Huei-Wen Chen

MicroRNA Signature Predicts Survival and Relapse in Lung Cancer

We investigated whether microRNA expression profiles can predict clinical outcome of NSCLC patients. Using real-time RT-PCR, we obtained microRNA expressions in 112 NSCLC patients, which were divided into the training and testing sets. Using Cox regression and risk-score analysis, we identified a five-microRNA signature for the prediction of treatment outcome of NSCLC in the training set. This microRNA signature was validated by the testing set and an independent cohort. Patients with high-risk scores in their microRNA signatures had poor overall and disease-free survivals compared to the low-risk-score patients. This microRNA signature is an independent predictor of the cancer relapse and survival of NSCLC patients.

Titanium dioxide nanoparticles induce emphysema-like lung injury in mice

Titanium dioxide nanoparticles (nanoTiO2) have been widely used as a photocatalyst in air and water cleaning. However, these nanoparticles inhalation can induce pulmonary toxicity and its mechanism is not fully understood. In this study we investigated the pulmonary toxicity of nanoTiO2 and its molecular pathogenesis. The adult male ICR mice were exposed to intratracheal single dose of 0.1 or 0.5 mg nanoTiO2 (19–21 nm) and lung tissues were collected at 3rd day, 1st wk, and 2nd wk for morphometric, microarray gene expression, and pathway analyses. NanoTiO2 can induce pulmonary emphysema, macrophages accumulation, extensive disruption of alveolar septa, type II pneumocyte hyperplasia, and epithelial cell apoptosis. NanoTiO2 induced differential expression of hundreds of genes include activation of pathways involved in cell cycle, apoptosis, chemokines, and complement cascades. In particular, nanoTiO2 up‐regulates placenta growth factor (PlGF) and other chemokines (CXCL1, CXCL5, and CCL3) expressions that may cause pulmonary emphysema and alveolar epithelial cell apoptosis. Cultured human THP‐1 cell‐derived macrophages treated with nanoTiO2 in vitro also resulted in up‐regulations of PlGF, CXCL1, CXCL5, and CCL3. These results indicated that nanoTiO2 can induce severe pulmonary emphysema, which may be caused by activation of PlGF and related inflammatory pathways.—Chen, H‐W., Su, S‐F., Chien, C‐T., Lin, W‐H., Yu, S‐L., Chou, C‐C., Chen, J. J. W., Yang, P. C. Titanium dioxide nanoparticles induce emphysema‐like lung injury in mice. FASEB J. 20, E1732–E1741 (2006)

Single-walled carbon nanotubes can induce pulmonary injury in mouse model.

Carbon nanotubes are a nanomaterial that is extensively used in industry. The potential health risk of chronic carbon nanotubes exposure has been raised as of great public concern. In the present study, we have demonstrated that intratracheal instillation of 0.5 mg of single-walled carbon nanotubes (SWCNT) into male ICR mice (8 weeks old) induced alveolar macrophage activation, various chronic inflammatory responses, and severe pulmonary granuloma formation. We then used Affymetrix microarrays to investigate the molecular effects on the macrophages when exposed to SWCNT. A biological pathway analysis, a literature survey, and experimental validation suggest that the uptake of SWCNT into the macrophages is able to activate various transcription factors such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), and this leads to oxidative stress, the release of proinflammatory cytokines, the recruitment of leukocytes, the induction of protective and antiapoptotic gene expression, and the activation of T cells. The resulting innate and adaptive immune responses may explain the chronic pulmonary inflammation and granuloma formation in vivo caused by SWCNT.

Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells

1 The possible mechanisms of the antiproliferative and apoptotic effects of curcumin (diferuloylmethane), a polyphenol in the spice turmeric, on vascular smooth muscle cells were studied in rat aortic smooth muscle cell line (A7r5). 2 The proliferative response was determined from the uptake of [3H]‐thymidine. Curcumin (10−6–10−4 m) inhibited serum‐stimulated [3H]‐thymidine incorporation of both A7r5 cells and rabbit cultured vascular smooth muscle cells in a concentration‐dependent manner. Cell viability, as determined by the trypan blue dye exclusion method, was unaffected by curcumin at the concentration range 10−6 to 10−5 m in A7r5 cells. However, the number of viable cells after 10−4 m curcumin treatment was less than the basal value (2×105 cells). 3 To analyse the various stages of the cell cycle, [3H]‐thymidine incorporation into DNA was determined every 3 h. After stimulation with foetal calf serum, quiescent A7r5 cells started DNA synthesis in 9 to 12 h (G1/S phase), then reached a maximum at 15 to 18 h (S phase). Curcumin (10−6–10−4 m) added during either the G1/S phase or S phase significantly inhibited [3H]‐thymidine incorporation. 4 Following curcumin (10−6–10−4 m) treatment, cell cycle analysis utilizing flow cytometry of propidium iodide stained cells revealed a G0/G1 arrest and a reduction in the percentage of cells in S phase. Curcumin at 10−4 m also induced cell apoptosis. It is suggested that curcumin arrested cell proliferation and induced cell apoptosis, and hence reduced the [3H]‐thymidine incorporation. 5 The apoptotic effect of 10−4 m curcumin was also demonstrated by haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling (TUNEL), and DNA laddering. Curcumin (10−4 m) induced cell shrinkage, chromatin condensation, and DNA fragmentation. 6 The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by curcumin at the concentration range 10−5 to 10−4 m. On the other hand, the cytosolic protein kinase C activity stimulated by phorbol ester was reduced by 10−4 m curcumin, but unaffected by lower concentrations (10−6–10−5 m). 7 The levels of c‐myc, p53 and bcl‐2 mRNA were analysed using a reverse transcription‐polymerase chain reaction (RT‐PCR) technique. The level of c‐myc mRNA was significantly reduced by curcumin (10−5–10−4 m) treatment. And, the level of bcl‐2 mRNA was significantly reduced by 10−4 m curcumin. However, the alteration of the p53 mRNA level by curcumin (10−5–10−4 m) treatment did not achieve significance. The effects of curcumin on the levels of c‐myc and bcl‐2 mRNA were then confirmed by Northern blotting. 8 Our results demonstrate that curcumin inhibited cell proliferation, arrested the cell cycle progression and induced cell apoptosis in vascular smooth muscle cells. Curcumin may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post‐angioplasty restenosis. Our results suggest that the antiproliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c‐myc mRNA expression. And, the apoptotic effect may partly be mediated through inhibition of protein tyrosine kinase activity, protein kinase C activity, c‐myc mRNA expression and bcl‐2 mRNA expression.

Curcumin inhibits lung cancer cell invasion and metastasis through the tumor suppressor HLJ1.

Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. In this study, we found that curcumin can inhibit cancer cell invasion and metastasis through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1). Human lung adenocarcinoma cells (CL1-5) treated with curcumin (1-20 mumol/L) showed a concentration-dependent reduction in cell migration, invasion, and metastatic ability, and this was associated with increased HLJ1 expression. Knockdown of HLJ1 expression by siRNA was able to reverse the curcumin-induced anti-invasive and antimetastasis effects in vitro and in vivo. The HLJ1 promoter and enhancer in a luciferase reporter assay revealed that curcumin transcriptionally up-regulates HLJ1 expression through an activator protein (AP-1) site within the HLJ1 enhancer. JunD, one of the AP-1 components, was significantly up-regulated by curcumin (1-20 mumol/L) in a concentration- and time-dependent manner. Knockdown of JunD expression could partially reduce the curcumin-induced HLJ1 activation and diminish the anti-invasive effect of curcumin, indicating that JunD would seem to be involved in curcumin-induced HLJ1 expression. Curcumin was able to induce c-Jun NH(2)-kinase (JNK) phosphorylation, whereas the JNK inhibitor (SP-600125) could attenuate curcumin-induced JunD and HLJ1 expression. Activation of HLJ1 by curcumin further leads to up-regulation of E-cadherin and a suppression of cancer cell invasion. Our results show that curcumin induces HLJ1, through activation of the JNK/JunD pathway, and inhibits lung cancer cell invasion and metastasis by modulating E-cadherin expression. This is a novel mechanism and supports the application of curcumin in anti-cancer metastasis therapy.

Cancer-associated fibroblasts regulate the plasticity of lung cancer stemness via paracrine signalling

Cancer stem cells (CSCs) are a promising target for treating cancer, yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro. Here we establish a sustainable primary culture of Oct3/4(+)/Nanog(+) lung CSCs fed with CD90(+) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically, we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover, this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness, and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC.

Cyclosporine A regulate oxidative stress‐induced apoptosis in cardiomyocytes: mechanisms via ROS generation, iNOS and Hsp70

Previous study suggested that cyclosporine A (CsA) could partially reduce ischaemia/reperfusion‐induced injury in isolated heart, but the mechanism was still unclear. In this study, the possible mechanisms of cyclosporine A in regulating oxidative stress‐induced cardiomyocyte apoptosis were examined. Morphological (cell shrinkage, apoptotic body formation, and DNA fragmentation) and biochemical (annexin‐V staining for exposed phosphatidylserine residues) evidences showed that both hydrogen peroxide (H2O2) and hypoxia/reoxygenation could induce apoptotic change in the embryonal rat heart myoblast‐derived cells (H9c2). These effects were inhibited by pre‐treatment with CsA at concentration of 0.01–1.0 μM for 24 h, but were increased with 10.0 μM CsA. While examining the mechanisms of CsA in protecting cardiomyocyte apoptosis, we found that the collapse of mitochondria membrane potential (ΔΨm) induced by oxidative stress was partially reversed by CsA (0.01–1.0 μM). Compared to the control, CSA at the concentration of 0.1 and 10.0 μM significantly increased the level of intracellular reactive oxygen species (ROS) to 117.2±12.4% and 234.4±9.3%, respectively. Co‐incubating with the antioxidant, ascorbic acid (10.0 μM), could partially reduce the protective effect of CsA (0.01–1.0 μM) and the toxic effect of 10.0 μM CsA. Pre‐treatment with CsA at concentration of 0.01–1.0 μM for 24 h produced up‐regulation of heat shock protein 70 (Hsp 70), inducible nitric oxide synthase (iNOS) and also induced NO production, indicating that these factors might be associated with the cell protective effects of CsA. These results suggest that CsA could protect the oxidative stress‐induced cardiomyocyte apoptosis not only by preventing the loss of ΔΨm in mitochondria, but also through ROS generation, Hsp70, and iNOS up‐regulation.

Anticancer effects of tanshinone I in human non-small cell lung cancer

Tanshinones are the major bioactive compounds of Salvia miltiorrhiza Bunge (Danshen) roots, which are used in many therapeutic remedies in Chinese traditional medicine. We investigated the anticancer effects of tanshinones on the highly invasive human lung adenocarcinoma cell line, CL1-5. Tanshinone I significantly inhibited migration, invasion, and gelatinase activity in macrophage-conditioned medium-stimulated CL1-5 cells in vitro and also reduced the tumorigenesis and metastasis in CL1-5-bearing severe combined immunodeficient mice. Unlike tanshinone IIA, which induces cell apoptosis, tanshinone I did not have direct cytotoxicity. Real-time quantitative PCR, luciferase reporter assay, and electrophoretic mobility shift assay revealed that tanshinone I reduces the transcriptional activity of interleukin-8, the angiogenic factor involved in cancer metastasis, by attenuating the DNA-binding activity of activator protein-1 and nuclear factor-κB in conditioned medium-stimulated CL1-5 cells. Microarray and pathway analysis of tumor-related genes identified the differentially expressed genes responding to tanshinone I, which may be associated with the Ras-mitogen-activated protein kinase and Rac1 signaling pathways. These results suggest that tanshinone I exhibits anticancer effects both in vitro and in vivo and that these effects are mediated at least partly through the interleukin-8, Ras-mitogen-activated protein kinase, and Rac1 signaling pathways. Although tanshinone I has a remarkable anticancer action, its potential anticoagulant effect should be noted and evaluated. [Mol Cancer Ther 2008;7(11):3527–38]

Nitric oxide as a regulator in preimplantation embryo development and apoptosis

OBJECTIVE To investigate the mechanisms of nitric oxide (NO) in the development and apoptosis of preimplantation mouse embryos. DESIGN Prospective, controlled study. SETTING Medical college laboratory. SUBJECT(S) Two-cell embryos from outbred ICR mice. INTERVENTION(S) Hyperstimulation protocol, two-cell embryos were collected, then treated with or without an NO synthase inhibitor (L-NAME) or an NO donor (SNP) and combined with a cGMP analogue (8-Br-cGMP) or a selective inhibitor of NO-sensitive soluble guanylyl cyclase (ODQ). MAIN OUTCOME MEASURE(S) The development of ICR mouse embryo from two cells to blastocyst stages in vitro. RESULT(S) The development of blastocyst was inhibited by L-NAME in a concentration-dependent manner (0.1-10 microM) and 0.1 microM SNP reversed this effect (80.5% of control). Annexin-V/propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling techniques demonstrated that excessive NO (> or =10 microM) might induce apoptosis in the mouse embryos. 8-Br-cGMP reversed the inhibitory effect of L-NAME and rescued the embryo growth. ODQ inhibited the embryo development in a dose-responsive fashion (0.1--100 microM) but had no effect in the NO-induced embryo apoptosis. P53 and Bax were found to be up-regulated during the embryo fragmentation. CONCLUSION(S) These results indicate that the cGMP pathway might be involved in the NO-regulated embryonic development, but not in NO-induced apoptosis, for which P53/Bax pathway might be involved.

The possible mechanisms of the antiproliferative effect of fullerenol, polyhydroxylated C60, on vascular smooth muscle cells

1 The possible mechanisms of the antiproliferative effect of polyhydroxylated fullerene (fullerenol), a novel free radical trapper, were studied in rat vascular smooth muscle cells (A7r5 cells) and compared with the effect of ascorbic acid. 2 Fullerenol‐1 and ascorbic acid inhibited the proliferative responses in a number of cells, including rat aortic smooth muscle cells (A7r5 cells), human coronary artery smooth muscle cells, and human CEM lymphocytes (CEM cells) in a concentration dependent manner. 3 At the concentration range of 10−6 to 10−2 M, fullerenol‐1 and ascorbic acid concentration‐dependently inhibited the proliferative responses stimulated by serum in A7r5 cells. Fullerenol‐1 was more potent than ascorbic acid. 4 The production of O2− induced by alloxan, a diabetogenic compound, was reduced by fullerenol‐1 (10−4 M) in the presence of A7r5 cells. 5 The cytosolic protein kinase C activity of A7r5 cells stimulated by phorbol ester was reduced by 10−3 M fullerenol‐1, but not ascorbic acid (10−4–10−2 M) and fullerenol‐1 at lower concentrations (10−6–10−4 M). 6 In contrast, the membraneous protein tyrosine kinase activity of A7r5 cells stimulated by foetal calf serum was significantly reduced by fullerenol‐1 (10−6–10−3 M) and ascorbic acid (10−4–10−2 M). Again, the inhibitory activity of fullerenol‐1 was greater than that of ascorbic acid. 7 Our results demonstrate that fullerenol‐1 and ascorbic acid exhibit inhibitory effects on transduction signals in addition to their antioxidative property. It is suggested that the antiproliferative effect of fullerenol‐1 on vascular smooth muscle cells may partly be mediated through the inhibition of protein tyrosine kinase.