Human Embryo Lung Fibroblast Cells Research Articles

High-content analysis of particulate matters-induced oxidative stress and organelle dysfunction in vitro

Oxidative stress is usually considered to be a common mechanism by which particulate matter (PM) exposure induces adverse effects. However, the further biological events such as organelle dysfunction following oxidative stress remain to be explored. In this study, we applied high-content screening (HCS) technique to investigate the toxicological effects of carbon black (CB), diesel exhaust particle (DEP) and PM2.5 on oxidative stress and organelle function in human bronchial epithelial cell (16HBE), human embryo lung fibroblast cell (HELF) and human umbilical vein endothelial cell (HUVEC) which were used to represent distinct regions of the lung, and compared the toxicity impacts of different PMs and the sensitiveness of cell lines. We found three types of PMs induced mitochondrial dysfunction in three cell lines and lysosomal alkalinization in HUVEC while only CB triggered endoplasmic reticulum (ER) stress in 16HBE and HUVEC, and oxidative stress might mediate these processes. Moreover, CB basically exhibited more potent toxicity compared with DEP and PM2.5, which might be attributed to its less oxygen content. Finally, the finding that PMs-induced toxicity impacts exhibited a cell-type dependent manner might provide some information to help to understand the sensitivity of different tissue in the lung.

Screening and identification of mRNAs targeted by human cytomegalovirus (HCMV)-encoded miR-US33-1-5p in HCMV-infected human embryo lung fibroblast cells

Objective To screen and identify mRNAs targeted by human cytomegalovirus-encoded miR-US33-1-5p (HCMV miR-US33-1-5p) for understanding the biological functions of HCMV miR-US33-1-5p. Methods Potential target mRNAs of HCMV miR-US33-1-5p were screened out by using hybrid-PCR, a simple and effective method. Dual-Luciferase Reporter Assay System was used to identify the binding abilities of HCMV miR-US33-1-5p to 3′-UTRs of these potential target mRNAs. Results Twelve potential mRNAs targeted by HCMV miR-US33-1-5p were screened out in human embryo lung fibroblast cells infected with HCMV. Luciferase activities of 3′-UTRs of seven target mRNAs were significantly down-regulated. Conclusion Proteins encoded by these target mRNAs are involved in virus replication, immune system regulation, protein synthesis, cell cycle regulation, energy metabolism and so on. Identification of mRNAs targeted by HCMV miR-US33-1-5p is helpful for further studies on biological functions of miR-US33-1-5p. Key words: Human cytomegalovirus; miRNA; Target mRNA

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately.

Subcellular quantitative proteomic analysis reveals host proteins involved in human cytomegalovirus infection

Viral replication requires host cell macromolecules and energy, although host cells can alter their protein expression to restrict viral replication. To study the host cell response to human cytomegalovirus (HCMV) infection, a stable isotope labeling by amino acids in cell culture (SILAC)-based subcellular quantitative proteomic study of HCMV-infected human embryo lung fibroblast (HEL) cells was performed, and a total of 247 host proteins were identified as differentially regulated by HCMV. Western blotting and immunofluorescence confocal microscopy were performed to validate the data sets. Gene Ontology analysis indicated that cellular processes involving the metabolism, localization and immune system were regulated as a result of HCMV infection. Functional analysis of selected regulated proteins revealed that knockdown of HNRPD, PHB2 and UB2V2 can increase HCMV replication, while knockdown of A4 and KSRP resulted in decreased HCMV replication. Our study may improve our understanding of the dynamic interactions between HCMV and its host and provide multiple potential targets for anti-HCMV agent research.

A novel human interleukin-24 peptide created by computer-guided design contributes to suppression of proliferation in esophageal squamous cell carcinoma Eca-109 cells.

Based on the three-dimensional modeling structure of human interleukin-24 (hIL-24) and its most likely active position predicted by solvent accessibility and apparent electrostatic properties, a novel hIL-24 peptide M1 was created by computer-guided molecular design. The cytotoxicity and cell selectivity of M1 were examined in three human carcinoma cell lines and one normal human embryo lung fibroblast cell line (HEL). MTT assay showed that M1 induced growth arrest in two IL-20 receptor complex-positive cancer cell lines (the esophageal squamous cell carcinoma cell line Eca-109 and the melanoma cell line A375), and antibodies against IL-24 or IL-20 receptor complexes significantly neutralized the inhibitory activity. Moreover, M1 had almost no cytotoxicity on the lung cancer A549 cell line, which lacks a full complement of the IL-20 receptor complexes, or on HEL cells that express the IL-20 receptor complexes. These findings demonstrate that M1 could act as an excellent candidate for the induction of growth arrest on receptor complex-positive cancer cells. In summary, the M1 peptide may represent a novel anticancer agent for esophageal squamous cell carcinoma therapy due to its cancer cell selectivity and its relatively low cytotoxicity to normal cells.

S100A9 promotes human lung fibroblast cells activation through receptor for advanced glycation end-product-mediated extracellular-regulated kinase 1/2, mitogen-activated protein-kinase and nuclear factor-κB-dependent pathways

S100A9 belongs to the S100 family of calcium-binding proteins and plays a key role in many inflammatory conditions. Recent studies have found that S100A9 was elevated significantly in the bronchoalveolar lavage fluid of idiopathic pulmonary fibrosis patients, and might be a biomarker for fibrotic interstitial lung diseases. However, the exact function of S100A9 in pulmonary fibrosis needs further studies. We performed this study to investigate the effect of S100A9 on human embryo lung fibroblast (HLF) proliferation and production of cytokines and collagen, providing new insights into the possible mechanism. S100A9 promoted proliferation of fibroblasts and up-regulated expression of both proinflammatory cytokines interleukin (IL)-6, IL-8, IL-1β and collagen type III. S100A9 also induced HLF cells to produce α-smooth muscle actin (α-SMA) and receptor for advanced glycation end-product (RAGE). In addition, S100A9 caused a significant increase in extracellular-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) phosphorylation, while the status of p38 and c-Jun N-terminal kinase (JNK) phosphorylation remained unchanged. Treatment of cells with S100A9 also enhanced nuclear factor kappa B (NF-κB) activation. RAGE blocking antibody pretreatment inhibited the S100A9-induced cell proliferation, cytokine production and pathway phosphorylation. S100A9-mediated cell activation was suppressed significantly by ERK1/2 MAPK inhibitor and NF-κB inhibitor. In conclusion, S100A9 promoted HLF cell growth and induced cells to secret proinflammatory cytokines and collagen through RAGE signalling and activation of ERK1/2 MAPK and NF-κB pathways.

Preferential expansion of umbilical cord blood‐derived CD34‐positive cells on human leukemia inhibitory factor transgenic feeder cells cultured on regenerated silk fibroin film

In vitro expansion of transplantable hematopoietic stem cells (HSCs) is a very promising approach for different clinical applications. We have recently developed a new culture system that facilitates in vitro expansion of transplantable cord blood HSCs. In our study, we constructed a recombinant adenovirus Ad-GFP/human leukemia inhibitory factor (hLIF) expressing hLIF. The hLIF gene was delivered into human embryo lung fibroblast cell line WI-38 via infection with Ad-GFP/hLIF. Then, the transgenic cells were cultured on regenerated silk fibroin (SF) films as feeder layer cells for expansion of cord blood CD34(+) cells. Our results showed that the hLIF transgenic WI-38 cells cultured on SF could express hematopoiesis-related cytokines at higher levels compared with control groups. The hLIF-expressing feeder layer cells cultured on SF in combination with cytokines more efficiently expanded CD34(+) cells and CD34(+) CD38(-) cells. The percentages of adhesion molecules on the expanded CD34(+) cells in transgenic feeder layer cells cultured on SF were higher than those of control groups. Interestingly, the migration rate assessed by transwell assay was also significantly higher than those of control groups, which suggests that transgenic feeder layer cells cultured on SF has powerful ability to maintain the homing capacity of expanded CD34(+) cells.

Feedback Regulations of miR-21 and MAPKs via Pdcd4 and Spry1 Are Involved in Arsenite-Induced Cell Malignant Transformation

ObjectiveTo establish the functions of miR-21 and the roles of two feedback regulation loops, miR-21-Spry1-ERK/NF-κB and miR-21-Pdcd4-JNK/c-Jun, in arsenite-transformed human embryo lung fibroblast (HELF) cells.MethodsFor arsenite-transformed HELF cells, apoptosis, clonogenicity, and capacity for migration were determined by Hoechst staining, assessment of their capacity for anchorage-independent growth, and wound-healing, respectively, after blockage, with inhibitors or with siRNAs, of signal pathways for JNK/c-Jun or ERK/NF-κB. Decreases of miR-21 levels were determined with anti-miR-21, and the up-regulation of Pdcd4 and Spry1 was assessed in transfected cells; these cells were molecularly characterized by RT-PCR, qRT-PCR, Western blots, and immunofluorescence assays.ResultsMiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels. However, there were no significant changes in mRNA levels for Pdcd4 and Spry1, which suggested that miR-21 regulates the expressions of Pdcd4 and Spry1 through translational repression. In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1. Down-regulation of miR-21 and up-regulations of Pdcd44 or Spry1 blocked the arsenite-induced activations of JNK/c-Jun or ERK/NF-κB, indicating that knockdown of miR-21 inhibits feedback of ERK activation and JNK activation via increases of Pdcd4 and Spry1 protein levels, respectively. Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.ConclusionThe results indicate that miR-21 is both a target and a regulator of ERK/NF-κB and JNK/c-Jun and the feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1, respectively, are involved in arsenite-induced malignant transformation of HELF cells.

Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells

Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type.

Regulation of miRNA-21 by reactive oxygen species-activated ERK/NF-κB in arsenite-induced cell transformation

After acute exposure of cells to arsenic, reactive oxygen species mediate changes in cell behavior, including activation of proliferative signaling. For chronic exposure to arsenic, however, the function of reactive oxygen species in cell transformation remains poorly understood. Although microRNA-21 (miR-21) has been implicated in various aspects of carcinogenesis, its functions and molecular mechanisms in carcinogen-induced tumorigenesis are unclear. The purpose of this study was to determine if miR-21 is involved in arsenite-induced malignant transformation and to characterize the associated signaling pathways. During arsenite-induced transformation of human embryo lung fibroblast (HELF) cells, miR-21 was upregulated, and the extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) signal pathway was activated. Moreover, superoxide radical dismutase (a scavenger of superoxide) and catalase (a scavenger of hydroperoxides) blocked the arsenite-induced effects in HELF cells and mouse embryonic fibroblasts. Blockage of ERK by the inhibitor U0126 or inhibition of NF-κB p65 by siRNA or Bay 11–7082 prevented the increases in miR-21 and the decreases in Spry1, Pten, and Pdcd4, the target proteins of miR-21, induced by arsenite. As determined by a ChIP-qPCR assay, NF-κB p65 regulated miR-21 expression by binding directly to the promoter of miR-21. Further, anti-miR-21 downregulated miR-21 expression and prevented the arsenite-induced activation of ERK via the increase in Spry1, indicating that miR-21 has a feedback effect in regulating ERK activation. Overexpression of miR-21 with an miR-21 mimic and feedback activation of ERK and NF-κB via the decrease in Spry1 promoted the malignancy of HELF cells exposed to arsenite, but knockdown of miR-21 with anti-miR-21 and feedback blockage of ERK and NF-κB activation through an increase in Spry1 decreased anchorage-independent growth of arsenite-transformed cells. Thus, the transformation of HELF cells induced by chronic exposure to arsenite is mediated by increased miR-21 expression, which, in turn, is mediated by reactive oxygen species activation of the ERK/NF-κB pathway.

DNA-PKcs-mediated stabilization of p53 by JNK2 is involved in arsenite-induced DNA damage and apoptosis in human embryo lung fibroblast cells

When cells encounter genotoxic stress, sensors for DNA lesions stabilize and activate p53; the signals involved, however, are largely unclear. Inorganic arsenite is a ubiquitous environmental contaminant associated with an increased risk of lung and skin damage and cancer. Although DNA double-strand breaks and apoptosis may relate to arsenite-induced damage and carcinogenesis, the mechanism of action remains obscure. Here, we find that, in human embryo lung fibroblast (HELF) cells, arsenite induces the activation of dependent protein kinase catalytic subunit (DNA-PKcs), which then phosphorylates and activates c-Jun N-terminal kinases 2 (JNK2), but not JNK1. As a positive regulator of p53, JNK2 binds to p53 and prevents p53 from murine double minute 2 (mdm2)-mediated, ubiquitin-proteasome-dependent degradation. Knockdown of DNA-PKcs/JNK2 signal pathway or p53 reduces apoptosis but elevates the DNA damage induced by a high level of arsenite. These results suggest that DNA-PKcs-mediated stabilization of p53 by JNK2 is involved in arsenite-induced DNA damage and apoptosis.

Roles of the ERK, JNK/AP‐1/cyclin D1–CDK4 pathway in silica‐induced cell cycle changes in human embryo lung fibroblast cells

Silica is a potent occupational fibrogenic agent capable of inducing lung fibrosis and many other lung diseases. Our current study focused on the signalling pathways regulating cell cycle changes in HELF (human embryo lung fibroblast) after silica (α-quartz) exposure. Our results showed silica exposure could lead to cell cycle changes. The cell cycle alternations were accompanied with overexpression of cyclin D1 and CDK4 (cyclin-dependent kinase 4) in a time-dependent manner. Silica exposure also decreased E2F-4 expression in HELF. These changes were blocked by overexpression of dominant-negative mutants of ERK (extracellular signal-regulated protein kinase) or the JNK (stress-activated c-Jun NH2-terminal kinase), respectively. Moreover, pretreatment of cells with curcumin, an activation of AP-1 (activator protein-1) inhibitor, inhibited silica-induced cell cycle alteration, the decreased expression of E2F-4 and overexpression of cyclin D1 and CDK4. Furthermore, both antisense cyclin D1 and antisense CDK4 can block silica-induced cell cycle changes. These results suggest that silica exposure can induce cell cycle changes, which may be mediated through ERK, JNK/AP-1/cyclin D1-CDK4-dependent pathway.

Up-regulation of cyclin D1 by JNK1/c-Jun is involved in tumorigenesis of human embryo lung fibroblast cells induced by a low concentration of arsenite

Inorganic arsenic, a ubiquitous environmental contaminant, is associated with an increased risk of cancer. There are several hypotheses regarding arsenic-induced carcinogenesis. The mechanism of action remains obscure, although hyper-proliferation of cells is involved. In the present study, the molecular mechanisms underlying the proliferation and malignant transformation of human embryo lung fibroblast (HELF) cells induced by a low concentration of arsenite were investigated. The results reveal that a low concentration of arsenite induces cell proliferation and promotes cell cycle transition from the G1 to the S phase. Moreover, arsenite activates the JNK1/c-Jun signal pathway, but not JNK2, which up-regulates the expression of cyclin D1/CDK4 and phosphorylates the retinoblastoma (Rb) protein. Blocking of the JNK1/c-Jun signal pathway suppresses the increases of cyclin D1 expression and Rb phosphorylation, which attenuates cell proliferation, reduces the transition from the G1 to the S phase, and thereby inhibits the neoplastic transformation of HELF cells induced by a low concentration of arsenite. Thus, activation of the JNK1/c-Jun pathway up-regulates the expression of cyclin D1, which is involved in the tumorigenesis caused by a low concentration of arsenite.

Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols

Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene.

Silica induces cell cycle changes through PI‐3K/AP‐1 pathway in human embryo lung fibroblast cells

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein-1 (AP-1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant-negative mutants of phosphatidylinositol-3 kinase (Δp85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica-induced cell cycle alteration, AP-1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol-3 kinase (PI-3K)/AP-1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI-3K, Akt, or p70S6K could inhibit silica-induced overexpression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) and decreased expression of E2F-4. Taken together, silica could induce cell cycle changes through PI-3K/ AP-1 pathway in HELFs.

Mot-2–Mediated Cross Talk between Nuclear Factor-κB and p53 Is Involved in Arsenite-Induced Tumorigenesis of Human Embryo Lung Fibroblast Cells

BackgroundInactivation of p53 is involved in arsenite-induced tumorigenesis; however, the molecular mechanisms remain poorly understood.ObjectiveWe investigated the molecular mechanisms underlying the inactivation of p53 and neoplastic transformation induced by arsenite in human embryo lung fibroblast (HELF) cells.MethodsAnchorage-independent growth assays were performed, and tumorigenicity in intact animals was assessed to confirm arsenite-induced neoplastic transformation. We determined the levels and functions of p53, nuclear factor-kappa B (NF-κB; a key transcriptional regulator), and mot-2 (a p53 inhibitor) and their relationships in arsenite-induced transformed HELF cells by two-dimensional electrophoresis, reverse-transcriptase polymerase chain reaction, Western blot, immunofluorescence, and co-immunoprecipitation assays.ResultsExposure of HELF cells to low levels of arsenite increased their proliferation rate and anchorage-independent growth and disrupted normal contact inhibition. When introduced into nude mice, transformed cells were tumorigenic. We used proteomic analysis to identify proteins with altered expression between untreated and arsenite-exposed cells. We found decreased expression of NF-κB repressing factor (NKRF; an inhibitor of NF-κB–mediated gene transcription), increased expression of mot-2, and increased activation of NF-κB. Changes in cells exposed to 1.0 μM arsenite were more marked than changes in cells exposed to 0.5 or 2.0 μM arsenite. Inactivation of NF-κB prevented malignant transformation induced by 1.0 μM arsenite. Moreover, we also identified a mechanism whereby NF-κB regulated p53. Specifically, activation of NF-κB up-regulated mot-2 expression, which prevented nuclear translocation of p53 and switched the binding preference of the p53 and NF-κB coactivator CBP [cyclic AMP-responsive element binding protein (CREB) binding protein] from p53 to NF-κB.Conclusionsmot-2–mediated cross talk between NF-κB and p53 appears to be involved in arsenite-induced tumorigenesis of HELF cells.

Interaction between malignant transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust

To study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust. (1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry. (1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding. (1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

Biphasic effect of cadmium on cell proliferation in human embryo lung fibroblast cells and its molecular mechanism

Hormesis, a biphasic dose–response phenomenon, is characterized by a low-dose stimulation and a high-dose inhibition. However, the mechanisms underlying hormesis induced by environmental agents are not well elucidated. The present study was to investigate the relationship between the hormesis effect of cadmium (Cd) and activation of ERK1/2, JNK and p38 pathways in human embryo lung fibroblast cells. Results showed that Cd induced significant cell proliferation at low concentrations, but markedly inhibited cell growth at high concentrations. Our data indicated that cell proliferation promoted by low concentrations of Cd was blocked obviously by ERK1/2 inhibitor PD98059 and partly by JNK inhibitor SP600125; while the decreases of cell proliferation induced by high concentrations of Cd were significantly restored by p38 inhibitor SB203580. Further analysis showed that phospho-ERK1/2 and phospho-JNK activities were increased with different concentrations of Cd, whereas phospho-p38 activity was markedly increased at high concentrations. Our findings suggested that low concentration of Cd induces the ERK and JNK pathways and promotes cell proliferation; while high concentration of Cd induces p38 pathway and inhibits cell proliferation. Activation of the ERK1/2 pathways seems to play a more important role than the JNK pathway in the biphasic effect of Cd on cell proliferation.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) as an injectable implant system for prevention of post-surgical tissue adhesion

An injectable implant system that immediately forms a film around the injection site of an animal was successfully developed by dissolving microbial polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in not harmful organic solvents including N-methyl pyrrolidone (NMP), dimethylacetamide (DMAC), 1,4-dioxane (DIOX), dimethyl sulfoxide (DMSO) and 1,4-butanolide (BL), respectively. The formation of the PHBHHx film was the result of contact between aqueous body fluids and the amphiphilic PHBHHx solvents, leading to the controllable precipitation (film formation) of PHBHHx around the contact site. The resultant PHBHHx film assumed the shapes of its surrounding cavities. The resulting porous PHBHHx film was not favorable for attachment of Human Embryo Lung Fibroblast (HELF) cells. As a consequence, the fibroblasts cultured on the PHBHHx film exhibited a spheroid-like morphology. It was found that hydrophilicity of the PHBHHx film prepared using the above technique was significantly reduced compared with the poly(lactic acid) (PLA) film prepared for the same purpose and a PHBHHx film prepared from chloroform casting. This reduced hydrophilicity explains the poor attachment of fibroblast cells to the injectable PHBHHx film, suggesting that the PHBHHx injectable implant system can be developed as a tissue adhesion prevention film for surgical operations.

Effects of long-term low-dose cadmium exposure on genomic DNA methylation in human embryo lung fibroblast cells

Cadmium is a toxic transition metal of continuing occupational and environmental concern. As a well-recognized human carcinogen, its carcinogenic mechanisms are still poorly understood. Cadmium has long been considered a non-genotoxic carcinogen and thought to act through epigenetic mechanisms. In the present study, we tested the effects of long-term low-dose cadmium exposure on DNA methylation in human embryo lung fibroblast (HLF) cells. After 2 months of exposure to 0–1.5 μmol/L cadmium, both the level of genomic DNA methylation and the enzyme activity of DNA methyltransferases (DNMTs) were increased in a concentration-related manner. Moreover, our results showed that cadmium exposure up-regulated the mRNA levels of DNMT1, DNMT3a and DNMT3b at higher concentrations. We further tested the growth dynamics of HLF cells, and observed significantly elevated growth rates, decreased cell population of G0/G1-phase and increased cell population of S-phase at 0.9, 1.2, and 1.5 μmol/L concentrations. Our study indicated that long-term low-dose cadmium exposure could disrupt DNA methylation, which may be one of the possible underlying carcinogenic mechanisms of cadmium.