Tsung-Ching Lai

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

MicroRNA-122 (miR-122), which accounts for 70% of the livers total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

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.)

Enhancement of cell radiation sensitivity by pegylated gold nanoparticles

Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

Biocompatibility of Fe(3)O(4) nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells.

In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia.

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.

Seedless, silver-induced synthesis of star-shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent

This work demonstrates a simple method for synthesizing a shape-controllable bimetallic gold/silver nanostructured material. Spiky star-shaped gold/silver nanoparticles are obtained by mixing HAuCl4, AgNO3 and ascorbic acid with shaking for 20 s. The wide range of star shapes and irregular quasi-spherical nanoparticles is tailored by tuning the ratio of metal precursors. The wavelengths absorbed by the nanoparticles can be tuned from visible light to near infrared by controlling their shape. To maintain the morphology of the nanoparticles, enhance their thermal stability and support their application in biological systems, modified chitosan was utilized for the properties and to keep the material well dispersed in solution in deionized water. The moderate concentration of modified chitosan capped bimetallic star-shaped nanoparticles not only ensured non-toxicity to normal cells and cancer cells, but also promoted high efficiency photothermal ablation of cancer cells. Ultimately, this nanotechnology-driven assay has huge potential for application in rapid synthesis, tunable absorption and non-cytotoxic photothermal therapy for the effective treatment of cancer.

Essential roles of caspases and their upstream regulators in rotenone-induced apoptosis

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.

Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

The significance of ANXA7 expression and its correlation with poor cellular differentiation and enhanced metastatic potential of gastric cancer.

Annexin‐A7 (ANXA7) exhibits biological and genetic properties expected of a tumor suppressor gene and may play a role in cancer progression. However, the ANXA7 expression in different histological subtypes of gastric adenocarcinomas and its correlation with invasive potentials has not been elucidated.

Defective Phosphatidylinositol 3-Kinase Signaling in Central Control of Cardiovascular Effects in the Nucleus Tractus Solitarii of Spontaneously Hypertensive Rats

Recently we have shown functional involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt-nitric oxide synthase (NOS) signaling pathway in central control of cardiovascular effects in the nucleus tractus solitarii (NTS) of normotensive Wistar-Kyoto (WKY) rats. In this study we determined whether PI3K/Akt signaling was defective in spontaneously hypertensive rats (SHR). WKY rats and SHR were anesthetized with urethane. Mean blood pressure (MBP) and heart rate (HR) were monitored intra-arterially. Unilateral microinjection (60 nL) of insulin (100 IU/mL) into the NTS produced prominent depressor and bradycardic effects in 8- and 16-week-old normotensive WKY and 8-week-old SHR. However, no significant cardiovascular effects were found in 16-week-old SHR after insulin injection. Furthermore, pretreatment with PI3K inhibitor LY294002 and NOS inhibitor L-NAME into the NTS attenuated the cardiovascular response evoked by insulin in WKY and 8-week-old SHR but not in 16-week-old SHR. Unilateral microinjection of 1 mmol/L of PI(3,4,5)P3 (phosphatidylinositol 3,4,5-triphosphate), a phospholipids second messenger produced by PI3K, into the NTS produced prominent depressor and bradycardic effects in 8- or 16-week-old WKY rats as well as 8-week-old SHR but not in 16-week-old SHR. Western blot analysis showed no significant increase in Akt phosphorylation in 8-week-old pre-hypertensive SHR after insulin injection. Similar results were also found in hypertensive 16-week-old SHR. Our results indicate that the Akt-independent signaling pathway is involved in NOS activation to regulate cardiovascular effects in the NTS of 8-week-old pre-hypertensive SHR. Both Akt-dependent and Akt-independent signaling pathways are defective in hypertensive 16-week-old SHR.