Human Liver Tumor Cells Research Articles

The NADPH oxidase NOX4 inhibits hepatocyte proliferation and liver cancer progression

The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates transforming growth factor-β-induced intracellular signals that provoke liver fibrosis, and preclinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are yet unknown. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knockdown of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, downregulation of p21(CIP1/WAF1), increase in cyclin D1 protein levels, and nuclear localization of β-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was downregulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethylnitrosamine-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred an advantage to human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth-inhibitory role in liver cells.

Anticancer Activity of Binary Toxins from Lysinibacillus sphaericus IAB872 against Human Lung Cancer Cell Line A549

The inhibitory effect of binary toxic (Bin) protein produced by Lysinibacillus sphaericus IAB872 on cell proliferation of human lung, liver, stomach and cervical tumor cell lines was assessed using MTT assay. The effect of Bin protein on A549 cell proliferation, apoptosis, cell cycle, migration and invasion were examined by MTT assay, Western blotting, Immunocytochemical staining, flow cytometry assay and wound-healing assay. Results showed that Bin protein inhibits proliferation of a range of human cancer cells in vitro. The anti-proliferative effect of Bin is associated with cell apoptosis as a result of an increased ratio of cellular Bax/bcl-2, up-regulated CyclinB1 and down-regulated Cdc25c expression, and its anti-proliferative action was associated with cell cycle arrest in the G2/M-phase. Bin protein could promote apoptosis and inhibit motility and invasion of A549 cancer cells. The anti-proliferative effect of Bin protein was associated with the induction of apoptotic cell death and cell cycle disruption. These results show that Bin protein has the potential to be developed as a chemotherapeutic agent by induction of human tumor cell apoptosis.

Overactivation of the TGF-β pathway confers a mesenchymal-like phenotype and CXCR4-dependent migratory properties to liver tumor cells

Transforming growth factor-beta (TGF-β) is an important regulatory suppressor factor in hepatocytes. However, liver tumor cells develop mechanisms to overcome its suppressor effects and respond to this cytokine by inducing other processes, such as the epithelial-mesenchymal transition (EMT), which contributes to tumor progression and dissemination. Recent studies have placed chemokines and their receptors at the center not only of physiological cell migration but also of pathological processes, such as metastasis in cancer. In particular, CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α) / chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we show that autocrine stimulation of TGF-β in human liver tumor cells correlates with a mesenchymal-like phenotype, resistance to TGF-β-induced suppressor effects, and high expression of CXCR4, which is required for TGF-β-induced cell migration. Silencing of the TGF-β receptor1 (TGFBR1), or its specific inhibition, recovered the epithelial phenotype and attenuated CXCR4 expression, inhibiting cell migratory capacity. In an experimental mouse model of hepatocarcinogenesis (diethylnitrosamine-induced), tumors showed increased activation of the TGF-β pathway and enhanced CXCR4 levels. In human hepatocellular carcinoma tumors, high levels of CXCR4 always correlated with activation of the TGF-β pathway, a less differentiated phenotype, and a cirrhotic background. CXCR4 concentrated at the tumor border and perivascular areas, suggesting its potential involvement in tumor cell dissemination. A crosstalk exists among the TGF-β and CXCR4 pathways in liver tumors, reflecting a novel molecular mechanism that explains the protumorigenic effects of TGF-β and opens new perspectives for tumor therapy.

Novel Combination of Sorafenib and Celecoxib Provides Synergistic Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer Cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies.

A new flavan-3-ol dimer from Ficus spragueana leaves and its cytotoxic activity.

Background:Isolation and structure elucidation of flavan-3-ol constituents from the leaves of Ficus spragueana and their cytotoxic activity.Materials and Methods:Different open silica gel column chromatographic techniques with different solvent systems were used for the separation of the constituents of the ethyl acetate-soluble fraction of the alcoholic extract of Ficus spragueana leaves. The structures of these compounds were assigned on the basis of spectroscopic analyses and comparison with literature data. MTT colorimetric assay method (Viability assay) was used for the evaluation of cytotoxic activity of compound 1 against human breast cancer (MCF-7) and human liver cancer (HepG2) cell lines.Results:The isolation of one flavan-3-ol dimer and was identified as (-)-afzelechin-(4α→8)-epicatechin 1, and two flavan-3-ol monomers and were identified as (-)-epiafzelechin 2 and (-)-epicatechin 3. Compound 1 was relatively inactive against human breast cancer (MCF-7) cell line at the tested concentrations as compared with the standard. However, at a concentration (50 ΅g) it was found to give inhibition upon the proliferation of examined human liver (HepG2) tumor cell line.Conclusions:Compound 1 is a new flavan-3-ol dimer and it showed a potent cytotoxic activity against human liver (HepG2) tumor cell line.

Isolation, Characterization, and Bioactivity Evaluation of 3-((6-Methylpyrazin-2-yl)methyl)-1H-indole, a New Alkaloid from a Deep-Sea-Derived Actinomycete Serinicoccus profundi sp. nov.

One new alkaloid, 3-((6-methylpyrazin-2-yl)methyl)-1H-indole (1) was obtained from the deep-sea actinomycete Serinicoccus profundi sp. nov., along with five known compounds (2–6). Their structures were determined on the basis of detailed analysis of the 1D and 2D NMR as well as MS data. The new indole alkaloid displayed weak antimicrobial activity against Staphylococcus aureus ATCC 25923 with an MIC value of 96 μg/mL. It showed no cytotoxicity on a normal human liver cell line (BEL7402) and a human liver tumor cell line (HL-7702).

Gene expression profiling of human liver carcinoma (HepG2) cells exposed to the marine toxin okadaic acid

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning. In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration-dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.

Abstract 2877: Perifosine alone and in combination with antimetabolites interferes with NF-kB pathway activation in colon cancer cell lines

Abstract Background: Perifosine, a novel, first-in-class, oral Akt inhibitor, in combination with the antimetabolite Capecitabine is currently in Phase III clinical trials for the treatment of refractory advanced colorectal cancer. We have shown previously that Perifosine in combination with antimetabolites displayed synergistic cytotoxicity and apoptosis in colon cancer and other cell lines [1]. Constitutive NF-kB activation is frequently observed in colorectal cancer and, moreover, is associated with resistance to chemotherapy [2]. Hence we analysed, wether downregulation of the NF-kB pathway contributes to Perifosine mechanism of action and to the observed synergism with antimetabolites in colon cancer cell lines. Methods: Cellular NF-kB activation in nuclear extracts was measured by the TransAm NF-kB p65 ELISA (Active Motif, USA) monitoring binding of NF-kB to its response element. To allow for higher throughput of NF-kB pathway analysis a monoclonal SW620 colon cancer cell line ectopically expressing a NF-kB response element driven reporter gene (Promega, USA) was established. Cellular cytotoxicity and apoptosis assays as well as western blotting were performed according to standard procedures. The analysis of drug combinations was conducted by CalcuSyn software (Biosoft, UK) [3]. Results: Perifosine pretreatment decreased TNFα-induced NF-kB activation in SW620 cells in both independent assay sytems with IC50 values of about 7-10 µM. This effect seems to be specific, since the results were not mimicked by cytotoxic effects of Perifosine. Some of the antimetabolites alone upregulated NF-kB activity in the reporter gene assay. In the presence of these antimetabolites Perifosine was still capable to downregulate NF-kB activity. Conclusions: In summary, these results demonstrate that downregulation of the NF-kB pathway may contribute to the mechanism of action of Perifosine. Moreover, cotreatment with Perifosine has the potential to interfere with the development of antimetabolite resistance due to NF-kB activation in colon cancer. Further experiments addressing Perifosine's mechanism of action in combination with antimetabolites in gastric cancer cell lines are ongoing.

Transient mTOR Inhibition Facilitates Continuous Growth of Liver Tumors by Modulating the Maintenance of CD133+ Cell Populations

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.

Development of optimized AAV3 serotype vectors: mechanism of high-efficiency transduction of human liver cancer cells

Our recent studies have revealed that among the 10 different commonly used AAV serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single-mutants in liver cancer cells in vitro. In immuno-deficient mouse xenograft models, direct intra-tumor injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intra-tumor and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing pro-apoptotic genes should prove useful for the potential gene therapy of human liver cancers.

The Structure of a New Cardenolide Diglycoside and the Biological Activities of Eleven Cardenolide Diglycosides from <i>Nerium oleander</i>

A new cardenolide diglycoside (1) was isolated from Nerium oleander together with ten known cardenolide diglycosides 2-11. The structure of compound 1 was established on the basis of their spectroscopic data. The in vitro anti-inflammatory activity of compounds 1-11 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 2-5 were active at an IC(50) value of less than 0.8 µM. The cytotoxicity of compounds 1-11 was evaluated against three human cell lines normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compound 3 was active toward VA-13 cells, and compounds 2-5 were active toward HepG2 cells at IC(50) values of less than 1.3 µM. The multidrug resistance (MDR)-reversal activity of compounds 1-11 was evaluated on the basis of the amount of calcein in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 1 and 8 showed moderate effects on calcein accumulation.

Polar cardenolide monoglycosides from stems and twigs of Nerium oleander and their biological activities

Twelve polar cardenolide monoglycosides, 1, 2, 4–13, and oleagenin (3) were isolated from the methanol extract of stems and twigs of Nerium oleander. Among these, oleagenin (3) and cardenolide monoglycosides named cardenolide B-1 (1) and cardenolide B-2 (2) were isolated from natural sources for the first time. The in vitro antiinflammatory activity of compounds 1–13 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 4–7 were active at an IC50 value of less than 0.4 μM. The cytotoxic activity of compounds 1–13 was evaluated against three human cell lines: normal human fibroblast cells (WI-38), malignant tumor cells derived from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 4, 6, and 7 were active toward these three cell lines at IC50 values of less than 0.7 μM, and compounds 5 and 8 were active toward the cell lines at IC50 values of less than 1.5 μM. The multidrug resistance (MDR) cancer-reversal activity of compounds 1–13 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compound 1 and 12 showed significant effects on calcein accumulation.

Platelet adhesion and fusion to endothelial cell facilitate the metastasis of tumor cell in hypoxia-reoxygenation condition

To investigate the relevant molecular mechanisms of platelet in promoting metastasis of tumor cell. The adhesion of fluorescence dye labeled-platelet to human liver sinusoidal endothelial cell (LSEC) line and tumor cell lines were detected by fluorescence microscope and fluorescence plate reader or laser scanning confocal microscope. The relevant adhesion molecules were analyzed by the antibody blockage experiment. The immune colloidal gold transmission electron microscope (TEM), flow cytometry and dye transfer were used to decipher the adhesion and fusion of platelet and LSEC. The tumor cells adhesion to vessels in ischemia condition was analyzed on mouse mesenteric vessels and the metastasis and neovascularization of metastatic foci in pulmonary tissue were also detected after tumor cells injected into nude mice via tail veil. After hypoxia-reoxygenation, tumor cell or LSEC markedly increased its adhesion with platelet, which could be blocked by different antibodies to platelet adhesion molecules. Platelet increased adhesion of tumor cell to LSEC in dose-dependent manner. The fusion of platelet and LSEC was demonstrated by translocation of fluorescent dye from platelet into the adherent LSEC; gpIIb emerged on the LSEC; and confirmed by TEM. The morphological examination found platelet presented between tumor cell and LSEC. Animal experiment indicated that the tumor adhesion to vessels was seldom in normal condition, but increased in ischemia-reperfusion condition, and further significantly enhanced by platelets. The number of tumor metastatic foci and the density of blood vessels within metastatic foci in lung were markedly increased by tumor cell pre-adhered with platelet. The adhesion or fusion of platelet to endothelial cell mediated by platelet surface adhesion molecules, which could promote the adhesion of tumor cell with endothelial cells and the tumor metastasis.

203 Perifosine in combination with antimetabolites induces synergistic effects on cytotoxicity and apoptosis in human colon, multiple myeloma, breast, renal, and liver tumor cell lines

203 Perifosine in combination with antimetabolites induces synergistic effects on cytotoxicity and apoptosis in human colon, multiple myeloma, breast, renal, and liver tumor cell lines

The biological activities of cardenolide triglycosides from stems, twigs, and leaves of Nerium oleander

Sixteen cardenolide triglycosides (1–16) were isolated from stems, twigs, and leaves of Nerium oleander. Among them, 3β-O-(4-O-gentiobiosyl-d-diginosyl)-7β,8-epoxy-14-hydroxy-5β,14β-card-20(22)-enolide, named cardenolide B-3 (16), was isolated from natural sources for the first time. The in vitro anti-inflammatory activities of compounds 1–16 were examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1). Compounds 1–5 were active at an IC50 value of less than 7 μM. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (W-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1–5 were active toward WI-38 cells; compounds 1, 3, and 5 were active toward VA-13 cells; and compounds 1–5 were active toward HepG2 cells at IC50 values of less than 10 μM. The multidrug-resistant (MDR) cancer-reversal activity of compounds 1–16 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 13 and 14 showed significant effects on calcein accumulation.

Novel combination of Celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Celecoxib (Celebrex®) exhibits antitumor effects in human HCC cells, and its mechanism of action is mediated either by its ability to inhibit cyclooxygenase 2 (COX-2) or by a number of various other COX-2 independent effects. Proteasome inhibitors (PIs) can exert cell growth inhibitory and apoptotic effects in different tumor cell types, including HCC cells. The present study examined the interaction between celecoxib and the PI MG132 in two human liver tumor cell lines HepG2 and HA22T/VGH. Our data showed that each inhibitor reduced proliferation and induced apoptosis in a dose-dependent manner in both cell lines. Moreover, the combination of celecoxib with MG132 synergistically inhibited cell viability and increased apoptosis, as documented by caspase 3 and 7 activation, PARP cleavage, and down-regulation of Bcl-2. Celecoxib and MG132, both alone and synergistically in combination, induced expression of the endoplasmic reticulum (ER) stress genes ATF4, CHOP, TRB3 and promoted the splicing of XBP1 mRNA. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased combination-induced cell death in HA22T/VGH cells, whereas it increased combination-induced cell death in HepG2 cells, suggesting that activation of the ER stress response might have either a detrimental or a protective role in liver tumor cell survival. In conclusion, our data indicate that combination treatment with celecoxib and MG132 resulted in synergistic antiproliferative and proapoptotic effects against liver cancer cells, providing a rational basis for the clinical use of this combination in the treatment of liver cancer.

Establishment and characterization of a cancer cell line derived from an aggressive childhood liver tumor

Hepatoblastoma is a rare malignancy of childhood. The scarcity of adequate cell models has limited our understanding of this tumor. Here we describe and characterize a new human liver tumor cell line, Hep293TT, derived from an aggressive childhood hepatoblastoma. Hep293TT cells were established using primary tumor tissues from a 5-year-old Caucasian female child. This cell line has been maintained for more than 34 months and over 20 subcultures, and was characterized by histopathology, ELISA, genotype, cytogenetics, CGH array, immunohistochemistry, and molecular sequence analyses. Cells were confirmed to originate from parental tumor cells, secrete alpha-fetoprotein, and express hepatic markers and beta-catenin. Hep293TT cells were able to form colonies in soft agar. Tumorigenicity was demonstrated by induction of solid tumors after subrenal capsule injection in immunodeficient mice. Hep293TT cells demonstrated a highly aneuploid karyotype, and a whole genome CGH analysis revealed chromosomal imbalances in every chromosome. Allelotype analysis demonstrated loss of alleles at distal 11p15.5 as is typical of embryonal tumors. Both Hep293TT cells and the primary tumor contain a deletion of 351 nucleotides in beta-catenin, as has been seen in other hepatoblastoma tumors. The cell line expressed beta-catenin protein in both full-length and partially deleted forms, and expressed NOTCH2 protein characteristic of hepatoblasts. No mutation was detected in the APC, MYH, MLH1, or MSH2 genes. This cell line, Hep293TT, is a valuable resource for the study of childhood liver cancer and may potentially provide a tool in the development of new agents.

Impact of d‐δ‐Tocotrienol on Human A2058 and A375 Melanoma Cells

The rate‐limiting activity of the mevalonate pathway, 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation or dolichylation of growth‐related proteins including nuclear lamins, Ras and growth factor receptors. d‐δ‐Tocotrienol, a post‐transcriptional down‐regulator of HMG CoA reductase, suppresses the proliferation of murine B16 melanoma cells and human blood, breast, cervix, colon, liver, lung, lymph gland, nerve, pancreas, and prostate tumor cells. Dietary d‐δ‐tocotrienol suppresses the growth of implanted B16 melanomas without toxicity to host mice. We evaluated d‐δ‐tocotrienol in human A2058 and A375 melanoma cells. d‐δ‐Tocotrienol induced dose‐dependent suppression of the cell proliferation following 72 h incubation in 96‐well plates with IC50 values of 48 (A2058) and 22 (A375) μmol/L, respectively. Morphological changes detected by fluorescence microscopy following acridine orange and ethidium bromide dual staining showed d‐δ‐tocotrienol‐induced apoptosis in A2058 cells. The impact of d‐δ‐tocotrienol on A2058 cell proliferation was potentiated by lovastatin (IC50=3 μmol/L), a competitive inhibitor of HMG CoA reductase. d‐δ‐Tocotrienol may have potential application in melanoma chemoprevention and/or therapy. TDA, TWU REP & Summer Stipend Award.

MicroRNA-like off-target transcript regulation by siRNAs is species specific

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand "seed region," similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.