Yat-Pang Chau

MicroRNA‐122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma

MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver‐specific microRNA‐122 (miR‐122) is significantly down‐regulated in liver cancers with intrahepatic metastastasis and negatively regulates tumorigenesis. Restoration of miR‐122 in metastatic Mahlavu and SK‐HEP‐1 cells significantly reduced in vitro migration, invasion, and anchorage‐independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR‐122 candidate target genes from two independent expression microarray datasets. Thirty‐two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell‐cell signaling, and transcription. We further showed that one of the miR‐122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR‐122. Conclusion: Our study suggests that miR‐122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR‐122 has a far‐reaching effect on the cell. Using the concomitant down‐regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR‐122 may prove to be beneficial for patients with hepatocellular carcinoma. (HEPATOLOGY 2009.)

Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma

A comprehensive microarray analysis of hepatocellular carcinoma (HCC) revealed distinct synexpression patterns during intrahepatic metastasis. Recent evidence has demonstrated that synexpression group member genes are likely to be regulated by master control gene(s). Here we investigate the functions and gene regulation of the transcription factor SOX4 in intrahepatic metastatic HCC. SOX4 is important in tumor metastasis as RNAi knockdown reduces tumor cell migration, invasion, in vivo tumorigenesis and metastasis. A multifaceted approach integrating gene profiling, binding site computation and empirical verification by chromatin immunoprecipitation and gene ablation refined the consensus SOX4 binding motif and identified 32 binding loci in 31 genes with high confidence. RNAi knockdown of two SOX4 target genes, neuropilin 1 and semaphorin 3C, drastically reduced cell migration activity in HCC cell lines suggesting that SOX4 exerts some of its action via regulation of these two downstream targets. The discovery of 31 previously unidentified targets expands our knowledge of how SOX4 modulates HCC progression and implies a range of novel SOX4 functions. This integrated approach sets a paradigm whereby a subset of member genes from a synexpression group can be regulated by one master control gene and this is exemplified by SOX4 and advanced HCC.

Involvement of Hydrogen Peroxide in Topoisomerase Inhibitor β-lapachone-Induced Apoptosis and Differentiation in Human Leukemia Cells

Beta-Lapachone a novel topoisomerase inhibitor, has been found to induce apoptosis in various human cancer cells. In this study we report that a dramatic elevation of hydrogen peroxide (H2O2) in human leukemia HL-60 cells following 1 microM beta-lapachone treatment and that this increase was effectively inhibited by treatment with antioxidant N-acetyl-L-cysteine (NAC), ascorbic acid, alpha-tocopherol. NAC strongly prevented beta-lapachone-induced apoptotic characteristics such as DNA fragmentation and apoptotic morphology. However, treatment of HL-60 cells with another topoisomerase inhibitor camptothecin (CPT) did not induce H2O2 production as compared to untreated cells. NAC also failed to block CPT-induced apoptosis. Correlated with these findings, we found that cancer cell lines K562, MCF-7, and SW620, contained high level of intracellular glutathione (GSH), were not elevated in H2O2 and were resistant to apoptosis after treatment with beta-lapachone. In contrast, cancer cell lines such as, HL-60, U937, and Molt-4 which have lower level of GSH, were readily increased of H2O2 and were sensitive to this drug. Furthermore, ectopic overexpression of Bcl-2 in HL-60 cells also attenuated beta-lapachone-induced H2O2 and conferred resistance to beta-lapachone-induced cell death. Beta-Lapachone at the concentration as low as 0.25 microM effectively induced HL-60 cells to undergo monocytic differentiation, as evidenced by CD14 antigenicity and alpha-naphthyl acetate esterase activity. Again, the beta-lapachone-induced monocytic differentiation was suppressed by NAC. These results suggest that intracellular H2O2 generation plays a crucial role in beta-lapachone-induced cell death and differentiation.

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

Abstract DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing α-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of β-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti‐angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. β‐Lapachone (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphtho‐[1,2‐b]pyran‐5,6‐dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti‐cancer and anti‐viral effects. Whether β‐lapachone can induce endothelial cell death or has an anti‐angiogenic effect is still an enigma. We investigated the in vitro effect of β‐lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during β‐lapachone‐induced endothelial cell death; (2) co‐treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD‐fmk, provided significant protection against apoptosis by preventing the β‐lapachone‐induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the β‐lapachone‐induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by β‐lapachone, but not the anti‐angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of β‐lapachone on human endothelial cells and suggest that β‐lapachone may have potential as an anti‐angiogenic drug. J. Cell. Physiol. 211: 522–532, 2007.

In vitro and in vivo wound healing-promoting activities of β-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.

Investigation of the Blood-Ganglion Barrier Properties in Rat Sympathetic Ganglia by Using Lanthanum Ion and Horseradish Peroxidase as Tracers

Vascular permeability in various rat sympathetic ganglia, including superior cervical ganglia, thoracic ganglia and the celiac-mesenteric ganglia (CMG) complex, was investigated by using lanthanum and horseradish peroxidase (HRP) as tracers with special attention to the neuronal and small granule-containing (SGC) cell area. After lanthanum perfusion, lanthanum tracer was present within the lumen of blood vessels. No lanthanum depositions were found in the extravascular space surrounding neurons in the superior cervical and thoracic ganglia. By contrast, an accumulation of lanthanum was observed in both luminal, abluminal and subendothelial surface of blood vessels in neuronal and SGC cell areas of the CMG complex and surrounding SGC cells in superior cervical ganglia. Injecting HRP revealed that all blood vessels of various sympathetic ganglia, either in neuronal or in SGC cell areas, were impermeable to HRP. HRP reaction product was limited to the vascular lumen and macrophages. The escape of HRP was obstructed by the junctional complex at intercellular clefts of endothelia and also by the diaphragms of the fenestrated capillaries associated with SGC cells. We conclude that there are different properties in the blood-ganglion barriers among rat sympathetic ganglia: (1) continuous capillaries in superior cervical ganglia and thoracic ganglia provide an efficient blood-ganglion barrier that prevents the penetration of tracers, and (2) capillaries in the CMG complex and in regions of the superior cervical ganglia that contain SGC cells possess a selective blood-ganglion barrier that discriminates between tracers based on their molecular sizes.

Circulating Wnt/β-catenin signalling inhibitors and uraemic vascular calcifications

BACKGROUND The process of vascular calcification has been associated with the canonical Wnt/β-catenin signalling pathway in cell cultures and animal studies. The relationship between circulating Wnt/β-catenin inhibitors and vascular calcification in dialysis patients is unknown. The aim of this study was to investigate the associations between serum dickkopf-1 (Dkk-1) and sclerostin, two circulating inhibitors of the Wnt/β-catenin signalling pathway, and the severity of aortic calcification (AoC) and cardiovascular outcomes in dialysis patients. METHODS This was a prospective observational cohort study. One hundred and twenty-five patients on maintenance haemodialysis participated in the study. Serum levels of Dkk-1 and sclerostin were measured. AoC scores were calculated from plain films of both posterior-anterior and lateral views. The patients were followed up for 2 years or until death or withdrawal. RESULTS The circulating sclerostin level was inversely associated with the severity of AoC (P = 0.035) and indicators of the bone turnover rate including serum alkaline phosphatase (ALP) (r = -0.235, P = 0.008) and intact parathyroid hormone (r = -0.523, P < 0.001). Furthermore, Cox regression analysis indicated that the patients with high circulating sclerostin levels were less likely to experience future cardiovascular events [1 pmol/L sclerostin increase, hazard ratio 0.982 (95% CI, 0.967-0.996), P = 0.015] after adjusting for a propensity score. In contrast, serum Dkk-1 was not associated with AoC and clinical outcomes. CONCLUSIONS In long-term haemodialysis patients, circulating sclerostin but not Dkk-1 is inversely associated with AoCs and future cardiovascular events. Our findings suggest that sclerostin, as a bone-related protein, might act as a communicator between uraemic bone and vasculature.

Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

β-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of β-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, β-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to β-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in β-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of β-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers.

Resistance to apoptosis induced by alkylating agents in v-Ha-ras-transformed cells due to defect in p53 function

In this study, we examined the susceptibility of various oncogene‐transformed NIH/3T3 cells to apoptosis induced by alkylating agents. Only v‐Ha‐ras‐transformed cells showed marked resistance to apoptotic death induced by these drugs. Upon treatment with methylmethane sulfonate (MMS), NIH/3T3 cells exhibited normal G1 checkpoint function accompanied by the accumulation of p53 and p21CIP1/WAF1 protein. However, no such effects were observed in v‐Ha‐ras‐transformed cells. To further examine the functional status of p53 in ras‐transformed cells, we determined the DNA sequence, protein half‐life, protein‐complexing activity, and specific DNA‐binding activity of p53. The results showed that ras transformants and parental NIH/3T3 cells had the same p53 protein half‐life of 40 min or less, the same normal wild‐type p53 cDNA sequence, and the same co‐immunoprecipitable cellular proteins complexed with p53. In electrophoretic mobility gel‐shift assays, however, nuclear extracts of cells treated with MMS, ras‐transformed cells, and normal cells displayed distinct patterns of binding between p53 ad its consensus binding site. Furthermore, western blot analysis showed that the bcl‐2 and bax proteins were constitutively elevated in ras‐transformed cells but not in parental NIH/3T3 cells. Heat‐shock protein 70 (hsp70), which has been found to be negatively regulated by wild‐type p53, was also dramatically induced in ras‐transformed cells but not in NIH/3T3 cells in response to MMS. Thus, our data suggest that an activated ras oncogene can suppress alkylating agent‐induced apoptotic cell death by means of a defect in the signal transduction pathway regulating p53 function and alteration in the expression of apoptotic (bax) or anti‐apoptotic proteins (bcl‐2 and hsp70). Mol. Carcinog. 18:221–231, 1997.