Low-Dose Arsenic Trioxide Modulates the Differentiation of Mouse

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Low dose arsenic trioxide modulates the differentiation of mouse embryonic stem cells Wenlin Yuan, Jun Chen, Hongren Huang, Zhihui Cai, Qinjie Ling, Feng Huang, and ZHI HUANG Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00027 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Chemical Research in Toxicology

Low dose arsenic trioxide modulates the differentiation of mouse embryonic stem cells Wenlin Yuan § 1), Jun Chen §1), Hongren Huang §, Zhihui Cai §, Qinjie Ling §, Feng Huang ¶ *, Zhi Huang § * §

Department of Biotechnology, School of Life Science and Technology, Jinan

University, Guangzhou, 510632, Guangdong Province, China ¶ Department

of Rehabilitation Medicine, School of Medical Engineering, Foshan

University, Foshan, 528000, Guangdong Province, China KEYWORDS: Arsenic trioxide (ATO); Embryonic stem cell (ESC); Differentiation; Caspase 3 (CASP3); Nanog

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ABSTRACT: ABSTRACT Arsenic (As) is a well-known environmental pollutant, while arsenic trioxide (ATO) has been proven to be an effective treatment for acute promyelocytic leukemia, however, the mechanism underlying its dual effects is not fully understood. Embryonic stem cells (ESCs) appear properties of stemness and serve as a popular model to investigate epigenetic modifiers including environmental pollutants. Herein, the effect of low dose ATO on differentiation were evaluated in vitro using a mouse ESCs (mESCs) cell line, CGR8. Cells treated with 0.2-0.5 µM ATO for 3-4 days appeared slight inhibition of proliferation while elevation of apoptosis, but obvious alterations of differentiation by morphological checking and alkaline phosphatase (AP) staining. Moreover, ATO exposure significantly decreased the mRNA expression of the stemness maintenance genes including Oct4, Nanog and Rex-1 (P < 0.01), whereas obviously increased some tissue-specific differentiation marker genes such as Gata4, Gata-6, AFP and IHH. These alterations were consistent with the differentiation phenotype induced by retinoic acid (RA) and the expression patterns of distinct pluripotency markers such as SSEA-1 and Oct4. Furthermore, low dose ATO led to a quantitative increase in Caspase 3 (CASP3) activation and subsequent cleavage of Nanog around 27 kDa, which was correspond with the mouse Nanog cleaved by CASP3 in tube cleavage assay. Take together, we suggest that low dose ATO exposure will induce differentiation otherwise apoptosis of ESCs, such effects might be tuned partially by ATO induced CASP3 activation and Nanog cleavage coupling with other differentiation related genes involved. The present findings provided a preliminary action mechanism of arsenic on the cell fate determination.

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INTRODUCTION Arsenic (As) is one of well-known environmental toxicants and carcinogens.1, 2 Contamination of groundwater with As has been recognized as a massive public health hazard and an estimated >140 million people worldwide are chronically exposed to As according to the WHO limitation of As in drinking water (10 μg/L, ~0.13 μM).3 A recent study reported that in the presence of arsenic trioxide (ATO) at 0.1-1.0 μM alters the differentiation of mouse embryonic stem cell into cardiomyocytes by the dysruption of the sarcomere and syncytium organisation.4 The paradoxical impact of arsenic species on human health is that ATO is recognized as one of the most effective arsenic agent for treatment acute promyelocytic leukemia (APL) and has been widely applied as a component of traditional Chinese medicine.5-7 The underlying mechanism of this dual effects, however, is not fully understood.8, 9 Numerous studies have revealed carcinogenicity of inorganic arsenic (iAs) involving its intracellular metabolism, oxidative stress, and epigenetic modulation etc.10-12 Because iAs is neither a classic initiator nor a promoter to specific genes, many investigators have concluded that alternative impacts of iAs in cell transformation and eventually in tumorigenesis needed to be postulated.13-15 One explanation of the carcinogenicity of iAs was that an imbalance induced by iAs exposure in the cellular homeostatic controls over two antagonistic processes-proliferation and differentiation.16 Embryonic stem cells (ESCs) with pluripotency will normally remain undifferentiated in the presence of proliferative stimuli, alternatively, these same cells will undergo differentiation and arrest of proliferation in the presence/absence of distinct biologic signals.17 ATO treatments exhibit to induce differentiation in APL cells18 and neuroblastoma cells.19 However, the disturbance of ATO to ESCs differentiation and underlying mechanism are still not clarified. With the properties of self-renewal and pluripotency, ESCs have been shown great promise in regeneration medicine. ESCs are maintained in undifferentiated state by the core transcriptional factors such as Nanog, Oct4, Rex-1, SSEA-1, Sox2, etc. Ectopic expression of Oct4, Sox2, Myc and Klf4 in terminal somatic cells can result in reprogramming and generation of induced pluripotent stem cells (iPS).20 Overexpression of Nanog was shown to mediate its self-renewal in ESCs, and 4


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endogenous deletion of Nanog induced ESCs differentiation.21, 22 Thus, Nanog plays central role in ESCs stemness sustainability. Therefore, the effect of ATO treatment on the expression of these core transcriptional factors needed to be identified. Cell differentiation is likely to be controlled by multiple signaling pathways. It is interesting that Caspase 3 (CASP3) is activated during differentiation but is limited in self-amplification of caspase cascade as seen during apotosis.23, 24 CASP3 activation was required for the functional differentiation of bone marrow stromal stem cells (BMSSCs), and also involved in the differentiation of ESCs and hematopoietic stem cells (HSCs).25-27 A suggested mechanism for the impacts of caspase activity on cell fate control in ESCs included the cleavage-resistant form of Nanog to enhance proliferation and the cleaved form of Nanog might exert functions of differentiation.28 The linkage between CASP3 activation and Nanog cleavage provides compelling evidence that the apoptotic response is also indispensable for the regulation of stem cell differentiation. It is well known that ATO exposure can trigger the activation of caspase cascade. Regarding clinical treatments with ATO caused differentiation and apoptosis of leukemic cells that was associated with caspase activation,29 implied that the influence of ATO on cell fate determination of ESCs might be also associated with the caspases signaling, which have not been explored yet. Up to now, no better cell model than ESCs is available to evaluate the effects and the molecular mechanisms of differentiation. Using the ESCs model, differentiation induced by RA and its mimic derivants have been evidenced.30-33 Effect of ATO during differentiation disruption from ESCs to cardiomyocytes has been analyzed recently to contribute the mechanistic comprehension of cardiac diseases caused by in utero arsenic exposure.4 Previously, we revealed that iAs modulates the expression of selenoproteins involving endoplasmic reticulum (ER) stress in mouse ESCs.34 Therefore, ESCs serve as a unique cell model for toxicity and carcinogenicity assessment of environmental toxicants including arsenic compounds. In this study, a mESCs cell line, CGR8, was used to study the effects and molecular mechanism of ATO on cell differentiation. The biological effects of arsenic compounds appeared in a dose-dependent manner.35 It has been reported that ATO induced partial differentiation at low concentrations (0.1-0.5 μM) in APL cells but induced apoptosis at relatively higher 5



concentrations (0.2-2.0 μM).36 Our previous survey in an arsenic exposed population showed that the total arsenic in serum of health controls and cases with skin lesions were 0.26 μM and 0.35 μM in average, respectively.2 Considering the various arsenic species and wide variation in concentrations of iAs determined in human body fluid/tissue sample and environment, the aim of the present study was focus on the modulation of low dose ATO (0.2–0.5 μM) to the differentiation of mESCs. At molecular and functional level, the effects of a continuous exposure to ATO for short term (3-4 days) in leading to CASP3 activation, and subsequent Nanog cleavage in mESCs were confirmed. MATERIALS AND METHODS Materials and Reagents. Reagents. ATO was obtained from Sigma (St. Louis, MO, USA) and is a highly toxic reactant that should be handled with extreme caution. Reagents including all-trans-retinoic acid (RA, Cat. No. R2625), β-mercaptoethanol (β-ME), tetrazolium dye of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and propidium iodide (PI), and chemicals including sodium dodecyl sulfate (SDS), Tween-20 and other commonly used solvents with analytical grade were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Proteinase inhibitor cocktail and Laemmli buffer were purchased from Bio-Rad (Hercules, CA). The bicinchoninic acid (BCA) kits for protein assay and the caspase activity assay kits were supplied by Beyotime (Shanghai, China). The pan-Caspase blocking peptide VAD was obtained from Peptide Institute (Osaka, Japan). Antibodies purchased from Cell Signaling Technology (Danvers, MA) included anti-SSEA-1, anti-Oct4, anti-Pro-CASP3, anti-CASP3, anti-PARP-1, anti-Nanog, anti-β-Actin, and anti-rabbit or anti-mouse secondary antibodies. The goat anti-mouse TRITC secondary antibodies were purchased from Neobioscience (Hong Kong, China). Cell Culture. ulture. The mouse ESC cell line, CGR8 was used in the present experiments as described in our previous study.34 CGR8 cells were cultured in ESCs medium consisting of GMEM from Sigma (St Louis, MO, USA), 10 % fetal calf serum (FBS) from GE Healthcare HyClone (Australia), 1 mM sodium pyruvate, 0.01 % β-ME, 1 × non-essential amino acids, and 1,000 U/mL of human recombinant Leukemia Inhibitory Factor (LIF, Chemicon, CA). Cells were passaged every 4-6 days. In all cases, cells were grown at 37 °C with 5 % CO2. 6


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Cell Proliferation Assay. ssay. ATO was dissolved in 0.1 M NaOH in Milli-Q water (Millipore) to a final concentration of 1.0 mM as a stock solution. The effect of ATO on cells proliferation was monitored by MTT assay as described previously.37 Briefly, CGR8 cells (1 × 103 cells/well) were seeded in 96-well plates overnight, after treated with increasing dosages of ATO ranging from 0-5.0 μM for 48 hours, the MTT solution (final 1.2 mM) was added 100 μL/well and incubated for another 4 hours. After replacement the medium by 200 μL/well of acidified β-isopropanol, the plate was stayed at room temperature for 30 min and the absorbance at 570 nm was detected by the microplate reader (BioTek) and the value of test groups was expressed as mean relative percent to solvent control in presence of 0.5 mM NaOH in the culture medium. Cells growth during a time course of 5 days were monitored by cell counting every day under the ATO treatments with various doses of 0, 0.2, 0.5 and 1.0 μM, respectively. Data was expressed as draft of the growth curve. Quantification of Apoptosis by FACS SubG1 Analysis. For quantification of apoptosis by subG1 analysis according to previous,38 cells were fixed in 70 % ethanol at -20°C and stained with propidium iodide (16.5 μg/mL) in PBS after RNase (0.03 μg/mL, QIAGEN, Hilden, Germany) digestion. About 1 × 104 cells were analyzed for each sample on a FACS-Aria flowcytometer (Becton-Dickinson, Franklin Lakes, NJ), and the number of apoptotic cells was calculated using the software FACS Diva (Becton-Dickinson). Alkaline Phosphatase Staining. taining. CGR8 cells were plated in 6-well plates (2 × 105 cells/well) with ES cell medium for 24 h, and then changed to medium contained either ATO (0.2 µM and 0.5 µM, respectively) or RA (2.0 µM) with or without VAD (10 µM) for another 3 days. Cells incubated with LIF served as controls. After rinsed with PBS, cells were fixed for 15 minutes in 4 % paraformaldehyde (Polyscience, cat. no. 18814) at room temperature. And then CGR8 cells were detected by alkaline phosphatase (AP) staining according to the manufacturer’s instructions (Vector Laboratories, Alkphos Substrate III Blue.). All photos were taken by using a Nikon digital camera with a 10 X objective lens of Olympus microscope. Cells morphology were followed that, no AP staining and no ES cell morphology, defined as totally differentiated colonies; colonies showed large areas of differentiated cells, only small areas of undifferentiated cells, defined as mostly differentiated; it showed more than 7



half undifferentiated cells, defined as partially differentiated; it showed almost no discernible signs of differentiation, defined as undifferentiated. RNA RNA Extraction and Realeal-time qPCR. qPCR. Cells were collected at 3rd day after treatments with ATO. Total RNA were extracted from cell lysates and treated by TRIzol reagent (Invitrogen, CA) and were quantified by spectrophotometry. Synthesis of cDNA was carried out using reverse transcriptase from Invitrogen (CA) with 1 µg RNA per 25 µL reaction. For real-time PCR, reaction systems were undertaken by the Real time PCR Master Mix (SYBR Green) Reagent Kit (TOYOBO). According to the manufacturer’s protocol, 1 µl of cDNA was used in 20 μL final volume for 45 cycles. Primer sequences were listed in Supporting Information Table S1. Specificity of every primer amplicons were confirmed by melting curve analyses. For quantification and statistical analyses, the expression of target gene mRNA was normalized to the expression levels of the housekeeping gene β-Actin, using the relative expression software tool (REST).39 Microscopy and Cell Staining. The morphologies of cells were captured using Nikon digital camera and then imported into PhotoShop 6.0 (Adobe Systems, San Jose, CA). For immunostaining, cells were fixed with 2 % paraformaldehyde (PFA) in PBS, washed, blocked in 10 % normal goat serum, and stained with first antibodies including: anti-SSEA-1 antibody, anti-Oct4 antibody then incubated with goat anti-mouse TRITC secondary antibodies. The images were captured by the Olympus FV500 confocal system. Western Blot Analysis. Analysis. Cells pellets were harvested and lysed in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 1 % Triton X-100, 0.5 % sodium deoxycholate) with 1 × proteinase inhibitor cocktail. The protein sample was mixed with equal volume of 2 × Laemmli buffer containing 10 % (v/v) glycerol, 5 % (v/v) 2-mercaptoethanol, 2 % (w/v) SDS, 0.002 % (w/v) bromophenol blue (BB) and 60 mM Tris base (pH 6.8), and boiled for 5 min. After loading 20 μL (20 μg protein) of each sample on 12 % polyacrylamide gel carried out using the Laemmli buffer system, electrophoresis was run at room temperature until the BB dye reaching the bottom of the gel. After electrophoresis separation, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), and then incubated using specific antibodies, including anti- Pro-CASP3 antibody, anti-CASP3 antibody, 8


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anti-PARP-1 antibody, anti-Nanog antibody, to detected target proteins. After the primary antibody reaction overnight at 4oC, the PVDF membrane was washed by TBST (137 mM NaCl, 2.7 mM KCl, 19 mM Tris base and 0.1 % Tween-20) three times and incubated with anti-rabbit or anti-mouse secondary antibody diluted 1/5000 in TBST. Blots were visualized according to the standard enhanced chemiluminescence system using LAS-3000 imaging system (Fujifilm, Tokyo, Japan). Densitometry of blots were analyzed and normalized by β-Actin or lamin A using Image J software (National Institutes of Health, USA). Detection of Nanog Cleavage by CASP3 CASP3. ASP3. According to the method previously reported,27 in brief, for the cleavage assay of endogenous Nanog and OCT4, Western blotting of Oct4 and Nanog were performed by using anti-Nanog and anti-OCT4 specific antibody respectively, after CGR8 cells (2 × 106/well cultured in 6-well plate) treated with ATO and RA for 3 days. For the in tube cleavage assay, Nanog were cloned into the pEF1-luciferase-IRES-NEO vector (kindly provided by Prf. Pei) by using XhoI and XbaI cloning sites, which together with a FLAG tag were added at the C-terminus by a PCR-based strategy with 5‘-ggactcgagatgagtgtggatccagcttgtcc-3’ and 5’-tcctctagatcacttatcgtcatccttgtaatc-3’ primers. The plasmid was transfected into 293T cells and CGR8 cells with lipofectamine 2000 (Invitrogen). CGR8 cells (1 × 106) were plated into 6-well plates and transfected on the following day with 10 μL lipofectamine 2000 plus (Invitrogen) with 4 μg plasmid DNA. After Nanog with FLAG tag was over expressed in 293T cells for 48 hours, Nanog was purified by immunoprecipitation (IP) and incubated with CASP3, and Western blotting was then performed to detect the cleavage state of Nanog. To measure the cleavage of Nanog in CGR8 cells in situ, Western blotting for the whole cell lysates was performed after the cells with over expression of Nanog were treated with CASP3. The anti-FLAG antibody was applied in these cleavage assay, and anti-OCT4 antibody was used to show the endogenous level and cleavage state of OCT4. Statistical Analysis. nalysis. All comparison analyses were done using the SPSS statistical software package (SPSS 20.0 for Windows; SPSS, Inc., Chicago, IL). A P-value < 0.05 was considered statistically significant. Comparison of mean values was done using analysis of variance. All other analyses of equal samples sizes for each group and equal variances among the groups were compared using one-way ANOVA with 9



the Tukey post-hoc test. RESULTS Effect of ATO on Proliferation of CGR8 Cells. ells. According to a previous study,36 ATO at 0.1-0.5 μM induced partial differentiation in APL cells, but induced apoptosis at 0.2-2.0 μM. Herein, we found the impact of ATO treatments on the proliferation of CGR8 cells in a dose-dependent manner. Overall, treatments with ATO for 48 h at 0.5 µM induced the relative cell viability descending to 91.6 ± 2.98 % in comparison with the solvent control in presence of 0.5 mM NaOH in the culture medium, whereas higher-dose ATO significantly inhibited the cells proliferation to 59.1 ± 5.31 % at 1.0 µM and 13.5 ± 2.06 % at 5.0 µM, respectively(P < 0.01) (Figure 1A). Although cell growth remained continuous increases after treatments with ATO at 0.2-1.0 µM within 5 days, cells number was inhibited to more than 20 % by 0.5 µM ATO treatment for 4-5 days and decreased 50-60 % with treatments of ATO increasing to 1.0 µM (P < 0.01) indicating increased cytotoxicity after 3-5 days exposure of ATO (Figure 1B). Therefore, in the present study we focus on the modulation of low dose ATO of 0.2-0.5 µM to the embryonic stem cells stemness of self-renewal and differentiation in mouse CGR8 cells model. Effect of ATO on Apoptosis of CGR8 Cells. ells. To determine the effects of ATO treatments on apoptosis of CGR8 cells in a dose dependent manner, we analyzed the apoptotic subG1 fraction of the treated cells with various ATO doses by FACS. In comparison with controls, treatments with ATO at 0.2-0.5 µM only caused about 0.5 % to1.5 % elevation of the ratios of apoptotic cells within 3 days (Supporting Information Table S2), and led to around 3-4 % increases of apoptotic ratios for 4-day treatments, however treatments with ATO at 1.0-2.0 µM for 4 days induced more than 36 % increases of the ratios of apoptotic cells (Figure 2A). This effect was consistent with the expression levels of the apoptosis related genes such as Bax was enhanced, while Bcl-2, Bcl-xl and Mcl-1 were reduced slightly in the ATO treated cells at present dosage, although the GADD-45 was increased significantly as shown in Figure2B (P < 0.05). Corresponding to our deduction, the cells with treatment of RA displayed no significance changes of apoptosis by detection of subG1 percentage and apoptotic marker genes (Figure 2). Effect of ATO on Differentiation of CGR8 Cells. ells. To investigate whether the induction 10


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of cell differentiation was a direct effect of low dose ATO, we cultured the CGR8 cells with treatments of 0.2-0.5 µM ATO. Data as shown in Figure 3A, ATO treatments at 0.2-0.5 µM caused differentiating morphological changes in CGR8 cells. Compared with the control cells, the ATO treated cells displayed much more spread out and dendritic like changes, which indicate the obvious differentiation phenotype of the treated mouse ESCs. Similarly, the ESCs show clear morphologic signs of differentiation after stimulation with RA (2.0 µM), a common used differentiation inducible reagent. However, the ESCs colonies co-treated with either ATO or RA and the pan-Caspase blocking peptide VAD (100 µM) both remained largely typical ES cell morphology, suggesting the induction of cell differentiation with low dose ATO treatments. To further confirm and quantify the differentiation of CGR8 cells induced by ATO treatments, AP staining was conducted and data as shown in Figure 3B. ESCs with low dose ATO treatments exhibited obviously higher percentage of differentiated cells than control, although this effect was lower than RA treatment. Interestingly, the pan-Caspase blocking peptide VAD co-treatment could block much of the cell differentiation induced by ATO treatment, although the blocking effect was not complete. These finding was similar to previous publications in which CASP3 activity has been reported involving in ESC differentiation, but it may not be the unique factor to contribute to the differentiation process.40 ATO Altered the Expression of Distinct Genes for Differentiation in CGR8 Cells. ells. To identify the potential molecular mechanism of the ATO induced cell differentiation, the expression pattern of differentiation related genes were detected by qPCR with folds differences measured between treated and control cells. As shown in Figure 4A, cells treated with ATO at 0.2-0.5 µM showed significant decreases in the transcriptional expression of Oct4, Nanog and Rex-1 (P < 0.01), which are required for maintenance the self-renewal of ESCs. However, cells with low dose ATO exposure displayed significant increases in the transcriptional expression of Gata-4 and Gata-6 (P < 0.01) as well as AFP and IHH (P < 0.05), which are well known in relation to differentiation. These results indicated that low dose ATO treatments led to the differentiation of CGR8 cells. ATO Altered Expression of the Pluripotency Markers in CGR8 Cells. ells. The pluripotency markers of SSEA-1 and Oct4 were monitored using immunofluorescence to define the differentiating CGR8 cells with ATO treatments. Data was shown in Figure 5, both 11



SSEA-1 and Oct4 were obviously decreased in ATO treated cells in comparison with the controls. Such effects were correspond to which occurred in RA induced differentiated CGR8 cells. In consistent with the changes of differentiation phenotype, co-treatment with VAD exhibited significant reversible effect on SSEA-1 and Oct4 expression in ATO treated cells. ATO Treatments Induced CASP3 Activation in CGR8 Cells. ells. Previously, CASP3 activity has been reported as a crucial factor to mediate the differentiation of ESC.27 Herein, we analyzed the CASP3 activity of CGR8 cells with ATO treatments to evidence the role of CASP3 activation in the ATO-induced cell differentiation. As shown in Figure 6A, CASP3 activity was increased significantly in the mouse ESCs after stimulated with 0.2-0.5 µM ATO and RA for 2-4 days. We also performed a Western Blot analysis for active CASP3 in lysates from these cultures. Antibodies that recognized both the pro-CASP3 and the cleaved active CASP3 subunits were employed in Western Blot analysis. Consistent with the CASP3 activity, our results indicate that the ATO treatments to the cells up-regulated the expression of the active CASP3 significantly at 0.2-0.5 µM, as well as RA stimulation (Figure 6B). Using antibodies against five major caspases, we failed to detect significant changes of other four cysteine proteases in the treated cells (data not shown). To substantiate whether the CASP3 activation was associated with cell differentiation, we detected the change of PARP-1, a recognized caspase target during the execution of proliferation, which might be cleaved after induction of differentiation. Western Blot analysis targeted to PARP-1 revealed that appearance cleavage of PARP-1 (the p85 fragment) in ESCs treated with ATO and RA for 3 days (Figure 6C). CASP3 Activation Induced Nanog Cleavage in CGR8 Cells by ATO Treatments. reatments. We also detected the ATO induced cleavage state of Nanog to reveal the role of CASP3 in differentiation. Nanog is a critical transcription factor involved in ESCs self-renewal and has been reported as one substrate of CASP3 during the process of cell differentiation. We performed Western Blot analysis to identify whether Nanog and Oct4 were cleaved by CASP3 induced by ATO treatments in mouse ESCs. As shown in Figure 7A, besides the full length Nanog (37 kDa) was detected, there was a smaller band around 27 kDa on the same blot of cells with 0.5 µM ATO exposure for 3 days. Similarly, ATO induced effect was consistent with the RA induced differentiation response of CASP3 activation and Nanog cleavage. However, the Oct4 was not 12


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detected to be cleaved in CGR8 cells with both ATO and RA treatments. Furthermore, in tube cleavage assay was performed by adding CASP3 to CGR8 cells lysate, and the data (Figure 7B) proved that the Nanog was completely cleaved resulting in a 27 kDa band, which was correspond with the cleaved form of mouse Nanog within deduced CASP3 cleavage site of amino residue 67(D) to 68(S).27 Additionally, CASP3 substrate cleavage assay revealed that CASP3 could split Nanog expressed in CGR8 cells to produce the 27 kDa band, while Oct4 remained in single band of the form of full length on the Western Blot (Figure 7C). DISCUSSION The dual effects of arsenic may be associated with its cellular homeostatic control over two antagonistic processes-proliferation and differentiation.13-16 ESCs play critical roles during fetal development, and show great promise in regeneration medicine due to its self-renewal and pluripotency. Mouse ESCs are popular to investigate biological processes of development and differentiation.4 In the present study, a mESCs cell line, CGR8, was used to evaluate the dual effects and the molecular mechanism of ATO on cellular differentiation in vitro. As expected, CGR8 cells exhibited sensitive to ATO by proliferation inhibition otherwise apoptosis induction in comparison with those effects of ATO on cancer cell lines.19, 41 The proliferation inhibition of CGR8 cells was induced by ATO treatments in a dose-dependent manner (Figure1), in which 0.2-0.5 µM ATO caused less than 10% decline of cells growth in comparison with controls, while 1.0 µM and higher ATO caused significant inhibition of the cells proliferation (P < 0.01). Moreover, 0.2-0.5 µM ATO treatments only enhanced about 0.5 % to 4 % increases of apoptotic ratios within 4 days treatments in CGR8 cells (Figure 2). Such exposure level was in the scope of the dosage of ATO treatment for APL in which 0.1-0.5 μM ATO induced partial differentiation of APL cells,36 but lower than previous study using 0.7-1.3 μM ATO to induce cardiac differentiation of mESCs.4 ESCs can retain in undifferentiated state in vitro in the presence of proliferative stimuli, and these cells will also undergo differentiation in response to appropriate biologic signals.17 With the aim of investigating the effects that low dose ATO exerts on ESCs differentiation, we cultured CGR8 cells with LIF that recapitulates the molecular events and the functional features of stemness during differentiation. Throughout 13



culture, CGR8 cells were continuously exposed to 0.2-0.5 μM ATO for 4 days. Our results described the induction of cell differentiation process by detection of morphological changes and AP staining in comparison with that signs of differentiation after RA stimulation. As shown in Figure 3, the frequency of differentiated clones was augmented in the presence of ATO. Furthermore, the mRNA expression pattern of differentiation related genes were detected to identify the molecular mechanism. Data (Figure 4) showed that ATO treatments caused significant decreases in the expression of Oct4, Nanog and Rex-1 (P < 0.01). In contrast, the up-regulation of Gata4, Gata-6, AFP and IHH were found, which are well known tissue-specific markers for ESC differentiation. Additionally, our finding confirmed that ATO treatments hampered the expression of distinct pluripotency markers both SSEA-1 and Oct4 in CGR8 cells (Figure 5). Above mentioned alterations in CGR8 cells with ATO treatments were consistent with the differentiation phenotype induced by RA. Previously, overexpression of Nanog has been proven to mediate its function in ESCs, and endogenous deletion of Nanog induced ESCs differentiation. Taken together, our results indicated that low dose ATO treatments led to the differentiation of CGR8 cells through in part the Nanog signaling. Besides observing alterations to the core transcriptional factors for ESCs stemness, we also describe that low dose ATO leads to a quantitative increase in CASP3 activation and subsequent molecular events involving cellular differentiation. As we well know that ATO treatments to the ESCs can up-regulate the expression of the active CASP3, such effect was found similar to that induced by RA stimulation (Figure 6A). Also, when ESCs colonies were co-treated with the pan-Caspase blocking peptide VAD and either ATO or RA, much cells still appeared in undifferentiated ESC phenotype by evaluation the morphology, AP staining and stemness markers such as SSEA-1 and Oct4 expression. These findings suggested a line clues between the CASP3 activation and cellular differentiation that was consistent with previous publications in which CASP3 activity involving ESC differentiation, but it may not be the unique factor to contribute to the differentiation process.27 Up to date, mounting evidence have shown that CASP3 serves as a crucial enzyme to control over cell fate of proliferation and differentiation.42 It is interesting that CASP3 activation during ESCs differentiation with low dose ATO was limited and did not self-amplify to cause further apoptosis (Figure 2), in opposite a continuous 14


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proliferation of CGR8 cells (Figure 1). Such effects implied that subsequent molecular events in ESCs with ATO exposure might be mediated by CASP3 activation and might be responsible for controlling the balance of proliferation and differentiation. Regarding to this point, we first found that the appearance cleavage of PARP-1 occurred in ESCs with either ATO or RA treatments for 3 days following CASP3 activation (Figure 6B). Although PARP-1 is recognized as one of major caspase targets, but up-expression and cleavage activation of PARP1 is involved in differentiation, proliferation, and tumor transformation. To some extent, its execution of proliferation might be more significant than induction of differentiation.43 Accordingly, some else CASP3 targets should be required in controlling ESCs fate with ATO stimulation. Herein, the effects of a continuous exposure to ATO for short term (3 days) leading to Nanog cleavage in mESCs were observed. Present data (Figure 7) proved that both ATO treated and RA induced CGR8 cells displayed the indicating Nanog cleavage form around 27 kDa in size, which was correspond with the mouse Nanog cleaved by CASP3.27 This finding was further confirmed by in tube cleavage assay with adding CASP3 to Nanog over-expressed cultures and cells lysate, respectively. ESCs include the cleavage-resistant form of Nanog to enhance proliferation and the cleaved form of Nanog might exert functions during differentiation.28 The linkage between CASP3 activation and Nanog cleavage state provides compelling evidence that the apparent apoptotic response to ATO is also a determination for cellular differentiation. To the best of our knowledge, this study is the first to demonstrate specific effects of low dose ATO on modulation mESCs differentiation through CASP3 activation and Nanog cleavage. Cell differentiation is likely to be controlled by an elaborate orchestration of multiple signaling pathways. Whilst the alterations of CASP3 and Nanog that we reported in the present study indicate the impaired stemness maintenance in ESCs with ATO exposure, other important molecular events induced by ATO on ESCs phenotype were limited and should be mentioned. First, ATO treatments at present low doses initiated apoptosis within 3-4 days (Supporting Information Table S2 and Figure 2) with hints of increased GADD-45, which is implicated as a stress sensor that modulates the response of mammalian cells to genotoxic/physiological stress. Stress responses to ATO have been studied intensively within a variety of malignant cell lines otherwise stem cells. For example, the remission in APL patients with ATO 15



treatment, arsenic targets to PML/RARα to mediate its degradation and then to exert apoptosis and partial differentiation has been suggested the basic mechanisms of ATO action.44, 45 However, little is known about stress signaling in ESCs in response to ATO exposure and arsenic metabolites in different time course. Second, we observed alterations to the expression of distinct genes for differentiation in CGR8 cells. Herein we described more about the negative impacts of ATO on the transcriptional expression of Oct4, Nanog and Rex-1, which play essential role in stemness maintenance. Regarding the influence of ATO to tissue-specific differentiation, transcriptional increases of Gata4, Gata-6, AFP and IHH also have been found in our study. Considering these genes involve in myocardial, endodermal, ectodermal and chondrocyte differentiation,46-49 quantification of these genes should be done in future embryotoxicity assays after arsenic exposure with ESCs. Third, as a cysteine protease, CASP3 plays critical roles in apoptosis by regulating activation of other caspases such as 6, 7 and 9.24 Although herein we showed that CASP3 activity was elevated in CGR8 cells with low dose ATO, but it did not amplify following caspases cascade activation in turn. It is worth to elucidate the molecular mechanism thereby ATO induced CASP3 activation discriminates apoptosis from differentiation in mESCs. Additionally, recent observing of the protease family of calpains played important roles in response to stresses and inflammation,50, 51 and ATO treatment exhibited to trigger the Ca++/calpain/caspase-12-mediated regulating the ER-mitochondrial crosstalk,52 implied such effects of ATO on protease activation mediated post translational cleavage in ESCs needed to be confirmed. Besides above mentioned, the molecular networks between RA and ATO induced ESCs differentiation including transcriptome and proteome levels also should be clarified in future. Altogether, further studies are required to explore if ATO as well as other arsenic compounds may serve as effective chemical candidates for inducing the differentiation of ESCs. In conclusion, the present study described the effects of low dose ATO exposure on cell fate determination in mESCs, CGR8. The major observation was that CGR8 cells with low doses ATO exposure appeared cell differentiation otherwise apoptosis. For the molecular mechanism, we preliminarily suggested that such effects might be tuned by ATO induced CASP3 activation and subsequent Nanog cleavage in mESCs. Reduced expression of Oct4, Nanog and Rex-1, but increased expression of Gata4, 16


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Gata6, AFP and IHH were also involved in low doses ATO induced differentiation of mESCs. All the findings provided partial understanding of the action mechanisms of arsenic on the cellular apoptosis and differentiation. Supporting Information Primer sequences used for real time quantitative PCR and percentage of cells with hypodiploid DNA content (subG1 fraction) following ATO treatment. AUTHOR INFORMATION Wenlin Yuan and Jun Chen contributed equally to this work. Corresponding Authors *(Z.H.) Department of Biotechnology, School of Life Science and Technology, Jinan University, Guangzhou, 510632, Guangdong Province, China. Tel: +86-20-85220219. E-mail: [email protected] *(F.H.) Department of Rehabilitation Medicine, School of Medical Engineering, Foshan University, Foshan, 528000, Guangdong Province, China. Tel: +86-757-82810464. E-mail: [email protected] ORCID Zhi Huang: 0000-0002-9698-8652 Funding This work was supported by grants of the National Nature Science Foundation of China (No.81570397), the Key Project of Marine Fishery Science and Technology of Guangdong Province (A201501C07), the Major Project of Science and Technology of Guangzhou (201604020142), the Project of Science and Technology of Guangdong Province (2014A050503044, 2017A0405067) and the Construction Project for Guangzhou Key Laboratory (201705030003). Notes The authors declare no competing financial interest. ABBREVIATIONS ABBREVIATIONS

ATO, arsenic trioxide; APL, acute promyelocytic leukemia; ESC, embryonic stem cell; CASP3, Caspase 3; As, arsenic; iAs, inorganic arsenic; LIF, Leukemia Inhibitory Factor; iPS, induced pluripotent stem cells; BMSSCs, bone marrow stromal stem cells; HSCs, hematopoietic stem cells; RA, retinoic acid; RAR, retinoic acid receptor; ERK2, activation of extracellular signal-regulated kinase 2; ER, endoplasmic 17



reticulum; PI, propidium iodide; DMEM, Dulbecco’s modified Eagle’s medium; SDS, sodium dodecyl sulfate; AP, alkaline phosphatase; REST, relative expression software tool; PVDF, polyvinylidene difluoride; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PFA, paraformaldehyde; IP, immunoprecipitation; FBS, fetal calf serum.

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Figure Legends Figure 1. 1. ATO treatment treatments reatments influence on the proliferation of CGR8 cells in a dosedose-dependent manner. manner. (A) CGR8 cells were treated with increased concentrations of ATO (0.2–5.0µM) for 48 hours, followed by MTT assays. The mean absorbance at 570 nm were obtained and expressed in relative percentage to the solvent control. (B) CGR8 cells were plated at 1 × 104 cells per well of 24-well plates, followed by treatment with increased concentrations of ATO (0.2–1.0µM). Cell numbers were counted after 1, 2, 3, 4 and 5 days. Results shown represent the mean ± SD (n=6) of pooled data obtained from three independent experiments. *P < 0.05, ***P < 0.001, significant different from value for the control. Figure 2. Low dose ATO treatments treatments led to slight increases of apoptosis in CGR8 CGR8 cells. cells. (A) The apoptotic subG1 fraction were analyzed by FACS. CGR8 cells were treated with the absence or presence of ATO (0.2-2 µM) and RA for 4 days. After fixed in 70% ethanol at -20°C and stained with propidium iodide, a total of 1 × 104 cells were analyzed for each sample on a FACS-Aria flowcytometer. (B) Transcriptional expression levels of GADD-45, Bax, Bcl-2, Mcl-1and Bcl-xl were determined by quantitative real-time PCR (qPCR). After cells treated with ATO for 3 days, total RNA were extracted and 2.0 μg of total RNA was applied to make cDNA, and then 1 μL of cDNA was used for qPCR analysis. Data was expressed as folds relative to housekeeping gene β-Actin. Statistical differences were determined by ANOVA (*P < 0.05) and referred to averages of three independent trials. Figure 3. Low dose ATO treatments induced differentiation differentiation of CGR8 cells. (A) 23



Morphology alterations of CGR8 cells treated with ATO or RA for 3 days in the absence or presence of the pan-Caspase blocking peptide VAD, and cells incubated with LIF served as controls. (B) Quantification of alkaline phosphatase (AP) staining colonies were shown. AP staining were performed on CGR8 cells treated as above, and all photos were taken by using a Nikon digital camera with a 10 x objective lens of Olympus microscope. Figure 4. Low dose ATO treatments reatments resulted resulted in alterations of mRNA levels of the distinct genes of CGR8 cells. cells. (A) mRNA levels of the genes including Rex-1, Oct4 and Nanog required for self-renewal; and (B) mRNA levels of the genes including

Gata4, Gata-6, AFP and IHH related to tissue-specific differentiation in ESCs were determined by qPCR. After cells treated with ATO for 3 days, total RNA were extracted and 2.0 μg of total RNA was applied to make cDNA, and then 1 μL of cDNA was used for qPCR analysis. Data was expressed as folds relative to housekeeping gene β-Actin. Statistical differences were determined by ANOVA (***P < 0.01) and referred to averages of three independent trials. Figure 5. Low dose ATO decreased the expression of pluripotency markers SSEASSEA-1 and Oct4 in CGR8 cells. cells. CGR8 cells were seeded on cover slides with ATO treatments at present doses or with RA stimulation for 3 days in the absence or presence of the pan-Caspase blocking peptide VAD. After fixed with 2% PFA in PBS, the expression levels of SSEA-1 (A) and Oct4 (B) in CGR8 cells were detected by immunofluorescence staining with anti-SSEA-1 and anti-Oct4 IgM respectively, followed by the TRITC-conjugated goat anti-mouse antibody. Cell images were 24


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scanned by Confocal Microscopy under the fluorescent field (a, b, c, d, e), bright field (f, g, h, i, j) and also merged together (k, l, m, n, o). Figure 6. Low dose ATO increased increased the activity activity of CASP3 CASP3 in CGR8 cells. cells. CGR8 cells were stimulated with ATO and RA for 2-4 days. (A) Caspase activity was measured in

vitro by caspase activity assay kit. (B) the levels of Pro-CASP3 and Active CASP3; and (C) the PARP-1 and its cleavage states were measured by Western Blot analysis with specific antibody. Densitometry assay for the blots were analyzed and normalized by β-Actin using Image J software. Figure 7. CASP3 was involved in Nanog cleavage induced by low dose ATO. After CGR8 cells treated with ATO and RA for the indicated time, (A) Western blotting of Oct4 and Nanog were then performed to show the cleavage of Nanog (around 27 kDa). After Nanog with FLAG tag was over expressed in 293T cells, (B) Western blotting was performed to show the cleavage of Nanog purified by immunoprecipitation (IP) and incubated with CASP3. When the CGR8 cells with Nanog over expression were treated with CASP3, (C) Western blotting was also performed to show the cleavage of Nanog in whole cell lysate, with showing the endogenous OCT4.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Low-Dose Arsenic Trioxide Modulates the Differentiation of Mouse

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