Apoptotic effects of cigarette smoke extracts on mouse embryonic stem cells via oxidative stress

Previous studies have reported that cigarette smoke and cigarette smoke extract (CSE) have negative effects on embryonic development. However, no studies have investigated the mechanism through which CSE affects the cellular signaling pathway leading to apoptosis and oxidative stress in embryonic cells, or how the two pathways are cross‐linked. Thus, we studied the effects of CSE on apoptosis and oxidative stress in mouse embryonic stem cells (mESCs). Specifically, we measured changes in cell viability in response to CSEs (3R4F and two domestic cigarettes CSE 1 and 2) using a water soluble tetrazolium (WST) assay and a neutral red uptake (NRU) assay, which revealed that cell viability decreased in a concentration‐dependent manner. Western blot analysis revealed that the expression of cyclin D1 and cyclin E1 was decreased and that of p21 and p27 was increased by CSE. Additionally, the number of terminal deoxynucleotidyl transferase (TUNEL)‐stained cells was increased by CSE, while the levels of Bax and Caspase‐3 increased and Bcl‐2 decreased. Moreover, a 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) assay and reactive oxygen species (ROS)‐Glo H₂O₂ assay confirmed that ROS were generated in response to CSE and that they were associated with up‐regulated Keaf‐1 and CHOP. Overall, the results revealed that cigarette smoke extract (CSE) inhibited cell proliferation by regulating cell cycle‐related protein expression and increased oxidative stress by regulating the expression of Kelch‐like ECH‐associated protein 1 (Keap‐1) and CCAAT/enhancer‐binding protein homologous protein (CHOP), resulting in apoptosis in mESCs.     

Inhibitory effects of cigarette smoke extracts on neural differentiation of mouse embryonic stem cells

Maternal smoking during the perinatal period is linked to adverse neonatal outcomes such as low birth weight and birth defects. Numerous studies have shown that cigarette smoke or nicotine exposure has a widespread effect on fetal nerve development. However, there exists a lack of understanding of what specific changes occur at the cellular level on persistent exposure to cigarette smoke during the differentiation of embryonic stem cells (ESCs) into neural cells. We previously investigated the effects of cigarette smoke extract (CSE) and its major component, nicotine, on the neural differentiation of mouse embryonic stem cells (mESCs). Differentiation of mESCs into neural progenitor cells (NPCs) or neural crest cells (NCCs) was induced with chemically defined media, and the cells were continuously exposed to CSE or nicotine during neural differentiation and development. Disturbed balance of the pluripotency state was observed in the NPCs, with consequent inhibition of neurite outgrowth and glial fibrillary acidic protein (Gfap) expression. These inhibitions correlated with the altered expression of proteins involved in the Notch-1 signaling pathways. The migration ability of NCCs was significantly decreased by CSE or nicotine exposure, which was associated with reduced protein expression of migration-related proteins. Taken together, we concluded that CSE and nicotine inhibit differentiation of mESCs into NPCs or NCCs, and may disrupt functional development of neural cells. These results imply that cigarette smoking during the perinatal period potentially inhibits neural differentiation and development of ESCs cells, leading to neonatal abnormal brain development and behavioral abnormalities.     

Effect of cigarette smoke components on vascular smooth muscle cell migration toward platelet-derived growth factor BB

Cigarette smoking is one of the factors causing accumulation of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. Changes in cell migration toward platelet-derived growth factor BB were investigated using a Boyden chamber after 48-h preincubation of GBaSM-4 VSMCs with nicotine or nicotine-free cigarette smoke extract (CSE). A nicotine concentration of 0.1 μM maximally promoted cell migration; 0.1% CSE also promoted cell migration, while high CSE concentrations damaged GBaSM-4 cells. Fetal bovine serum (FBS) long-depletion induced decrease in migration of GBaSM-4 cells. Our results suggest that nicotine and some CSE components can induce GBaSM-4 cell migration.     

Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway

Aim:To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs).Methods:Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 μmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging.Results:Compared with control, mESCs treated with ouabain (20 μmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 μmol/L) inhibited cardiac differentiation while ouabain (20 μmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR).Conclusion:Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.     

Effects of methylmercury exposure on neuronal differentiation of mouse and human embryonic stem cells

The establishment of more efficient in vitro approaches has been widely acknowledged as a critical need for toxicity testing. In this study, we examined the effects of methylmercury (MeHg), which is a well-known developmental neurotoxicant, in two neuronal differentiation systems of mouse and human embryonic stem cells (mESCs and hESCs, respectively). Embryoid bodies were generated from gathering of mESCs and hESCs using a micro-device and seeded onto ornithine–laminin-coated plates to promote proliferation and neuronal differentiation. The cells were exposed to MeHg from the start of neuronal induction until the termination of cultures, and significant reductions of mESCs and hESCs were observed in the cell viability assays at 1,10,100 and 1000 nM, respectively. Although the mESC derivatives were more sensitive than the hESC derivatives to MeHg exposure in terms of cell viability, the morphological evaluation demonstrated that the neurite length and branch points of hESC derivatives were more susceptible to a low concentration of MeHg. Then, the mRNA levels of differentiation markers were examined using quantitative RT-PCR analysis and the interactions between MeHg exposure and gene expression levels were visualized using a network model based on a Bayesian algorithm. The Bayesian network analysis showed that a MeHg-node was located on the highest hierarchy in the hESC derivatives, but not in the mESC derivatives, suggesting that MeHg directly affect differentiation marker genes in hESCs. Taken together, effects of MeHg were observed in our neuronal differentiation systems of mESCs and hESCs using a combination of morphological and molecular markers. Our study provided possible, but limited, evidences that human ESC models might be more sensitive in particular endpoints in response to MeHg exposure than that in mouse ESC models. Further investigations that expand on the findings of the present paper may solve problems that occur when the outcomes from laboratory animals are extrapolated for human risk evaluation.     

Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

Aim:

Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells. The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and, if so, whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α).

Methods:

Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol. The cumulative percentage of beating EBs was calculated. The expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) was detected using RT-PCR, real-time PCR and Western blot. The dispersed beating EBs were examined using immunofluorescent staining.

Results:

The percentage of beating EBs and the expression of cTnI were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium. From 6 to 18 d of differentiation, the increased expression of cTnI and α-MHC by ghrelin (1 nmol/L) was time-dependent, and in line with the alteration of the percentages of beating EBs. Furthermore, the dispersed beating EBs were double-positively immunostained with antibodies against cTnI and α-actinin. However, blockage of GHS-R1α with its specific antagonist D-[lys³]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation.

Conclusion:

Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells, which is not mediated via GHS-R1α.     

胚胎干细胞体外分化为肝细胞和心肌细胞的研究进展

胚胎干细胞是从早期胚胎或原始性腺分离后能在体外长期传代培养的全能细胞系。胚胎干细胞体外可以诱导分化为各种体细胞,并且用这些体细胞治疗相应的疾病,一直以来是生物医药领域的研究焦点。目前,在该领域用于诱导胚胎干细胞分化的方法很多,其中的一些方法就用到化合物和部分中药提取物。整理近5年来胚胎干细胞体外通过拟胚体途径、细胞单层诱导分化为肝细胞和心肌细胞的有关文献,做一简要综述。     

Expression profiles of histone lyslne demethylases during cardiomyocyte differentiation of mouse embryonic stem cells

Aim： Histone lysine demethylases （KDMs） control the lineage commitments of stem cells. However, the KDMs involved in the determination of the cardiomyogenic lineage are not fully defined. The aim of this study was to investigate the expression profiles of KDMs during the cardiac differentiation of mouse embryonic stem cells （mESCs）. Methods： An in vitro cardiac differentiation system of mESCs with Brachyury （a mesodermal specific marker） and FIk-1^＋/Cxcr4^＋ （dual cell surface biomarkers） selection was used. The expression profiles of KDMs during differentiation were analyzed using Q-PCR. To understand the contributions of KDMs to cardiomyogenesis, the mESCs on differentiation d 3.5 were sorted by FACS into Brachyury^＋ cells and Brachyury cells, and mESCs on d 5.5 were sorted into FIk-1^＋/Cxcr4^＋ and FIk-1-/Cxcr4- cells. Results： mESCs were differentiated into spontaneously beating cardiomyocytes that were visible in embryoid bodies （EBs） on d 7. On d 12-14, all EBs developed spontaneously beating cardiomyocytes. Among the 16 KDMs examined, the expression levels of Phf8, Jaridla, Jhdmld, Utx, and Jmjd3 were increased by nearly 2-6-fold on d 14 compared with those on d O. Brachyury^＋ cells showed higher expression levels of Jmjd3, Jmjd2a and Jhdmld than Brachyury- cells. A higher level of Jmjd3 was detected in FIk-1^＋/Cxcr4^＋ cells, whereas the level of Jmjd2c was lower in both Brachyury^＋ cells and FIk-1^＋/Cxcr4^＋ cells. Conclusion： KDMs may play important roles during cardiomyogenesis of mESCs. Our results provide a clue for further exploring the roles of KDMs in the cardiac lineage commitment of mESCs and the potential interference of cardiomyogenesis.     

羧甲司坦对香烟烟雾提取物诱导A549细胞炎性损伤的影响

目的：探讨羧甲司坦抑制香烟烟雾提取物（CSE）诱导A549细胞炎性损伤的作用。方法：传代培养人A549细胞并分为5组：对照组,CSE组,羧甲司坦低、中、高剂量组（予不同浓度羧甲司坦孵育和CSE诱导）。qRT-PCR、ELISA检测主要细胞因子的合成和释放,免疫蛋白印迹（Western-blot）、免疫荧光（IF）观察NF-κB及MAPK相关信号通路的活化情况。结果：以羧甲司坦预处理或后处理,均可降低CSE诱导的A549细胞的IL-6、IL-8和MIP-1β mRNA的表达;降低IL-6及IL-8的释放。羧甲司坦预处理可抑制p65入核。Western-blot结果显示,对照组,CSE组,羧甲司坦低、中、高剂量组的P-p65蛋白相对表达量分别为（0.17±0.05）、（0.90±0.19）、（0.68±0.15）、（0.64±0.12）和（0.57±0.13）,pERK1/2蛋白相对表达量分别为（0.30±0.10）、（1.25±0.33）、（1.01±0.19）、（0.89±0.22）和（0.81±0.18）,CSE组均明显高于对照组,羧甲司坦低、中、高剂量组均明显低于CSE组（P<0.05）。结论：羧甲司坦通过抑制NF-κB p65及ERK1/2 MAPK活化,发挥对CSE诱导的A549细胞炎性损伤的保护作用。     

葡萄糖对小鼠胚胎干细胞生长的影响

目的：目前小鼠胚胎干细胞（mouse embryonic stem cells,mESC）常规应用高浓度葡萄糖（25mmol／L）培养基培养,但是在应用干细胞向糖尿病胰岛beta细胞分化的研究中发现,慢性高糖培养可促进干细胞的凋亡、降低细胞分化的效率及分化后胰岛beta细胞对葡萄糖的反应性,因此本研究拟选择合适的较低浓度的葡萄糖以优化胚胎干细胞的培养基、提高胚胎干细胞的生长、分化效率。方法：mESC传代4或12h后,将传统的25mmol／L葡萄糖培养基分别换为5、10、15、25mmol／L葡萄糖培养基培养,均用碱性磷酸酶（AP）染色计数细胞集落形成情况、台盼兰染色测定细胞数目,MTT法测定细胞活力及用4’,6-联脒-2-苯基吲哚（4’,6-diamidino-2-phenylindole,DAPI）染色检测细胞凋亡情况。结果：mESC传代4h后即调换葡萄糖浓度：（1）各葡萄糖浓度组（5、10、15mmol／L）与25mmol／L组相比,集落形成明显减少。（2）各葡萄糖浓度组与25mmol／L组相比,mESC增殖及细胞活力均受到不同程度明显影响。mESC传代12h后调换葡萄糖浓度：（1）各葡萄糖浓度组（5、10、15mmol／L）与25mmol／L组相比,集落形成无明显变化,且AP染色呈强阳性。（2）15mmol／L葡萄糖组对mESC增殖及细胞活力均无明显影响。（3）15mmol／L葡萄糖组与25mmol／L葡萄糖组细胞核形态正常,均未见明显凋亡。结论：ESC传代12h后将培养基中传统的高糖（25mmol／L）降低为15mmol／L,不影响mESC细胞活力及多能分化潜能和未分化状态。     

全反式视黄酸促进胚胎神经干细胞诱导分化的研究

目的本研究观察全反式视黄酸（atRA）对于体外分离培养的胚胎神经干细胞（ENSCs）生长增殖和诱导分化的作用及其可能的分子机制。方法分离培养孕15dSD大鼠（E15 d SD Rattus）海马回ENSCs,采用不同组合成分的神经干细胞培养基培养ENSCs,利用MTF法检测其对于ENSCs存活和增殖的影响;采用不同组合成分的神经细胞诱导分化培养基培养ENSCs,利用细胞免疫荧光染色法鉴定ENSCs及atRA对于其诱导分化的影响。结果MTT实验结果表明,atRA^＋组、atRA^-组和DMSO组均出现ENSCs的增殖效果,而对照组则没有明显的增殖现象,其中atRA^-组最明显,DMSO组次之,atRA^＋组最弱。AtRA^＋组与atRA^-的ENSCs经过诱导分化后产生的神经细胞类型有较明显差异,并且atRA组处理产生的神经元是对照组的3倍左右。结论atRA能够抑制ENSCs的增殖,并且抵消神经生长因子对于ENSCs的促进有丝分裂作用,atRA还可以促进ENSCs定向诱导分化为神经元。     

Comparison of two in vitro models of cigarette smoke exposure

Cigarette smoke is associated with a high morbidity and mortality, and affects particularly the respiratory tract. Various in vitro models have been developed to study the effects of cigarette smoke on bronchial epithelial cells. To identify an adequate exposure model of cigarette smoke, we analysed the effects of cigarette smoke extract (CSE) and a smoking chamber on bronchial epithelial cells. The release of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-10, and vascular endothelial growth factor (VEGF) was measured. Bronchial epithelial cells isolated from Sprague-Dawley rat (NRBE) were exposed to 3% CSE or air control every day for 3 days. In the second model, NRBE were placed in an air/liquid interface and exposed, in a smoking chamber, to whole smoke from 2 cigarettes, twice daily for 3 days. Levels of MCP-1, IL-10, and VEGF were measured by enzyme-linked immunosorbent assay (ELISA), 24?h after the last exposure. The pattern of MCP-1 production by bronchial epithelial cells was different between the two models. MCP-1 release was increased after 3 days of exposure in the CSE model, but was inhibited using the smoking chamber model. Production of IL-10 by NRBE was reduced after 3 days in both models. Finally, no difference was observed in the production of VEGF between the two models. CSE and the smoking chamber differently modulate bronchial epithelial cell mediator production, demonstrating that the model of cigarette smoke exposure used can influence the data obtained.     

Effect of cigarette smoke on protein synthesis in brain and liver

The rates of protein synthesis in brain and liver were determined in 2 inbred mouse strains (BALB/c and CXBH) during 1 hr of smoke exposure, and after 3 and 6 days of cigarette smoke treatment. Exposure to cigarette smoke reduced valine incorporation into brain and liver protein by 12 and 30% respectively. The greatest part of this reduction in synthesis was due to the hypothermie effect of smoke exposure, which was an approx. 8% change in the synthesis rate for each degree of reduction in body temperature, though a significant smoke effect was still evident. Two major components of cigarette smoke, nicotine and carbon monoxide, were individually tested. Injections of nicotine produced a similar inhibition of brain protein synthesis, with no effect on liver protein synthesis. After extraction of nicotine from the smoke by use of a Cambridge filter, only slight inhibition of brain protein synthesis was observed, which was due to the decrease in body temperature; there was still a significant inhibition in the liver. Incorporation measured, not during but after smoke exposure, was still significantly reduced, although reduction in the liver was smaller than that measured during smoke exposure. Carboxyhemoglobin at levels 50% higher than that achieved by smoke exposure had no effect on brain or liver protein synthesis; higher carboxyhemoglobin levels (300–400% higher than levels during smoke exposure) produced inhibition of liver protein synthesis. The results suggest that the slight and significant inhibition of brain protein synthesis is due to nicotine, whereas the effect on the liver is probably due to anoxia. Smoke treatment for 3–6 days suggests that there is no adaptation to these effects. Strain differences in smoke sensitivity are not related to the effect of nicotine on protein synthesis, suggesting that other mechanisms are involved in smoke sensitivity.     

Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation

Aim:

Some small molecules can induce mouse embryonic stem (ES) cells to differentiate into neuronal cells. Here, we explored the effect of isobavachin (IBA), a compound with a prenyl group at position 8 of ring A, on promoting neuronal differentiation and the potential role of its protein prenylation.

Methods:

The hanging drop method was employed for embryonic body (EB) formation to mimic embryo development in vivo. The EBs were treated with IBA at a final concentration of 10⁻⁷ mol/L from EB stage (d 4) to d 8+10. Geranylgeranyltransferase I inhibitor GGTI-298 was subsequently used to disrupt protein prenylation. Neuronal subtypes, including neurons and astrocytes, were observed by fluorescence microscopy. Gene and protein expression levels were detected using RT-PCR and Western blot analysis, respectively.

Results:

With IBA treatment, nestin was highly expressed in the neural progenitors generated from EBs (d 4, d 8+0). EBs then further differentiated into neurons (marked by β-tubulin III) and astrocytes (marked by GFAP), which were both up-regulated in a time-dependent manner on d 8+5 and d 8+10. Co-treatment with GGTI-298 selectively abolished the IBA-induced neuronal differentiation. Moreover, in the MAPK pathway, p38 and JNK phosphorylation were down-regulated, while ERK phosphorylation was up-regulated after IBA treatment at different neuronal differentiation passages.

Conclusion:

IBA can facilitate mouse ES cells differentiating into neuronal cells. The mechanism involved protein prenylation and, subsequently, phos-ERK activation and the phos-p38 off pathway.     

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA‐4, TRA‐1‐60 and TRA‐1‐81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5‐fluorouracil, indomethacin and non‐embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT‐4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma‐derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down‐regulated in a dose‐dependent manner after treatment with embryotoxic chemicals. After treatment with 5‐fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up‐regulation of development, cell cycle and apoptosis‐related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development‐, cell cycle‐ and apoptosis‐related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage. Copyright © 2014 John Wiley & Sons, Ltd.     

Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α‐actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L ‐threonine dehydrogenase, X‐ray repair cross‐complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system. Copyright © 2015 John Wiley & Sons, Ltd.     

Cigarette smoke extracts induced the colon cancer migration via regulating epithelial mesenchymal transition and metastatic genes in human colon cancer cells

There was considerable evidence that exposure to cigarette smoke is associated with an increased risk for colon cancer. Nevertheless, the mechanism underlying the relationship between cigarette smoking and colon cancer remains unclear. Moreover, there were only a few studies on effects of complexing substance contained in cigarette smoke on colon cancer. Thus, we further investigated whether cigarette smoke extract (CSE) affects the cell cycle, apoptosis and migration of human metastatic colon cancer cells, SW‐620. MTT assay revealed that SW‐620 cell proliferation was significantly inhibited following treatments with all CSEs, 3R4F, and two‐domestic cigarettes, for 9 days in a concentration‐dependent manner. Moreover, CSE treatments decreased cyclin D1 and E1, and increased p21 and p27 proteins by Western blot analysis in SW‐620 cells. Additionally, the treatment of the cells with CSE contributed to these effects expressing by apoptosis‐related proteins. An increased migration or invasion ability of SW‐620 cells following CSE treatment was also confirmed by a scratch or fibronectin invasion assay in vitro. In addition, the protein levels of E‐cadherin as an epithelial maker were down‐regulated, while the mesenchymal markers, N‐cadherin, snail, and slug, were up‐regulated in a time‐dependent manner. A metastatic marker, cathepsin D, was also down‐regulated by CSE treatment. Taken together, these results indicate that CSE exposure in colon cancer cells may deregulate the cell growth by altering the expression of cell cycle‐related proteins and pro‐apoptotic protein, and stimulate cell metastatic ability by altering epithelial‐mesenchymal transition (EMT) markers and cathepsin D expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 690–704, 2017.