Identification of FN1BP1 as a novel cell cycle regulator through modulating G1 checkpoint in human hepatocarcinoma Hep3B cells.

A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

Acetylcholinesterase, a key prognostic predictor for hepatocellular carcinoma, suppresses cell growth and induces chemosensitization.

UNLABELLED: Acetylcholinesterase (ACHE) plays important roles in the cholinergic system, and its dysregulation is involved in a variety of human diseases. However, the roles and implications of ACHE in hepatocellular carcinoma (HCC) remain elusive. Here we demonstrate that ACHE was significantly down-regulated in the cancerous tissues of 69.2% of HCC patients, and the low ACHE expression in HCC was correlated with tumor aggressiveness, an elevated risk of postoperative recurrence, and a low survival rate. Both the recombinant ACHE protein and the enhanced expression of ACHE significantly inhibited HCC cell growth in vitro and tumorigenicity in vivo. Further study showed that ACHE suppressed cell proliferation via its enzymatic activity of acetylcholine catalysis and degradation. Moreover, ACHE could inactivate mitogen-activated protein kinase and phosphatidyl inositol-3'-phosphate kinase/protein kinase B pathways in HCC cells and thereby increase the activation of glycogen synthase kinase 3ß and lead to ß-catenin degradation and cyclin D1 suppression. In addition, increased ACHE expression could remarkably sensitize HCC cells to chemotherapeutic drugs (i.e., adriamycin and etoposide). CONCLUSION: For the first time, we describe the function of ACHE as a tumor growth suppressor in regulating cell proliferation, the relevant signaling pathways, and the drug sensitivity of HCC cells. ACHE is a promising independent prognostic predictor for HCC recurrence and the survival of HCC patients. These findings provide new insights into potential strategies for drug discovery and improved HCC treatment.

Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA.

Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

MicroRNA-30d promotes tumor invasion and metastasis by targeting Galphai2 in hepatocellular carcinoma.

UNLABELLED: The pathological relevance and significance of microRNAs (miRNAs) in hepatocarcinogenesis have attracted much attention in recent years; however, little is known about the underlying molecular mechanisms through which miRNAs are involved in the development and progression of hepatocellular carcinoma (HCC). In this study, we demonstrate that miR-30d is frequently up-regulated in HCC and that its expression is highly associated with the intrahepatic metastasis of HCC. Furthermore, the enhanced expression of miR-30d could promote HCC cell migration and invasion in vitro and intrahepatic and distal pulmonary metastasis in vivo, while silencing its expression resulted in a reduced migration and invasion. Galphai2 (GNAI2) was identified as the direct and functional target of miR-30d with integrated bioinformatics analysis and messenger RNA array assay. This regulation was further confirmed by luciferase reporter assays. In addition, our results, for the first time, showed that GNAI2 was frequently suppressed in HCC by way of quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining assays. The increase of the GNAI2 expression significantly inhibits, whereas knockdown of the GNAI2 expression remarkably enhances HCC cell migration and invasion, indicating that GNAI2 functions as a metastasis suppressor in HCC. The restoration of GNAI2 can inhibit miR-30d-induced HCC cell invasion and metastasis. CONCLUSION: The newly identified miR-30d/GNAI2 axis elucidates the molecular mechanism of HCC cell invasion and metastasis and represents a new potential therapeutic target for HCC treatment.

A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry.

Liver cirrhosis and hepatocellular carcinoma (HCC) are fatal sequelaes of chronic hepatitis B in China. The sera from HCC and cirrhosis were profiled by rapid resolution liquid chromatography coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry. Reversed-phased (RP) liquid chromatography and hydrophilic interaction chromatography (HILIC) were used for the data acquisition. The normalized and combined data were handled by chemometric analysis, and the combination proved to be effective and reliable for the orthogonal projection to latent structures (OPLS) analysis. Metabonomic profiles and the potential biomarkers were found based on the OPLS models. Shared and unique structure (SUS) plots were used for the evaluation of the potential biomarkers. Glycocholic acid, glycochenodeoxycholic acid, taurocholic acid and taurochenodesoxycholic acid were found to be potential biomarkers related to liver cirrhosis, while dihydrosphingosine and phytosphingosine were potential diagnostic biomarkers of HCC. The other identified metabolites were considered as common potential biomarkers for the two liver diseases. Correlation networks based on these metabolites were also built for the systemic understanding of these diseases and the possible biological implications are discussed. This metabonomic approach may provide insight into discovery and identification of new diagnostic biomarkers for liver cancer and associated diseases.

Role of hepatitis B surface antigen in the development of hepatocellular carcinoma: regulation of lymphoid enhancer-binding factor 1.

BACKGROUND: There are around 350 million of hepatitis B surface antigen (HBsAg) carriers worldwide, and among them, high risk of developing hepatocellular carcinoma (HCC) has been identified by epidemiological studies. To date, the molecular role of HBsAg in HCC development has not been fully studied. We have previously reported that in cell cultures, HBsAg up-regulated the expression of lymphoid enhancer-binding factor 1 (LEF-1), a key component of the Wnt pathway. In this study we aimed to study this effect of HBsAg on LEF-1 in the development of HCC. METHODS: Expression of HBsAg, LEF-1 and its downstream effector genes were compared among 30 HCCs, their peritumor tissue counterparts and 9 normal control liver tissues by quantitative real-time PCR. In addition, immunohistochemical staining studies on HBsAg and LEF-1 expression were conducted among these samples. RESULTS: The expression of LEF-1 was compared between 13 HBsAg positive HCC tissues and 17 HBsAg negative HCC tissues. Simultaneous detection of LEF-1 and HBsAg was observed in HBsAg positive HCC tissues and, additionally, the simultaneous detection of HBsAg and LEF-1 was more pronounced in peritumor tissues, compared to that in the tumor tissues. The distribution of cellular LEF-1 in peritumor tissues was predominantly in the cytoplasm; while LEF-1 in the tumor tissues was located either exclusively in the nucleus or both in the nucleus and cytoplasm. By real-time PCR, the expression levels of LEF-1 downstream effector genes cyclin D1 and c-myc were higher in peritumor cells compared to that of the tumor cells. However, a 38 kDa truncated isoform of LEF-1, rather than the 55 kDa wild-type LEF-1, was significantly elevated in the HBsAg positive tumor cells. CONCLUSION: Data indicate that deregulation of the Wnt pathway by HBsAg occurred in HBV-associated HCCs, but was more pronounced in the peritumor cells. It is speculated that HBsAg could stimulate proliferation and functional modification of hepatocytes via LEF-1 through the Wnt pathway at the pre-malignant stage.

Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma.

MicroRNAs (miRNAs) are important gene regulators, which are often deregulated in cancers. In this study, the authors analyzed the microRNAs profiles of 78 matched cancer/noncanerous liver tissues from HCC patients and 10 normal liver tissues and found that 69 miRNAs were differentially expressed between hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues (N). Then the expressions of 8 differentially expressed miRNAs were validated by real time RT PCR. The set of differentially expressed miRNAs could distinctly classify HCC, N and normal liver tissues (NL). Moreover, some of these differentially expressed miRNAs were related to the clinical factors of HCC patients. Most importantly, Kaplan-Meier estimates and the log-rank test showed that high expression of hsa-miR-125b was correlated with good survival of HCC patients (hazard ratio, 1.787, 95% confidence interval, 1.020-3.133, p = 0.043). The transfection assay showed that overexpression of miR-125b in HCC cell line could obviously suppress the cell growth and phosporylation of Akt. In conclusion, the authors have demonstrated the diagnostic miRNA profile for HCC, and for the first time, identified the miR-125b with predictive significance for HCC prognosis.

Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.

Transforming growth factor-beta (TGF-beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF-beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR-23a approximately 27a approximately 24, is induced in an early stage by TGF-beta in Huh-7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR-23a approximately 27a approximately 24 to TGF-beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR-23a approximately 27a approximately 24 can function as an antiapoptotic and proliferation-promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF-beta could induce specific miRNA expression to escape from tumor-suppressive response in HCC cells.

Metabonomics study on the effects of the ginsenoside Rg3 in a beta-cyclodextrin-based formulation on tumor-bearing rats by a fully automatic hydrophilic interaction/reversed-phase column-switching HPLC-ESI-MS approach.

The goal of this study was the application of a novel, fully automatic column-switching approach in a metabonomics study combining the orthogonal selectivities of hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography. The temporal, pharmacodynamic effects of the ginsenoside Rg3 on the metabonome in urine of healthy and liver-tumor-bearing rats have been investigated. Within a total analysis time of 52 min we detected 5686 polar, and on the second column an additional 1808 apolar, urinary metabolite ions. The administration of a single, high dose of Rg3 in a beta-cyclodextrin-based formulation led to a considerable change of the metabolic pattern in cancer rats during 3 days studied. Seventeen biomarker candidates including three apolar metabolites, which were not retained on the HILIC column, were detected. Overall, the results suggest that the developed liquid chromatography-mass spectrometry strategy is a promising tool in metabonomics studies for global analysis of highly complex biosamples. It may not only increase the number of discovered biomarkers but consequently improve the comprehensive information on metabolic changes in a fully automatic manner.

Dysfunction of murine dendritic cells induced by incubation with tumor cells.

In vivo studies showed that dendritic cell (DC) dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs (imDCs) are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CD11b, CD11a and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation.

Analysis of acetylcholine, choline and butyrobetaine in human liver tissues by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The strong polar quaternary ammoniums, acetylcholine (ACh), choline (Ch) and butyrobetaine (BB, (3-carboxypropyl)trimethylammonium), are believed playing important roles in liver metabolism. These metabolites are at low levels and are weakly retained on reversed-phase liquid chromatographic (RP-LC) columns. Several hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) methods have been reported to analyze these compounds from different samples. However, no application to human liver tissues has been published. In this study, HILIC-MS/MS method was developed to simultaneously determine these three metabolites in human liver tissues. They were simply extracted from tissue, separated on a HILIC column, and detected by tandem MS in the mode of multiple reaction monitoring (MRM). Further studies on the recovery and repeatability based on real samples indicated the method was accurate and reliable. This method was successfully applied to measure the levels of ACh, Ch and BB in 61 human liver tissue samples including normal, hepatocellular carcinoma (HCC) and matched non-cancerous liver tissues. By comparison of Ch and ACh contents in 29 HCC with their matched non-cancerous liver tissues, it was found that ACh content increased in 11/29 HCC cases and decreased in 13/29 cases. Furthermore, the ACh/Ch ratio increased in 16/29 HCC cases, while it decreased in 8/29 cases. These results strongly indicated that there exist different patterns of ACh content in cancer tissues among HCC patients, thus highlighting the understanding of ACh and its relevant signal pathways in hepatic carcinogenesis and HCC progression.

Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.

Ciliary neurotrophic factor receptor alpha subunit-modulated multiple downstream signaling pathways in hepatic cancer cell lines and their biological implications.

UNLABELLED: Ciliary neurotrophic factor (CNTF) plays important roles in a variety of tissues including neural and non-neural systems, but the function of CNTF and its receptor (CNTFR) in liver remains unclear. In this study, we demonstrate that CNTFRalpha is expressed heterogeneously in normal human liver and hepatocellular carcinoma (HCC) specimens but not in hepatoblastoma specimens. We choose the CNTFRalpha(+)/CNTFRalpha(-) (CNTFRalpha positive/ CNTFRalpha negative) cell models of hepatic origin to study multiple downstream pathways of CNTFRalpha. We show that the presence of CNTFRalpha determines the temporal activation patterns of downstream signaling molecules and serves as a key modulator in regulating PI3K and AMP-activated protein kinase (AMPK) dynamically under CNTF stimulation, thus resulting in the increase of glucose uptake and translocation of glucose transporter 4 (GLUT4). Furthermore, CNTF-induced mitogen-activated protein kinase (MAPK) activation suppresses AMPK activity in the early phase of CNTF stimulation. Moreover, the protective role of CNTF against cell-cycle arrest is dependent on the presence of CNTFRalpha and is modulated by the glucose concentration of the culture medium. CONCLUSION: Our results demonstrate the importance of CNTFRalpha-mediated downstream signaling pathways and their functional implications in hepatic cancer cells, thus highlighting a better understanding of the biological roles of CNTFRalpha in human liver abnormalities, including metabolic diseases and hepatocarcinogenesis.

A protein-protein interaction network of transcription factors acting during liver cell proliferation.

Liver regeneration is a complex process that involves a multitude of cellular functions, including primarily cell proliferation, apoptosis, inflammation, and metabolism. A number of signaling pathways that control these processes have been identified, and cross communication between them by direct protein-protein interactions has been shown to be crucial in orchestrating liver regeneration. Previously, we have identified a group of transcription factors capable of regulating liver cell growth and that may be involved in liver cancer development. The expression of some of their mouse counterpart genes was altered dramatically after liver injury and regeneration induced by CCl(4) in mice. In an effort to elucidate the molecular basis for liver regeneration through protein-protein interactions (PPI), a matrix mating Y2H approach was produced to generate a PPI network between a set of 32 regulatory proteins. Sixty-four interactions were identified, including 4 that had been identified previously. Ten of the interactions were further confirmed with GST pull-down and coimmunoprecipitation assays. Information provided by this PPI network may shed further light on the molecular mechanisms that regulate liver regeneration at the protein interaction level and ultimately identify regulatory factors that may serve as candidate drug targets for the treatment of liver diseases.

Candidate genes responsible for human hepatocellular carcinoma identified from differentially expressed genes in hepatocarcinogenesis of the tree shrew (Tupaia belangeri chinesis).

AIM: To explore gene expression profiles during hepatocarcinogenesis of the tree shrew, and to find the genes responsible for human hepatocellular carcinoma (HCC). METHODS: Tree shrews were used as an animal model for HCC induction employing aflatoxin B(1) (AFB(1)) alone or AFB(1) plus hepatitis B virus (HBV) as etiological factors. Gene expression profiles from the tissues of HCC, HCC-surrounding liver tissues (para-HCC) and the corresponding biopsies taken from the same animals before HCC had developed (pre-HCC) were analyzed by cDNA microarray assay to identify differentially expressed genes. Two genes, CuZn-superoxide dismutase (SOD1) and glutathione S-transferase A1 (GSTA1), were further investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical (IHC) assays were done on tree shrew and human HCC samples. RESULTS: RESULTS from the cDNA microarray analysis indicated that the gene expression profiles of HCC between AFB(1)and AFB(1) + HBV treatment groups were markedly different. A total of 11 genes, including SOD1 and GSTA1, were found changing in expression levels in all detected samples from both groups. RESULTS from RT-PCR and IHC assays indicated that mRNA and protein levels of SOD1 and GSTA1 were markedly downregulated in both tree shrew and human HCC, and downregulation of SOD1 and GSTA1 proteins in human HCC samples was closely correlated with the histopathological grading (P < 0.05). CONCLUSION: The differentially expressed genes found in all HCC cases induced by different etiological factors among different species should be considered as good candidate genes responsible for HCC. Downregulation of SOD1 and GSTA1 might play an important role in hepatocarcinogenesis.

Choline transporters in human lung adenocarcinoma: expression and functional implications.

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential choline-starvation strategy of cancer interference through targeting choline transporters, especially CTL1.

Cloning, expression and characterization of human tissue-specific DNA polymerase lambda2.

DNA polymerase (POL) lambda plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL lambda variant was cloned from a human liver cDNA library and named POL lambda2 (GenBank Accession No. AY302442). POL lambda2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL lambda2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL lambda2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL lambda and POL lambda2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL lambda. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL lambda2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope a-(32)P-dCTP incorporation in vitro showed that the purified recombinant POL lambda2 exhibited DNA polymerase activity.

BNIPL-2 promotes the invasion and metastasis of human hepatocellular carcinoma cells.

Enhanced cell migration and invasion play key roles in cancer metastasis. However, the molecules involved in this process are not fully understood. In this study, a full-length human BNIPL-2 (Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-2) cDNA was transfected into human hepatocellular carcinoma cells with low metastatic potential (MHCC97-L). The in vitro and in vivo effects of BNIPL-2 on cell invasion and metastasis were examined. In vitro analysis showed that the overexpression of BNIPL-2 increases cell invasion and promotes cell migration. The rates of intrahepatic and pulmonary metastasis in nude mice were also increased. Cdc42 activation assays and immunoblot analysis indicated that the activation of Cdc42 and the upregulation of CD44 were involved in the metastasis of cancer cells. The overexpression of BNIPL-2 promotes the invasion and metastasis of MHCC97-L cells. Thus, BNIPL-2 is a gene related with cancer metastasis.

CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity.

Recently increasing reported data have suggested that only a small subset of cancer cells possess capability to initiate malignancies including leukemia and solid tumors, which was based on investigation in these cells displaying a distinct surface marker pattern within the primary cancers. CD133 is a putative hematopoietic and neuronal stem-cell marker, which was also considered as a tumorigenic marker in brain and prostate cancer. We hypothesized that CD133 was a marker closely correlated with tumorigenicity, since it was reported that CD133 expressed in human fetal liver and repairing liver tissues, which tightly associated with hepatocarcinogenesis. Our findings showed that a small population of CD133 positive cells indeed exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. From SMMC-7721 cell line, CD133+ cells isolated by MACS manifested high tumorigenecity and clonogenicity as compared with CD133- HCC cells. The implication that CD133 might be one of the markers for HCC cancer stem-like cells needed further investigation.

Identification of carboxypeptidase of glutamate like-B as a candidate suppressor in cell growth and metastasis in human hepatocellular carcinoma.

PURPOSE: We have previously done large-scale cDNA transfection screening on human hepatocellular carcinoma (HCC) cells and have identified 3,806 cDNA genes that possess the ability of either stimulating or inhibiting cell growth. In this study, we characterized one of these growth suppressor genes, carboxypeptidase of glutamate like-B (CPGL-B), in HCC. EXPERIMENTAL DESIGN: Semiquantitative reverse-transcription PCR was used to examine the expression levels of CPGL-B. The cellular localization and functions of CPGL-B were investigated by enforced expression of CPGL-B in HCC cells. RESULTS: From our previous cDNA transfection screening, we identified a gene named CPGL and its isoform, CPGL-B. With computational analysis, CPGL was located at chromosome 18q22.3 and was a homologue of peptidase family M20. CPGL was expressed in all adult and fetal tissues, whereas its isoform, CPGL-B, lacking exons 3 and 4, was expressed in all fetal tissues but only in liver and placenta of adult tissues. In HCC, CPGL-B was frequently underexpressed (35 of 90, 38.9%) in tumorous tissues compared with the corresponding nontumorous livers. Intriguingly, the underexpression was significantly associated with the presence of venous invasion (P=0.018) and tumor microsatellite formation (P=0.004). Stable transfection of CPGL-B in SMMC7721 HCC cells showed significant inhibition in cell viability, colony formation, cell invasion, and tumor formation in nude mice. CPGL-B also down-regulated CXCR3, matrix metalloproteinase 11, and CD44s, which are involved in cell growth and cell migration. CONCLUSIONS: These findings suggest that the frequent underexpression of CPGL-B may be associated with cell growth and metastasis of HCC.