Zhixiong Zhuang

SiO2 nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells.

BackgroundNanometer silicon dioxide (nano-SiO2) has a wide variety of applications in material sciences, engineering and medicine; however, the potential cell biological and proteomic effects of nano-SiO2 exposure and the toxic mechanisms remain far from clear.ResultsHere, we evaluated the effects of amorphous nano-SiO2 (15-nm, 30-nm SiO2). on cellular viability, cell cycle, apoptosis and protein expression in HaCaT cells by using biochemical and morphological analysis, two-dimensional differential gel electrophoresis (2D-DIGE) as well as mass spectrometry (MS). We found that the cellular viability of HaCaT cells was significantly decreased in a dose-dependent manner after the treatment of nano-SiO2 and micro-sized SiO2 particles. The IC50 value (50% concentration of inhibition) was associated with the size of SiO2 particles. Exposure to nano-SiO2 and micro-sized SiO2 particles also induced apoptosis in HaCaT cells in a dose-dependent manner. Furthermore, the smaller SiO2 particle size was, the higher apoptotic rate the cells underwent. The proteomic analysis revealed that 16 differentially expressed proteins were induced by SiO2 exposure, and that the expression levels of the differentially expressed proteins were associated with the particle size. The 16 proteins were identified by MALDI-TOF-TOF-MS analysis and could be classified into 5 categories according to their functions. They include oxidative stress-associated proteins; cytoskeleton-associated proteins; molecular chaperones; energy metabolism-associated proteins; apoptosis and tumor-associated proteins.ConclusionsThese results showed that nano-SiO2 exposure exerted toxic effects and altered protein expression in HaCaT cells. The data indicated the alterations of the proteins, such as the proteins associated with oxidative stress and apoptosis, could be involved in the toxic mechanisms of nano-SiO2 exposure.

SiO2 nanoparticles induce global genomic hypomethylation in HaCaT cells

The increasing amount of nanotechnological products, found in our environment and those applicable in engineering, material sciences and medicine has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic response to nm-SiO(2) particles in human HaCaT cells and methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) induced by nano-SiO(2) particles. Nm-SiO(2) treatment induced global hypoacetylation implying a global epigenomic response. The levels of DNMT1, DNMT3a and methyl-CpG binding protein 2 (MBD2) were also decreased in a dose dependent manner at mRNA and protein level. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.

Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G0–G1 phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST–PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.

Comparison of global DNA methylation profiles in replicative versus premature senescence

DNA methylation is considered to play an essential role in cellular senescence. To uncover the mechanism underlying cellular senescence, we established the model of premature senescence induced by hydrogen peroxide (H(2)O(2)) in human embryonic lung fibroblasts and investigated the changes of genome methylation, DNA methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) in comparison with those observed during normal replicative senescence. We found that premature senescence triggered by H(2)O(2) exhibited distinct morphological characteristics and proliferative capacity which were similar to those of replicative senescence. The genome methylation level decreased gradually during the premature as well as replicative senescence, which was associated with the reduction in the expression of DNMT1, reflecting global hypomethylation as a distinct feature of senescent cells. The levels of DNMT3b and methyl-CpG binding protein 2 (MeCP2) increased in both mid-aged and replicative senescent cells, while DNMT3a and MBD2 were upregulated in the mid-aged cells. Only DNMT3b was elevated in the cells in the premature senescence persistence status. Additionally, the expression for DNMTs, MBD2 and MeCP2 was increased rapidly upon H(2)O(2) treatment. These results indicate that H(2)O(2)-induced premature senescence share some features of replicative senescence, such as basic biological characteristics and global hypomethylation while there are slight differences in the profile of methylation-associated enzyme expression. Oxidative damage may hence be a causative factor in epigenetic alteration partly responsible for cellular senescence.

Effect of low dose bisphenol A on the early differentiation of human embryonic stem cells into mammary epithelial cells

It has been previously reported that bisphenol A (BPA) can disturb the development of mammary structure and increase the risk of breast cancer in experimental animals. In this study, an in vitro model of human embryonic stem cell (hESC) differentiation into mammary epithelial cells was applied to investigate the effect of low dose BPA on the early stages of mammogenesis. A newly established hESC line was directionally differentiated into mammary epithelial cells by a well-established three-dimensional (3D) culture system. The differentiated mammary epithelial cells were characterized by immunofluorescence and western blotting assay, and were called induced differentiated mammary epithelial cells (iDMECs) based on these data. The hESCs were treated with low doses of BPA range 10(-9)-10(-6)M during the differentiation process, with DMSO as the solvent control and 17-β-estrodiol (E2) as the estrogen-positive control. Our results showed that low dose BPA and E2 could influence the mammosphere area of iDMECs and upregulate the expression level of Oct4 and Nanog proteins, while only BPA could downregulate the expression of E-cadherin protein. Taken together, this study provides some insights into the effects of low dose BPA on the early differentiation stage of mammary epithelial cells and suggests an easier canceration status of iDMECs under the effect of low dose BPA during its early differentiation stage.

α4 is highly expressed in carcinogen-transformed human cells and primary human cancers.

A regulator of the protein phosphatase 2A (PP2A), α4, has been implicated in a variety of functions that regulate many cellular processes. To explore the role of α4 in human cell transformation and tumorigenesis, we show that α4 is highly expressed in human cells transformed by chemical carcinogens including benzo(a)pyrene, aflatoxin B1, N-methyl-N′-nitro-N-nitrosoguanidine, nickel sulfate and in several hepatic and lung cancer cell lines. In addition, overexpression of α4 was detected in 87.5% (74/80) of primary hepatocellular carcinomas, 84.0% (21/25) of primary lung cancers and 81.8% (9/11) of primary breast cancers, indicating that α4 is ubiquitously highly expressed in human cancer. Functional studies revealed that elevated α4 expression results in an increase in cell proliferation, promotion of cell survival and decreased PP2A-attributable activity. Importantly, ectopic expression of α4 permits non-transformed human embryonic kidney cells (HEKTER) and L02R cells to form tumors in immunodeficient mice. Furthermore, we show that the highly expressed α4 in transformed cells or human tumors is not regulated by DNA hypomethylation. A microRNA, miR-34b, that suppresses the expression of α4 through specific binding to the 3′-untranslated region of α4 is downregulated in transformed or human lung tumors. Taken together, these observations identify that α4 possesses an oncogenic function. Reduction of PP2A activity due to an enhanced α4–PP2A interaction contributes directly to chemical carcinogen-induced tumorigenesis.

Development of human cell models for assessing the carcinogenic potential of chemicals.

To develop human cell models for assessing the carcinogenic potential of chemicals, we established transgenic human cell lines and tested the sensitivity of known carcinogens using a cell transformation assay. A retroviral vector encoding an oncogenic allele of H-Ras (HBER) or c-Myc (HBEM) was introduced into human bronchial epithelial cells (HBE) immortalized by SV40 large T (LT) antigen, leading to increased cell proliferation but failing to confer a transformed phenotype characterized by anchorage-independent cell growth and tumor formation of immunodeficient mice. When these pre-transformed cells were treated with nickel sulfate (NiSO4), we found that it shortened the latency of malignant transformation at least by 19 wk in HBER cells or 16 wk in HBEM cells compared to vector control cells. Similarly, the latency of cell transformation was shorter by 15 wk in HBER cells or 9 wk in HBEM cells when cells were treated with benzo(a)pyrenediol epoxide (BPDE). HBER cells appeared to be more sensitive to TPA, NiSO4 or BPDE-induced cell transformation compared to human embryonic kidney cells expressing H-Ras (HEKR), implying that cell-type specificity is one of important factors determining the effectiveness of the assay. Using AFB1 and BaP as the representative pro-carcinogens, we also compared the efficiency of three different metabolic conditions in mediating cell transformation. Low dose chemical induction seems to be a prospective system used for metabolic activation of pro-carcinogens. Our findings provided direct evidence that a genetically modified human cell transformation model can be applied to the assessment of potent carcinogens.

Uptake of silica nanoparticles: Neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 μg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular β-amyloid 1-42 (Aβ(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimers disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-β-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3β at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3β. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

CpG Site–Specific Hypermethylation of p16INK4α in Peripheral Blood Lymphocytes of PAH-Exposed Workers

Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAH-associated biologic consequence. Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBL) from PAH-exposed workers and in benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells. The relationships between p16INK4α methylation and the level of urinary 1-hydroxypyrene (1-OHP) or the frequency of cytokinesis block micronucleus (CBMN) were analyzed. Results: Compared with the control group, PAH-exposed workers exhibited higher levels of urinary 1-OHP (10.62 vs. 2.52 μg/L), p16INK4α methylation (7.95% vs. 1.14% for 22 “hot” CpG sites), and CBMN (7.28% vs. 2.92%) in PBLs. p16INK4α hypermethylation in PAH-exposed workers exhibited CpG site specificity. Among the 35 CpG sites we analyzed, 22 were significantly hypermethylated. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-OHP and CBMN in PBLs. Moreover, the hypermethylation and suppression of p16 expression was also found in BaP-transformed HBER cells. Conclusion: PAH exposure induced CpG site–specific hypermethylation of p16INK4α gene. The degree of p16INK4α methylation was associated with the levels of DNA damage and internal exposure. Impact: p16INK4α hypermethylation might be an essential biomarker for the exposure to PAHs and for early diagnosis of cancer. Cancer Epidemiol Biomarkers Prev; 21(1); 182–90. ©2011 AACR.