Article pubs.acs.org/crt
Cite This: Chem. Res. Toxicol. 2018, 31, 472−481
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*,† †
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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 S Supporting Information *
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) exhibit properties of stemness and serve as a popular model to investigate epigenetic modifiers including environmental pollutants. Herein, the effects 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 had slight inhibition of proliferation with 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 corresponded with the mouse Nanog cleaved by CASP3 in a tube cleavage assay. Taken together, we suggest that low-dose ATO exposure will induce differentiation, other than 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 provide a preliminary action mechanism of arsenic on the cell fate determination.
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INTRODUCTION Arsenic (As) is a well-known environmental toxicant and carcinogen.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 the presence of arsenic trioxide (ATO) at 0.1−1.0 μM alters the differentiation of mouse embryonic stem cells into cardiomyocytes by the dysruption of the sarcomere and syncytium organization.4 The paradoxical impact of arsenic species on human health is that ATO is recognized as one of the most effective arsenic agents for treatment of acute promyelocytic leukemia (APL) and has been widely applied as a component of traditional Chinese medicine.5−7 The underlying mechanism of these 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 © 2018 American Chemical Society
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 was 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, and alternatively, these same cells will undergo differentiation and arrest of proliferation in the presence/absence of distinct biologic signals.17 ATO treatments induce differentiation in APL cells18 and neuroblastoma cells.19 However, the disturbance of ATO to ESCs differentiation and the underlying mechanism are still not clarified. With the properties of self-renewal and pluripotency, ESCs have shown great promise in regeneration medicine. ESCs are maintained in an undifferentiated state by the core transcripReceived: February 3, 2018 Published: May 16, 2018 472
DOI: 10.1021/acs.chemrestox.8b00027 Chem. Res. Toxicol. 2018, 31, 472−481
Chemical Research in Toxicology
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tional 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 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 selfamplification 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 that 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 the caspase cascade. Clinical treatments with ATO caused differentiation and apoptosis of leukemic cells that was associated with caspase activation,29 implying 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 The 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 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 was 0.26 μM and 0.35 μM on 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 is to focus on the modulation of low-dose ATO (0.2−0.5 μM) to the differentiation of mESCs. At molecular and functional levels, the effects of a continuous exposure to ATO for short-term (3−4 days) leading to CASP3 activation and subsequent Nanog cleavage in mESCs were confirmed.
Article
MATERIALS AND METHODS
Materials and 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-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 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, antiPARP-1, anti-Nanog, anti-β-Actin, and antirabbit or antimouse secondary antibodies. The goat antimouse TRITC secondary antibodies were purchased from Neobioscience (Hong Kong, China). Cell Culture. 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 × nonessential amino acids, and 1000 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. Cell Proliferation Assay. 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 being treated with increasing dosages of ATO ranging from 0 to 5.0 μM for 48 h, the MTT solution (final 1.2 mM) was added 100 μL/well and incubated for another 4 h. After replacement of the medium with 200 μL/well of acidified β-isopropanol, the plate was kept at room temperature for 30 min, 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 the 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 were 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. CGR8 cells were plated in 6well plates (2 × 105 cells/well) with ES cell medium for 24 h and then changed to a medium containing 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 rinsing with PBS, cells were fixed for 15 min 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× objective lens of Olympus microscope. Cells morphology was followed with 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 were defined as mostly differentiated. It showed more than half undifferentiated cells, defined as partially differentiated, and it showed 473
DOI: 10.1021/acs.chemrestox.8b00027 Chem. Res. Toxicol. 2018, 31, 472−481
Article
Chemical Research in Toxicology
Statistical Analysis. All comparison analyses were done using the SPSS statistical software package (SPSS 20.0 for Windows; SPSS, Inc., Chicago, IL). A P-value
