase-3 and PARP (Fig. 7D). These data indicate that inhibition of CCT128930-induced autophagy drives HepG2 cells to die of apoptosis. 3.7. Inhibition of autophagy enhances the CCT128930-induced phosphorylation of H2AX Last, we determined the changes of CCT128930-induced H2AX expression in the presence of CQ. From the western blotting results,
The poor results of chemotherapy in HCC combined with the success of sorafenib have led to intensifying research efforts aiming on identification of novel molecular targets for HCC therapy [20]. CCT128930, a novel ATP-competitive Akt inhibitor, is discovered by fragment-based in silico screening and structure-based design, and shows selectivity for Akt over PKA by targeting a single amino acid difference [21]. Previous studies have showed the antitumor activity of CCT128930 in U87MG and BT474 human breast cancer xenografts. Here, we report the mechanism that CCT128930 inhibits cell cycle procession and induces DNA damage and autophagy in a human hepatoma cell line HepG2. Abnormal type of excessive proliferation is a key characteristic of tumorigenesis, and inhibiting the proliferative signals in tumor cells is an effective way in anti-cancer therapy. In this study, we showed that exposure of HepG2 cells to CCT128930 resulted in the accumulation of cells in the G1 phase. CCT128930 treatment decreased the expression of cyclinD1 and Cdc25A, and increased the levels of p21, p27 and p53. In addition to inhibiting the proliferation of HepG2 cells, high dose of CCT128930 showed an ability to induce apoptosis. Caspase activation is generally considered to be a key mark of apoptosis, playing an important role in the induction and regulation of apoptosis. Our study clearly showed that after 24 h of treatment with CCT128930, the expression of cleaved caspase-9, -3 and PARP was enhanced significantly. One of the biochemical hallmarks of apoptotic cell death is the formation of DNA double-strand breaks (DSB), which produces oligonucleosomal DNA fragments. Under conditions unrelated to apoptosis, DSB induce the rapid activation of conserved DNA damage response (DDR) pathways [22]. The biological goal of DDR
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 7. Inhibition of autophagy enhanced anticancer activity of CCT128930 in HepG2 cells. (A) Representative images of RFP-GFP-LC3 in HepG2 cells transfected with RFP-GFP-LC3 plasmid. 24 h after transfection, cells were treated with 20 mM CCT128930 for another 24 h. (B) Cells were treated for 24 h with indicated concentrations of CCT128930. Protein expression of LC3 and Beclin-1 was analyzed by western blotting analysis. b-actin served as loading control. Representative experiments of three independent experiments are shown. (C) HepG2 cells were exposed to 20 mM CCT128930 for 24 h in the presence or absence of CQ. Cell apoptosis was determined by Annexin V/PI FACS assay. (D) HepG2 cells were treated with 20 mM CCT128930 and/or with 20 mM CQ for 24 h. Protein expression of caspase-3 and PARP was assessed by western blotting analysis.
is to protect the damaged cell, if the damage cannot be repaired, cells undergo apoptosis. DNA damage sensor Chk1/Chk2 becomes phosphorylated by several members of the phosphatidylinositol-3kinase family, i.e., ATM, ATR, and DNA-PK (DNA-dependent protein kinase) [23,24]. In order to further elucidate the molecular mechanism leading to CCT128930-mediated cell cycle arrest, we determine the effects of CCT128930 on the activation of DNA damage signaling pathway. One prominent chromatin modification in response to DNA damage is phosphorylation of histone H2AX on serine 139, which is referred to as g-H2AX [25]. We quantified the phosphorylation levels and g-H2AX nuclear foci formation efficiency after cells were exposed to CCT128930, because g-H2AX plays an important role in recruiting DNA repair proteins to nuclear foci containing the sites of damage. We found that the CCT128930 not only upregulated the phosphorylation of H2AX but also increased g-H2AX formation of nuclear foci in HepG2 cells. Activation of ATM by phosphorylation at Ser-1981 was enhanced after CCT128930 treatment, while there was no change in the levels of ATR phosphorylation, suggesting the possible involvement of ATM signaling in CCT128930-induced DNA damage and cell cycle arrest.
Consistent with ATM activation, the phosphorylation of Chk1, Chk2 and p53 were also increased in CCT128930-stimulated HepG2 cells. Recent reports suggest that autophagy plays an important role in determining cell fate although apoptosis has been widely studied as a cellular response to DNA damage [26]. Autophagy is a ubiquitous highly conserved pathway in eukaryotic cells that takes place as a response to a variety of conditions, such as nutrient deprivation, growth factors withdrawal, and oxidative stress [27]. Conversion of LC3 from cytosolic form (LC3-I) to the proteolytic and lipidated form (LC3-II) is a characteristic hallmark of autophagy [28]. We found that CCT128930 could induce autophagy evidenced by increased levels of LC3-II in HepG2 cells. It will also be interesting to investigate whether inhibitors of autophagy can enhance the anticancer activity of CCT128930. We noted that combination of CCT128930 and CQ led to the desired effects on apoptosis in HepG2 cells. Targeting the autophagy pathway may be a promising therapeutic strategy to enhance CCT128930 lethality via apoptosis for HCC treatment. In conclusion, our results suggest that CCT128930 inhibits cancer cell growth. CCT128930 can induce cell cycle arresting, DNA damage and autophagy in a dose dependent manner. Treatment of the cells with CCT128930 significantly enhanced phosphorylation of ERK1/2 and JNK1/2. Inhibition of autophagy with a lysosomal protease inhibitor CQ potentiates CCT128930’s apoptosis-inducing activity and anticancer activity in cancer cells. Conflict of interest statement The authors declare that they have no conflicts of interest. Acknowledgments
Fig. 8. Inhibition of autophagy enhanced CCT128930-induced phosphorylation of H2AX. HepG2 cells were treated with 20 mM CCT128930 and/or with 20 mM CQ for 24 h. Expressions of LC3-II and g-H2AX were determined by western blotting analysis. b-actin served as loading control.
This work was supported by grants from the National Natural Science Foundation of China (No 81272683 to Feng-Ze Wang, No 81271275 and No 81070947 to Bao-Liang Sun).
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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References [1] E. Aksamitiene, A. Kiyatkin, B.N. Kholodenko, Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance, Biochem. Soc. Trans. 40 (2012) 139e146. [2] J.A. Engelman, J. Luo, L.C. Cantley, The evolution of phosphatidylinositol 3kinases as regulators of growth and metabolism, Nat. Rev. Genet. 7 (2006) 606e619. [3] S. Saiki, Y. Sasazawa, Y. Imamichi, S. Kawajiri, T. Fujimaki, I. Tanida, H. Kobayashi, F. Sato, S. Sato, K. Ishikawa, M. Imoto, N. Hattori, Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition, Autophagy 7 (2011) 176e187. [4] H.Y. Chang, M.H. Shih, H.C. Huang, S.R. Tsai, H.F. Juan, S.C. Lee, Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells, PLoS One 8 (2013) e54117. [5] H.J. Cho, Y.J. Oh, S.H. Han, H.J. Chung, C.H. Kim, N.S. Lee, W.J. Kim, J.M. Choi, H. Kim, Cdk1 protein-mediated phosphorylation of receptor-associated protein 80 (RAP80) serine 677 modulates DNA damage-induced G2/M checkpoint and cell survival, J. Biol. Chem. 288 (2013) 3768e3776. [6] J. Smith, L.M. Tho, N. Xu, D.A. Gillespie, The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer, Adv. Cancer Res. 108 (2010) 73e112. [7] A. Kulkarni, K.C. Das, Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation, Am. J. Physiol. Lung Cell. Mol. Physiol. 294 (2008) L998e L1006. [8] A. Choudhury, A. Cuddihy, R.G. Bristow, Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome, Semin. Radiat. Oncol. 16 (2006) 51e58. [9] Z.J. Yang, C.E. Chee, S. Huang, F.A. Sinicrope, The role of autophagy in cancer: therapeutic implications, Mol. Cancer Ther. 10 (2011) 1533e1541. [10] X. Sui, R. Chen, Z. Wang, Z. Huang, N. Kong, M. Zhang, W. Han, F. Lou, J. Yang, Q. Zhang, X. Wang, C. He, H. Pan, Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment, Cell Death Dis. 4 (2013) e838. [11] C. Seitz, M. Hugle, S. Cristofanon, A. Tchoghandjian, S. Fulda, The dual PI3K/ mTOR inhibitor NVP-BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial-lysosomal cross-talk, Int. J. Cancer 132 (2013) 2682e2693. [12] V. Papadimitrakopoulou, Development of PI3K/AKT/mTOR pathway inhibitors and their application in personalized therapy for non-small-cell lung cancer, J. Thorac. Oncol. 7 (2012) 1315e1326. [13] P. Wu, Y.Z. Hu, PI3K/Akt/mTOR pathway inhibitors in cancer: a perspective on clinical progress, Curr. Med. Chem. 17 (2010) 4326e4341. [14] M. Mazzoletti, M. Broggini, PI3K/AKT/mTOR inhibitors in ovarian cancer, Curr. Med. Chem. 17 (2010) 4433e4447. [15] H.R. Fei, L.Y. Cui, Z.R. Zhang, Y. Zhao, F.Z. Wang, Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through
[16]
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
modulating GSK3b/b-catenin pathway, J. Cell. Biochem. 113 (2012) 3403e 3410. T.A. Yap, M.I. Walton, L.J. Hunter, M. Valenti, A. de Haven Brandon, P.D. Eve, R. Ruddle, S.P. Heaton, A. Henley, L. Pickard, G. Vijayaraghavan, J.J. Caldwell, N.T. Thompson, W. Aherne, F.I. Raynaud, S.A. Eccles, P. Workman, I. Collins, M.D. Garrett, Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930, Mol. Cancer Ther. 10 (2011) 360e371. J. Wang, L. Yuan, H. Xiao, C. Xiao, Y. Wang, X. Liu, Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways, Apoptosis 18 (2013) 751e765. Y. Shiloh, Y. Ziv, The ATM protein kinase: regulating the cellular response to genotoxic stress, and more, Nat. Rev. Mol. Cell. Biol. 4 (2013) 197e210. C.B. McLeland, J. Rodriguez, S.T. Stern, Autophagy monitoring assay: qualitative analysis of MAP LC3-I to II conversion by immunoblot, Methods Mol. Biol. 697 (2011) 199e206. A. Psyrri, N. Arkadopoulos, M. Vassilakopoulou, V. Smyrniotis, G. Dimitriadis, Pathways and targets in hepatocellular carcinoma, Expert Rev. Anticancer Ther. 12 (2012) 1347e1357. J.J. Caldwell, T.G. Davies, A. Donald, T. McHardy, M.G. Rowlands, G.W. Aherne, L.K. Hunter, K. Taylor, R. Ruddle, F.I. Raynaud, M. Verdonk, P. Workman, M.D. Garrett, I. Collins, Identification of 4-(4-aminopiperidin-1-yl)-7H-pyrrolo [2,3-d]pyrimidines as selective inhibitors of protein kinase B through fragment elaboration, J. Med. Chem. 51 (2008) 2147e2157. S. Solier, O. Sordet, K.W. Kohn, Y. Pommier, Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways, Mol. Cell. Biol. 29 (2009) 68e82. P. Bouwman, J. Jonkers, The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance, Nat. Rev. Cancer 12 (2012) 587e598. S. Solier, Y.W. Zhang, A. Ballestrero, Y. Pommier, G. Zoppoli, DNA damage response pathways and cell cycle checkpoints in colorectal cancer: current concepts and future perspectives for targeted treatment, Curr. Cancer Drug. Targets 12 (2012) 356e371. H. Cha, J.M. Lowe, H. Li, J.S. Lee, G.I. Belova, D.V. Bulavin, A.J. Fornace Jr., Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response, Cancer Res. 70 (2010) 4112e4122. N.S. Deen, S.J. Huang, L. Gong, T. Kwok, R.J. Devenish, The impact of autophagic processes on the intracellular fate of Helicobacter pylori: more tricks from an enigmatic pathogen? Autophagy 9 (2013) 639e652. B.J. Altman, S.R. Jacobs, E.F. Mason, R.D. Michalek, A.N. MacIntyre, J.L. Coloff, O. Ilkayeva, W. Jia, Y.W. He, J.C. Rathmell, Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis, Oncogene 30 (2011) 1855e1867. O. Florey, M. Overholtzer, Autophagy proteins in macroendocytic engulfment, Trends Cell. Biol. 22 (2012) 374e380.
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Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition Q4
Feng-Ze Wang a, b, Zheng-Yao Chang a, Hong-Rong Fei c, Ming-Feng Yang b, Xiao-Yi Yang b, Bao-Liang Sun b, d, * a
Q1
School of Biological Science, Taishan Medical University, Taian 271016, PR China Key Lab of Cerebral Microcirculation in Universities of Shandong, Taishan Medical University, Taian 271000, PR China School of Pharmacology, Taishan Medical University, Taian 271016, PR China d Department of Neurology, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2014 Accepted 22 April 2014 Available online xxx
PI3K/Akt/mTOR pathway plays an important role in tumor progression and anti-cancer drug resistance. The aim of the present study is to determine the antitumor effect of CCT128930, a novel small molecule inhibitor of Akt, in the HepG2 hepatoma cancer cells. Our results showed that at low concentrations, CCT128930 increased, but not inhibited, the phosphorylation of Akt in HepG2 and A549 cells. CCT128930 inhibited cell proliferation by inducing cell cycle arrest in G1 phase through downregulation of cyclinD1 and Cdc25A, and upregulation of p21, p27 and p53. A higher dose (20 mM) of CCT128930 triggered cell apoptosis with activation of caspase-3, caspase-9, and PARP. Treatment with CCT128930 increased phosphorylation of ERK and JNK in HepG2 cells. CCT128930 activated DNA damage response of HepG2 cell characterized by phosphorylation of H2AX, ATM (ataxia-telangiectasia mutated), Chk1 and Chk2. Upon exposure to CCT128930 at a higher concentration, HepG2 cells exhibited autophagy was accompanied by an increase the levels of LC3-II and Beclin-1. Blocking autophagy using chloroquine magnified CCT128930-induced apoptotic cell death and the phosphorylation of H2AX. The results in this study have advanced our current understandings of the anti-cancer mechanisms of CCT128930 in cancer cells. Ó 2014 Published by Elsevier Masson SAS.
Keywords: CCT128930 Akt Cell cycle DNA damage Autophagy
1. Introduction It is widely accepted that PI3K/Akt/mTOR pathway plays a key role in controlling cell growth, apoptosis and cell differentiation. Mutations or genetic alterations of many components of the PI3K/ Akt pathway frequently occur in human cancers [1]. Signaling through PI3K drives a number of cell functions by the activation of the Akt protein (also known as protein kinase B, PKB). The downstream effector of PI3K, Akt, increases the cell cycle progression by blocking FOXO-mediated transcription of cell cycle inhibitors that lead to the accumulation and activation of the mTOR-raptor kinase complex, which in turn mediates protein kinase p70S6K1 activation to regulate protein synthesis and cell proliferation [2]. mTOR functions as a central element in a signaling pathway involved in the control of cell proliferation, apoptosis and autophagy, an ubiquitous and evolutionarily conserved process that degrades
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* Corresponding author. Department of Neurology, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, PR China. E-mail addresses: [email protected], [email protected] (B.-L. Sun).
cytosolic components via the lysosomes and allows cells to survive various forms of stress [3]. Many studies have shown that DNA damage or replication errors results in the inhibition of Cdks activity via activation of the cell cycle checkpoint, which is essential for preventing entry into mitosis in the presence of genomic defects [4,5]. The key regulators of the checkpoint pathways in eukaryotic cell DNA damage response are ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases, which are activated in response to DNA damaging agents such as ionizing radiation, ultraviolet light and DNA replication inhibitors [6]. The downstream target kinases of ATR/ATM in response to DNA damage are Chk1 and Chk2. ATR/ATM activate Chk1 and Chk2 protein kinases responsible for the inhibition via phosphorylation of the Cdc25 phosphatase. Although Chk1 or Chk2 are known to trigger G2/M phase cell cycle arrest, these kinases are also known to mediate ATM-dependent G1 checkpoint due to activating p53 tumor suppressor protein by phosphorylation [7,8]. Autophagy is a conservative process of protein degradation through autophagosome and lysosome system, which generally
http://dx.doi.org/10.1016/j.biochi.2014.04.008 0300-9084/Ó 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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functions as a homeostasis mechanism to maintain a balance between the synthesis and degradation of cellular products. The role of autophagy in cancer and cell death is apparently quite complex and poorly understood. Evidence indicates that the predominant function of autophagy in cancer cells is to confer stress tolerance, which contributes to tumor cell survival during cancer progression. On the other hand, induction of autophagic cell death has been proposed as a mechanism of cell death, given that characteristics of autophagy have been discovered in dying cells [9]. Similar to its potential to either promote cell survival or induce cell death, a large body of research shows a paradoxical role of autophagy during chemotherapy [10]. There are increasing evidences indicates that PI3K/Akt/mTOR inhibitors initiate autophagy as a survival program that may interfere with their antitumor activity [11]. Therefore, autophagy may be a potential resistance mechanism and its abrogation may be increase the cytotoxicity of mTOR inhibition. The prevalence of PI3K/Akt/mTOR signaling abnormalities in cancer cells has suggested the potential use of this pathway as a novel therapeutic target. Inhibitors of this signaling pathway are being actively developed and tested for tumor therapy [12e14]. In this study, we investigate a potential antitumor activity of CCT128930 in vitro models. We demonstrate that CCT128930 inhibits the cell proliferation by inducing G1 cell cycle arrest. At high concentrations, CCT128930 causes apoptosis. Activation of g-H2AX, ATM, Chk1 and Chk2 may be responsible for the DNA damage in CCT128930-treated HepG2 cells. CCT128930 also triggers the autophagy, as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by chloroquine (CQ) further increases the cytotoxicity of CCT128930. Our results indicate that CCT128930 might represent a promising drug for the treatment of cancer.
medium was then removed, and cells were treated with various concentrations of CCT128930. An amount of 20 mL MTT (5 mg/mL) was added for 4 h. After removing supernatant, 150 mL DMSO was added to resolve formazan crystals, and the absorbance was detected at 490 nm. For cell colony formation assay, HepG2 cells were seeded at 1500 cells per well (6-well cell culture plate) and treated with different concentrations of CCT128930 for 2 weeks. Cells were fixed in 1% glutaraldehyde and stained with 0.5% crystal violet. Colonies with >30 cells were counted under an inverted microscope. 2.4. Cell cycle analysis Cells were treated with CCT128930 for 24 h. At the end of the treatment, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS), and fixed overnight in cold 70% ethanol at 4 C. After washing with PBS, the cells was digested with RNase A and stained with PI. Samples were analyzed for their DNA content by using a FACSCalibur flow cytometry with CellQuest software (Becton Dickinson, USA). 2.5. Annexin V-FITC/PI staining Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. To assess the extent of apoptosis induction, cells were harvested (adherent and suspended cells) and stained according to the manufacturer’s instructions. Samples were analyzed with the FACScan flow cytometry and CellQuest analysis software (Becton Dickinson). 2.6. Western blot analysis
2. Materials and methods 2.1. Reagent CCT128930 was purchased from Sata Cruz Biotechnology (Shanghai, China), and dissolved with DMSO. Crystal violet and 3[4,5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) and chloroquine were obtained from SigmaeAldrich (St. Louis, USA). PD98059 was purchased from Promega (Madison, USA). SP600129 was obtained from Calbiochem (CA, USA). Annexin V-FITC apoptosis detection kit was obtained from KeyGEN Biotech (Nanjing, China). Western Lightning Plus ECL was purchased from perkinelmer (MA, USA). Antibodies specific to b-actin, Beclin-1 and LC3-II were purchased from SigmaeAldrich. Rabbit anti-Akt, p-Akt, p70SK6, caspase-9, caspase-3, PARP, ATM, p-ATM, ATR, p-ATR, p-Chk2, Chk1, p-Chk1, g-H2AX, p-p53, p-p38, JNK and Cdc2 polyclonal antibodies were obtained from Cell Signaling Technology (Shanghai, China). Mouse anti-Chk2, p21, p27 and p-JNK were purchased from BD Biosciences. Anti-cyclinD1, Cdc25A, Cdc25B, Cdc25C, cyclinB1, p53, ERK and p-ERK were purchased from Santa cruz Biotechnology.
After cells were lysed with ice-cold lysis buffer (150 mM NaCl, 20 mM TriseHCl, 1% NP-40, 0.5% Na-DOC, 0.1% SDS, 0.2 mmol/L PMSF, and protease inhibitor cocktails) for 20 min on ice. Cell supernatants were obtained by centrifugation and the protein concentration was determined. 30 mg protein was used for western blotting analysis. Detection of the target proteins on the membranes was performed using the ECL Western Blotting Detection Reagents. 2.7. Immunoflourescence assay Cultured cells were fixed with cold 100% methanol at 4 C for 10 min and permeabilized with PBS-0.1%Triton X-100 for 10 min. After blocked with 3% BSA, the cells were incubated with anti-gH2AX antibody. After washing, cells were incubated with FITCconjugated secondary antibodies (Santa cruz, 1:200 dilution), and DAPI was used to stain the nuclei. Fluorescence images were obtained using a fluorescence microscope with appropriate filter sets. 2.8. Statistical analysis
2.2. Cells and cell culture HepG2 cell and A549 cell were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and was cultured in RMPI 1640 medium containing 10% fetal calf serum (Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified atmosphere (37 C; 5% CO2).
All data were expressed as mean SD. Statistical analysis was performed by the student’s t-test. P < 0.05 was indicated to be statistical significant. 3. Results
2.3. Cell viability assay and cell colony formation assay
3.1. CCT128930 inhibits cell proliferation in a concentrationdependent manner
MTT assay was used to detect cell viability, as reported elsewhere [15]. Briefly, cells were plated in 96-well plates for 24 h. The
CCT128930 was reported to be a PI3K/mTOR inhibitor [16], and then we detect the effects of CCT128930 on the phosphorylation of
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 1. The effect of CCT128930 on the phosphorylation state of Akt and p70S6K in HepG2 and A549 cells. Cells were incubated with CCT128930 at the indicated concentrations for 24 h and then processed for western blotting analysis. b-actin was used as the loading control.
Akt, an activator of mTOR, and its downstream effector p70 ribosomal protein S6 kinase (p70S6K) in HepG2 and A549 cells. As shown in Fig. 1, CCT128930 treatment did not inhibit the phosphorylation of Akt (Ser473) and p70S6K (Thr389) at indicated concentrations. Much to our surprise, when the cells were treated with lower concentration of CCT128930, an increase in phosphorylation of Akt was observed. To investigate the effects of CCT128930 on the growth of cancer cells, MTT assay was used to evaluate the HepG2 and A549 cell viability. The results showed that CCT128930 decreased the viability of cancer cells in a dose-dependent manner (IC50 ¼ 30.03 mM in HepG2 cell and IC50 ¼ 32.94 mM in A549 cell) (Fig. 2A). Colony formation assay revealed that treatment with 10 mM CCT128930 totally blocked the formation of colonies,
confirming the anti-cancer activity of CCT128930 against cancer cells (Fig. 2B). Light microscopy observation of CCT128930-treated HepG2 cells revealed morphological changes accompanying shrinkage of the cell size and constriction of cell shape, which may be an early indicator of responses to cell growth inhibition or apoptotic stimuli (Fig. 2C). 3.2. CCT128930 induces G1 phase arrest in HepG2 and A549 cells For finding whether the growth inhibitory function of CCT128930 was associated with its effect on cell cycle progression, we investigated changes of cell cycle distribution in CCT128930treated cancer cells by flow cytometric analysis. As shown in Fig. 3A, CCT128930 treatment significantly increased G1 phase cells
Fig. 2. CCT128930 inhibited the growth of cancer cells. (A) CCT128930 reduced the viability of HepG2 and A549 cells. Cells were seeded at a density of 1 106 cells/mL in 96-well polystyrene culture plates at 37 C with 5% (v/v) CO2 for one day. After 24 h of incubation, cells were incubated with CCT128930 for 24 h and then processed for MTT assay. (B) CCT128930 inhibits the colony formation ability of HepG2 cell. The histogram represents the mean colony number of 3 independent experiments (mean SD). *P < 0.05, **P < 0.01 compared with the control. (C) Morphological changes in CCT128930-treated HepG2 cells. Morphological changes observed in the HepG2 cells after 24 h of treatment with various concentrations of CCT128930.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 3. CCT128930 induced G1 phase arrest in HepG2 and A549 cells. (A) CCT128930 was observed to induce G1 phase arrest. Upon exposure to CCT128930 at indicated concentrations for 24 h, both HepG2 cells and A549 cells all exhibited G1 phase arrest. (B) Expression of the cell cycle-associated proteins. HepG2 cells were treated with CCT128930 for the indicated concentrations. Cell lysates were fractionated on SDS-polyacrylamide gels and analyzed by western blotting with antibodies against cell cycle associated proteins. bactin was shown as loading control.
in HepG2 and A549 cells. Investigation of the pivotal proteins involved in cell cycle transition by CCT128930 showed that expression of p21, p27 and p53 was increased by CCT128930 treatment (Fig. 3B). 3.3. High doses of CCT128930-induced HepG2 cell apoptosis Next, we performed experiments to determine whether this inhibitory effect of CCT128930 on cell viability resulted from apoptotic cell death. Apoptotic or necrotic cells were detected after incubation with increasing concentrations of CCT128930 for 24 h.
Annexin V/PI flow cytometry results revealed that apoptotic rate in 20 mM CCT128930 treatment HepG2 cells increased from 5.33% to 12.62% (Fig. 4A), these results indicated that high dose of CCT128930 induced the cell death in HepG2 cells. To elucidate whether CCT128930-treated hepG2 cells express typical apoptotic markers, we used western blotting analysis to determine the cleavage of apical pro-caspase-3, pro-caspase-9, and PARP in HepG2 cells. As shown in Fig. 4B, HepG2 cells exhibited significantly increased the cleavage of caspase-3, caspase-9 and PARP after exposure to 20 mM CCT128930, suggesting enhanced apoptotic activity in HepG2 cells.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 4. High dose of CCT128930 induced cell apoptosis in HepG2 cells. (A) Annexin V/FITC-PI FACS assay of HepG2 cells exposed to various doses of CCT128930 (0e20 mM) for 24 h. The percentage of early apoptotic cells with exposed CCT128930 is located in the lower right quadrant and the percentage of necrotic/apoptosis in terminal stages is located in the upper right quadrant. Data are representative of one of three repeats. (B) Western blotting analysis of apoptosis-related proteins. HepG2 cells were exposed to CCT128930 for 24 h, and western blot analysis was carried out to detect the cleavage of caspases-9, caspase-3 and PARP.
3.4. ERK and JNK are required for CCT128930-induced cytotoxicity in HepG2 cells
3.5. CCT128930 activates DNA damage sensor kinases and associates cellular checkpoints in HepG2 cells
Mitogen-activated protein kinase (MAPK) signaling pathway activation contributes to decrease cell proliferation and inducing apoptosis in response to a variety of extra cellular stresses [17], we then determined whether the activity of ERK, JNK and p38 MAPK were upregulated in HepG2 cells after treatment with CCT128930. Using western blotting analysis, we found that phosphorylation of ERK and JNK protein was increased in HepG2 cells after exposure to CCT128930 for 24 h (Fig. 5A). However, no significant changes of phosphorylated p38 MAPK were observed from the western blotting results. To assess whether CCT128930-induced cell viability inhibition was dependent on ERK and JNK activation, HepG2 cells were cultured with CCT128930 (20 mM) either alone or in combination with inhibitors of ERK (PD98059) and JNK1/2 (SP600129) for 24 h, and MTT assays were carried out. As shown in Fig. 5B, inhibition of cell viability by CCT128930 was partially alleviated in the presence of PD98059 or SP600125, suggesting a dependence on ERK and JNK signaling for the CCT128930 mediates the cell growth and survival.
To further elucidate the possible mechanism of CCT128930induced cell cycle arrest and apoptosis, we detected the DNA damage after CCT128930 exposure by examined the expression of phosphorylation of H2AX (g-H2AX), an indicator of the presence of DNA damage. We observed an increased phosphorylation of H2AX at Ser-139 in CCT128930-treated HepG2 cells (Fig. 6A). Immunofluorescence staining also showed an increased formation of gH2AX foci in CCT128930-treated HepG2 cells (Fig. 6B). Both ATM and ATR play a central role in coordinating the DNA damage response, including cell cycle checkpoint control and apoptosis [18]. We therefore measured the effect of CCT128930 on the key signaling proteins of this pathway. Treatment of HepG2 cells with indicated concentrations of CCT128930 increased the phosphorylation of ATM at Ser-1981 without any change in the total levels of ATM. However, we did not observe any changes in the phosphorylation of ATR (Ser428) in CCT128930-treated cells (Fig. 6C). Since phosphorylation of Chk1 and Chk2 also serves as an indicator of checkpoint activation in response to DNA damage, we determined whether the phosphorylation of Chk1 and Chk2 were affected in CCT128930-
Fig. 5. Pretreatment ERK and JNK inhibitor partially reduced CCT128930-inhbited cell viability. (A) Cells were treated with different doses of CCT128930 for 24 h and protein expression was determined by western blotting analysis. (B) To investigate the effect of the ERK and JNK inhibitor on CCT128930-inhibited cell viability, HepG2 cells were pretreated as before with 20 mM PD98059 (PD) and 20 mM SP600125 (SP), respectively. After 24 h incubation with CCT128930 (20 mM), cell viability was analyzed using MTT assay. *Indicates that P < 0.05 versus control cells.
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Fig. 6. Effect of CCT128930 on DNA damage responses. (A) DNA double-strand breaks, as measured by g-H2AX, were induced by CCT128930 treatment. Whole cell lysates were prepared and subjected to western blotting using antibodies against indicated proteins. b-actin antibody was used as an internal control to show equal protein loading. (B) Detection of g-H2AX by immunofluorescence. Cells were treated with the indicated concentrations of CCT128930 for 24 h, stained with rabbit anti-g-H2AX (Ser139) antibody and goat antirabbit FITC-conjugated secondary antibody (green), and then counterstained with DAPI (blue). (C) CCT128930 regulates the phosphorylation of ATM, Chk1, and Chk2 in HepG2 cells. Cells were treated with different doses of CCT128930 for 24 h, and then western blotting analysis was performed with antibodies against the indicated proteins. b-actin antibody was used as a loading control.
treated cells. As shown in Fig. 6C, exposure to CCT128930 significantly increased the phosphorylation of Chk1 and Chk2 in HepG2 cells. 3.6. Inhibition of autophagy exacerbates the cytotoxic activity of CCT128930
we found that the inhibition of autophagy with CQ may be potentiating CCT128930-evoked DNA damage response, because the inhibition of autophagy significantly enhanced CCT128930induced phosphorylation of H2AX in HepG2 cells (Fig. 8). 4. Discussion
Autophagy is the basic catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes; we then determined whether CCT128930 induced autophagy in HepG2 cells. We detected the levels of LC3-II for monitoring autophagy because it has been widely used for estimating the abundance of the autophagosome or autophagy [19]. HepG2 cells were transfected with RFP-GFP-LC3 for 24 h; the cells were then treated with CCT128930 for 24 h. As presented in Fig. 7A, CCT128930 induced the redistribution of GFP-LC3 from a diffuse pattern to punctate structures. Moreover, an increase in LC3-II expression was observed after treatment of CCT128930 in HepG2 cells. Beclin1 (Atg6), a wellknown key regulator of autophagy, was also upregulated in CCT128930-treated HepG2 cells (Fig. 7B). To determine whether induction of autophagy by CCT128930 is a survival or death mechanism, we detected the CCT128930induced apoptosis in the presence of CQ, a lysosomal protease inhibitor. As shown in Fig. 7C, the presence of 20 mM CQ markedly enhanced the HepG2 cells apoptosis, indicating that the inhibition of autophagy enhanced CCT128930-induced apoptosis of HepG2 cells. In agreement, the combination of CCT128930 with CQ was also more potent than CCT128930 alone in inducing apoptosis evidenced by increased cleavage of caspase-3 and PARP (Fig. 7D). These data indicate that inhibition of CCT128930-induced autophagy drives HepG2 cells to die of apoptosis. 3.7. Inhibition of autophagy enhances the CCT128930-induced phosphorylation of H2AX Last, we determined the changes of CCT128930-induced H2AX expression in the presence of CQ. From the western blotting results,
The poor results of chemotherapy in HCC combined with the success of sorafenib have led to intensifying research efforts aiming on identification of novel molecular targets for HCC therapy [20]. CCT128930, a novel ATP-competitive Akt inhibitor, is discovered by fragment-based in silico screening and structure-based design, and shows selectivity for Akt over PKA by targeting a single amino acid difference [21]. Previous studies have showed the antitumor activity of CCT128930 in U87MG and BT474 human breast cancer xenografts. Here, we report the mechanism that CCT128930 inhibits cell cycle procession and induces DNA damage and autophagy in a human hepatoma cell line HepG2. Abnormal type of excessive proliferation is a key characteristic of tumorigenesis, and inhibiting the proliferative signals in tumor cells is an effective way in anti-cancer therapy. In this study, we showed that exposure of HepG2 cells to CCT128930 resulted in the accumulation of cells in the G1 phase. CCT128930 treatment decreased the expression of cyclinD1 and Cdc25A, and increased the levels of p21, p27 and p53. In addition to inhibiting the proliferation of HepG2 cells, high dose of CCT128930 showed an ability to induce apoptosis. Caspase activation is generally considered to be a key mark of apoptosis, playing an important role in the induction and regulation of apoptosis. Our study clearly showed that after 24 h of treatment with CCT128930, the expression of cleaved caspase-9, -3 and PARP was enhanced significantly. One of the biochemical hallmarks of apoptotic cell death is the formation of DNA double-strand breaks (DSB), which produces oligonucleosomal DNA fragments. Under conditions unrelated to apoptosis, DSB induce the rapid activation of conserved DNA damage response (DDR) pathways [22]. The biological goal of DDR
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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Fig. 7. Inhibition of autophagy enhanced anticancer activity of CCT128930 in HepG2 cells. (A) Representative images of RFP-GFP-LC3 in HepG2 cells transfected with RFP-GFP-LC3 plasmid. 24 h after transfection, cells were treated with 20 mM CCT128930 for another 24 h. (B) Cells were treated for 24 h with indicated concentrations of CCT128930. Protein expression of LC3 and Beclin-1 was analyzed by western blotting analysis. b-actin served as loading control. Representative experiments of three independent experiments are shown. (C) HepG2 cells were exposed to 20 mM CCT128930 for 24 h in the presence or absence of CQ. Cell apoptosis was determined by Annexin V/PI FACS assay. (D) HepG2 cells were treated with 20 mM CCT128930 and/or with 20 mM CQ for 24 h. Protein expression of caspase-3 and PARP was assessed by western blotting analysis.
is to protect the damaged cell, if the damage cannot be repaired, cells undergo apoptosis. DNA damage sensor Chk1/Chk2 becomes phosphorylated by several members of the phosphatidylinositol-3kinase family, i.e., ATM, ATR, and DNA-PK (DNA-dependent protein kinase) [23,24]. In order to further elucidate the molecular mechanism leading to CCT128930-mediated cell cycle arrest, we determine the effects of CCT128930 on the activation of DNA damage signaling pathway. One prominent chromatin modification in response to DNA damage is phosphorylation of histone H2AX on serine 139, which is referred to as g-H2AX [25]. We quantified the phosphorylation levels and g-H2AX nuclear foci formation efficiency after cells were exposed to CCT128930, because g-H2AX plays an important role in recruiting DNA repair proteins to nuclear foci containing the sites of damage. We found that the CCT128930 not only upregulated the phosphorylation of H2AX but also increased g-H2AX formation of nuclear foci in HepG2 cells. Activation of ATM by phosphorylation at Ser-1981 was enhanced after CCT128930 treatment, while there was no change in the levels of ATR phosphorylation, suggesting the possible involvement of ATM signaling in CCT128930-induced DNA damage and cell cycle arrest.
Consistent with ATM activation, the phosphorylation of Chk1, Chk2 and p53 were also increased in CCT128930-stimulated HepG2 cells. Recent reports suggest that autophagy plays an important role in determining cell fate although apoptosis has been widely studied as a cellular response to DNA damage [26]. Autophagy is a ubiquitous highly conserved pathway in eukaryotic cells that takes place as a response to a variety of conditions, such as nutrient deprivation, growth factors withdrawal, and oxidative stress [27]. Conversion of LC3 from cytosolic form (LC3-I) to the proteolytic and lipidated form (LC3-II) is a characteristic hallmark of autophagy [28]. We found that CCT128930 could induce autophagy evidenced by increased levels of LC3-II in HepG2 cells. It will also be interesting to investigate whether inhibitors of autophagy can enhance the anticancer activity of CCT128930. We noted that combination of CCT128930 and CQ led to the desired effects on apoptosis in HepG2 cells. Targeting the autophagy pathway may be a promising therapeutic strategy to enhance CCT128930 lethality via apoptosis for HCC treatment. In conclusion, our results suggest that CCT128930 inhibits cancer cell growth. CCT128930 can induce cell cycle arresting, DNA damage and autophagy in a dose dependent manner. Treatment of the cells with CCT128930 significantly enhanced phosphorylation of ERK1/2 and JNK1/2. Inhibition of autophagy with a lysosomal protease inhibitor CQ potentiates CCT128930’s apoptosis-inducing activity and anticancer activity in cancer cells. Conflict of interest statement The authors declare that they have no conflicts of interest. Acknowledgments
Fig. 8. Inhibition of autophagy enhanced CCT128930-induced phosphorylation of H2AX. HepG2 cells were treated with 20 mM CCT128930 and/or with 20 mM CQ for 24 h. Expressions of LC3-II and g-H2AX were determined by western blotting analysis. b-actin served as loading control.
This work was supported by grants from the National Natural Science Foundation of China (No 81272683 to Feng-Ze Wang, No 81271275 and No 81070947 to Bao-Liang Sun).
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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References [1] E. Aksamitiene, A. Kiyatkin, B.N. Kholodenko, Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance, Biochem. Soc. Trans. 40 (2012) 139e146. [2] J.A. Engelman, J. Luo, L.C. Cantley, The evolution of phosphatidylinositol 3kinases as regulators of growth and metabolism, Nat. Rev. Genet. 7 (2006) 606e619. [3] S. Saiki, Y. Sasazawa, Y. Imamichi, S. Kawajiri, T. Fujimaki, I. Tanida, H. Kobayashi, F. Sato, S. Sato, K. Ishikawa, M. Imoto, N. Hattori, Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition, Autophagy 7 (2011) 176e187. [4] H.Y. Chang, M.H. Shih, H.C. Huang, S.R. Tsai, H.F. Juan, S.C. Lee, Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells, PLoS One 8 (2013) e54117. [5] H.J. Cho, Y.J. Oh, S.H. Han, H.J. Chung, C.H. Kim, N.S. Lee, W.J. Kim, J.M. Choi, H. Kim, Cdk1 protein-mediated phosphorylation of receptor-associated protein 80 (RAP80) serine 677 modulates DNA damage-induced G2/M checkpoint and cell survival, J. Biol. Chem. 288 (2013) 3768e3776. [6] J. Smith, L.M. Tho, N. Xu, D.A. Gillespie, The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer, Adv. Cancer Res. 108 (2010) 73e112. [7] A. Kulkarni, K.C. Das, Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation, Am. J. Physiol. Lung Cell. Mol. Physiol. 294 (2008) L998e L1006. [8] A. Choudhury, A. Cuddihy, R.G. Bristow, Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome, Semin. Radiat. Oncol. 16 (2006) 51e58. [9] Z.J. Yang, C.E. Chee, S. Huang, F.A. Sinicrope, The role of autophagy in cancer: therapeutic implications, Mol. Cancer Ther. 10 (2011) 1533e1541. [10] X. Sui, R. Chen, Z. Wang, Z. Huang, N. Kong, M. Zhang, W. Han, F. Lou, J. Yang, Q. Zhang, X. Wang, C. He, H. Pan, Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment, Cell Death Dis. 4 (2013) e838. [11] C. Seitz, M. Hugle, S. Cristofanon, A. Tchoghandjian, S. Fulda, The dual PI3K/ mTOR inhibitor NVP-BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial-lysosomal cross-talk, Int. J. Cancer 132 (2013) 2682e2693. [12] V. Papadimitrakopoulou, Development of PI3K/AKT/mTOR pathway inhibitors and their application in personalized therapy for non-small-cell lung cancer, J. Thorac. Oncol. 7 (2012) 1315e1326. [13] P. Wu, Y.Z. Hu, PI3K/Akt/mTOR pathway inhibitors in cancer: a perspective on clinical progress, Curr. Med. Chem. 17 (2010) 4326e4341. [14] M. Mazzoletti, M. Broggini, PI3K/AKT/mTOR inhibitors in ovarian cancer, Curr. Med. Chem. 17 (2010) 4433e4447. [15] H.R. Fei, L.Y. Cui, Z.R. Zhang, Y. Zhao, F.Z. Wang, Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through
[16]
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
modulating GSK3b/b-catenin pathway, J. Cell. Biochem. 113 (2012) 3403e 3410. T.A. Yap, M.I. Walton, L.J. Hunter, M. Valenti, A. de Haven Brandon, P.D. Eve, R. Ruddle, S.P. Heaton, A. Henley, L. Pickard, G. Vijayaraghavan, J.J. Caldwell, N.T. Thompson, W. Aherne, F.I. Raynaud, S.A. Eccles, P. Workman, I. Collins, M.D. Garrett, Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930, Mol. Cancer Ther. 10 (2011) 360e371. J. Wang, L. Yuan, H. Xiao, C. Xiao, Y. Wang, X. Liu, Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways, Apoptosis 18 (2013) 751e765. Y. Shiloh, Y. Ziv, The ATM protein kinase: regulating the cellular response to genotoxic stress, and more, Nat. Rev. Mol. Cell. Biol. 4 (2013) 197e210. C.B. McLeland, J. Rodriguez, S.T. Stern, Autophagy monitoring assay: qualitative analysis of MAP LC3-I to II conversion by immunoblot, Methods Mol. Biol. 697 (2011) 199e206. A. Psyrri, N. Arkadopoulos, M. Vassilakopoulou, V. Smyrniotis, G. Dimitriadis, Pathways and targets in hepatocellular carcinoma, Expert Rev. Anticancer Ther. 12 (2012) 1347e1357. J.J. Caldwell, T.G. Davies, A. Donald, T. McHardy, M.G. Rowlands, G.W. Aherne, L.K. Hunter, K. Taylor, R. Ruddle, F.I. Raynaud, M. Verdonk, P. Workman, M.D. Garrett, I. Collins, Identification of 4-(4-aminopiperidin-1-yl)-7H-pyrrolo [2,3-d]pyrimidines as selective inhibitors of protein kinase B through fragment elaboration, J. Med. Chem. 51 (2008) 2147e2157. S. Solier, O. Sordet, K.W. Kohn, Y. Pommier, Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways, Mol. Cell. Biol. 29 (2009) 68e82. P. Bouwman, J. Jonkers, The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance, Nat. Rev. Cancer 12 (2012) 587e598. S. Solier, Y.W. Zhang, A. Ballestrero, Y. Pommier, G. Zoppoli, DNA damage response pathways and cell cycle checkpoints in colorectal cancer: current concepts and future perspectives for targeted treatment, Curr. Cancer Drug. Targets 12 (2012) 356e371. H. Cha, J.M. Lowe, H. Li, J.S. Lee, G.I. Belova, D.V. Bulavin, A.J. Fornace Jr., Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response, Cancer Res. 70 (2010) 4112e4122. N.S. Deen, S.J. Huang, L. Gong, T. Kwok, R.J. Devenish, The impact of autophagic processes on the intracellular fate of Helicobacter pylori: more tricks from an enigmatic pathogen? Autophagy 9 (2013) 639e652. B.J. Altman, S.R. Jacobs, E.F. Mason, R.D. Michalek, A.N. MacIntyre, J.L. Coloff, O. Ilkayeva, W. Jia, Y.W. He, J.C. Rathmell, Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis, Oncogene 30 (2011) 1855e1867. O. Florey, M. Overholtzer, Autophagy proteins in macroendocytic engulfment, Trends Cell. Biol. 22 (2012) 374e380.
Please cite this article in press as: F.-Z. Wang, et al., CCT128930 induces cell cycle arrest, DNA damage, and autophagy independent of Akt inhibition, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.04.008
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