HLE Cell Research Articles

Inhibition of Annexin A10 Contributes to ZNF281 Mediated Aggressiveness of Hepatocellular Carcinoma.

To investigate the involvement and transcriptional targets of zinc finger protein 281 (ZNF281) in the progression of hepatocellular carcinoma (HCC). The expression of ZNF281 in HCC was detected in tissue microarray and cell lines. The role of ZNF281 in aggressiveness of HCC was examined using wound healing, matrigel transwell, pulmonary metastasis model and assays for expression of EMT markers. RNA-seq was used to find potential target gene of ZNF281. Chromatin immunoprecipitation (ChIP) assay and co-immunoprecipitation (Co-IP) were employed to uncover the mechanism of the transcriptional regulation of ZNF281 on the target gene. ZNF281 was increased in tumor tissues and positively correlated with vascular invasion in HCC. Knockdown of ZNF281 suppressed the migration and invasion with significant alteration of EMT marker expression in HLE and Huh7 HCC cell lines. RNA-seq screening showed that the tumor suppressor gene Annexin A10 (ANXA10) was a most up-regulated gene in response to ZNF281 depletion and responsible for the attenuation of aggressiveness. Mechanistically, ZNF281 interacted with the ANXA10 promoter region harboring ZNF281 recognition sites, and recruited components of nucleosome remodeling and deacetylation (NuRD) complex. By knocking down such components like HDAC1 or MTA1, ANXA10 was released from transcriptional repression by ZNF281/NuRD, and in turn reversed the EMT, invasion and metastasis driven by ZNF281. ZNF281 drives invasion and metastasis of HCC partially through transcriptional repression of tumor suppressor gene ANXA10 by recruiting NuRD complex.

Comprehensive analysis of circulating microRNAs as predictive biomarkers for sorafenib therapy outcome in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Clinical management has improved the prognosis of early HCC, but that of advanced HCC remains poor. Sorafenib, an oral multikinase inhibitor, provided a treatment option for advanced-stage HCC, and prolonged the survival and inhibited tumor progression as first-line therapy in patients with advanced HCC. In this study, we investigated if specific microRNAs could act as predictive biomarkers of sorafenib effectiveness and indicate the best time to switch to second-line therapies. Sorafenib inhibited the proliferation of the Li-7, Hep3B, HepG2 and Huh7 liver cancer cell lines (effective group), but not that of the HLE, HLF and ALEX cancer cell lines (non-effective group). A microRNA (miRNA/miR) analysis was performed comparing sorafenib-effective and non-effective cells lines as well as serum samples from patients with HCC from sorafenib-effective (complete response/partial response) and -non-effective (progressive disease) groups before sorafenib administration and detected three differentially-expressed miRNAs that were common among the in vivo and in vitro samples. The increase rate (effective/non-effective) of hsa-miR-30d in the medium was higher than that in the cancer cells. hsa-miR-30d was highly expressed in the serum and exosomes of patients with HCC in the effective group when compared to those of the non-effective group. Additionally, the hsa-miR-30d expression in the medium of cancer cell lines was highly upregulated in the effective group compared with the non-effective group. These results suggested that hsa-miR-30d might be secreted by the cancer cells to the serum through the exosomes. We identified a specific circulating miRNA that is related to refractory HCC under sorafenib therapy. Therefore, hsa-miR-30d might serve as a predictive biomarker for the efficacy of sorafenib therapy in HCC.

ISOLATION OF CYTOTOXIC CONSTITUENT FROM BIOACTIVITY GUIDED FRACTION OF ALYSICARPUS MONILIFER L. (DC.)

Objective: Alysicarpus monilifer (Family Papilionaceae) has been used in the Indigenous system of medicine in tumor removal. The present study was designed to isolate and identify the constituent responsible for cytotoxic (anti-tumor) effects of the plant Alysicarpus monilifer.
 Methods: The plant was powdered and extracted to give a methanolic extract. Initially, Hexane, chloroform, ethyl acetate and methanolic fractions of the methanolic extract of the plant were subjected to cytotoxic screening using cell line based assay (MTT assay and NRU assay). The chloroform fraction showed significant cytotoxicity, so it was further subjected to column chromatography, to separate the cytotoxic phytoconstituent. The cell lines selected were breast cancer cells (MCF-7 and MDA-MB-468) and Liver cancer cells (HepG2 and HLE cell). Results were calculated as percentage growth inhibition with respect to untreated (control) cells versus treated cells.
 Result: A triterpene, Betulinic acid, was isolated from the aerial parts of Alysicarpus monilifer. The cytotoxic activity of the identified compound against MCF-7, MDA-MB-231, HLE and HepG2 cells was also found to be highly significant with 90% growth inhibition.
 Conclusion: The triterpene was identified to be betulinic acid, to which the cytotoxic activity can be attributed. It is a first report of isolation of betulinic acid from the Alysicarpus species.

Tumor-specific inhibitory action of decorin on different hepatoma cell lines

BackgroundIn spite of therapeutic approaches, liver cancer is still one of the deadliest type of tumor in which tumor microenvironment may play an active role in the outcome of the disease. Decorin, a small leucine-rich proteoglycan is not only responsible for assembly and maintenance of the integrity of the extracellular matrix, but a natural inhibitor of cell surface receptors, thus it exerts antitumorigenic effects. Here we addressed the question whether this effect of decorin is independent of the tumor phenotypes including differentiation, proliferation and invasion. MethodFour hepatoma cell lines HepG2, Hep3B, HuH7 and HLE, possessing different molecular backgrounds, were selected to investigate. After proliferation tests, pRTK arrays, WB analyses, and immunofluorescent examinations were performed on decorin treated and control cells for comparison. ResultsSignificant growth inhibitory potential of decorin on three out of four hepatoma cell lines was proven, however the mode of its action was different. Induction of p21WAF1/CIP1, increased inactivation of c-myc and β-catenin, and decrease of EGFR, GSK3β and ERK1/2 phosphorylation levels were observed in HepG2 cells, pathways already well-described in literature. However, in the p53 deficient Hep3B and HuH7, InsR and IGF-1R were the main receptors transmitting signals. In harmony with its receptor status, Hep3B cells displayed high level of activated AKT. As the cell line is retinoblastoma mutant, ATR/Chk1/Wee1 system might hinder the cell cycle in G2/M phase via phosphorylation of CDK1. In Huh7 cells, all RTKs were inhibited by decorin followed by downregulation of AKT. Furthermore, HuH7 cell line responded with concentration-dependent ERK activation and increased phospho-c-myc level. Decorin had only a non-significant effect on the proliferation rate of HLE cell line. However, it responded with a significant decrease of pAKT, c-myc and β-catenin activity. In this special cell line, the inhibition of TGFβ may be the first step of the protective effect of decorin. ConclusionsBased on our results decorin may be a candidate therapeutic agent in the battle against liver cancer, but several questions need to be answered. It is certain that decorin is capable to exert its suppressor effect in hepatoma cells without respect to their phenotype and molecular background.

Effect of Exogenous Zinc on MsrB1 Expression and Protein Oxidation in Human Lens Epithelial Cells.

Aging has been related to zinc deficiency, resulting in protein oxidation and age-related decline of methionine sulfoxide reductase (Msr) activity. This study was designed to investigate the levels of methionine sulfoxide reductase B1 (MsrB1) mRNA and oxidized proteins in human lens epithelial (hLE) cells after treatment with exogenous zinc. The role of exogenous zinc in regulation of MsrB1 gene expression and protein oxidation in hLE cells was studied by MTT assay, oxidized protein measurement kit, and real-time PCR. The results showed that hLE cell viability was significantly decreased by MsrB1 gene knockdown or peroxynitrite (ONOO-) treatment, while it was significantly increased after treatment with exogenous zinc (P < 0.05). Protein carbonyl content in hLE cell by MsrB1 gene knockdown or ONOO- treatment was significantly decreased after treatment with ZnSO4 (P < 0.01). And exogenous zinc could increase the level of MsrB1 in hLE cell under normal (P < 0.001) and oxidative stress (P < 0.01) conditions. In conclusion, exogenous zinc could protect hLE cells against MsrB1 gene knockdown or ONOO--induced cell death by upregulation of MsrB1 involved in the elimination of reactive oxygen species (ROS) and oxidized proteins.

Oct3/4 is potentially useful for the suppression of the proliferation and motility of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

Role of caveolin-1 in hepatocellular carcinoma arising from non-alcoholic fatty liver disease.

The molecular features of hepatocellular carcinoma arising from non‐alcoholic fatty liver disease (NAFLD‐HCC) are not well known. In this study, we investigated the mechanism by which NAFLD‐HCC survives in a fat‐rich environment. We found that caveolin (CAV)‐1 was overexpressed in clinical specimens from NAFLD‐HCC patients. HepG2, HLE, and HuH‐7 HCC cell lines showed decreased proliferation in the presence of the saturated fatty acids palmitic acid and stearic acid, although only HLE cells expressed high levels of CAV‐1. HLE cells treated with oleic acid (OA) showed robust proliferation, whereas CAV‐null HepG2 cells showed reduced proliferation and increased apoptosis. CAV‐1 knockdown in HLE cells attenuated the OA‐induced increase in proliferation and enhanced apoptosis. Liquid chromatography–tandem mass spectrometry analysis revealed that the levels of OA‐containing ceramide, a pro‐apoptotic factor, were higher in HepG2 and CAV‐1‐deficient HLE cells than in HLE cells, suggesting that CAV‐1 inhibits apoptosis by decreasing the level of OA‐containing ceramide. These results indicate that CAV‐1 is important for NAFLD‐HCC survival in fatty acid‐rich environments and is a potential therapeutic target.

Elevated GPC3 level promotes cell proliferation in liver cancer.

The aim of the present study was to investigate the biological role of glypican 3 (GPC3), and to identify its mechanism and clinical significance in the carcinogenesis of liver cancer. A total of 114 patients with liver cancer were involved. Their clinical data, hematoxylin and eosin-stained and Antigen Ki-67 protein (Ki-67) and GPC3 immunohistochemically-stained liver cancer tissue sections were analyzed to evaluate the correlation between the liver cancer proliferation, differentiation and GPC3 expression. Fluorescence microscopy, western blotting, MTT and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were performed in HepG2 and HLE cell lines to investigate the potential mechanisms of action. Among the 114 patients with liver cancer enrolled in the present study, 12 exhibited well-differentiated liver cancer, of which 6 (50%) were positive for GPC3. A total of 30 cases exhibited poorly differentiated liver cancer; 26 (87%) of these expressed GPC3 and 11 cases (37%) demonstrated strong positive expression levels. The other 72 liver cancer cases were moderately differentiated; 75% (54/72) of these expressed GPC3 and 12.5% (9/72) exhibited strong positive expression levels. There was a significant association between the levels of GPC3 expression and liver cancer differentiation (χ2=16.306, P=0.008). Ki-67 staining as the criteria of the liver cancer cell proliferation index also indicated a cross correlation between liver cancer proliferation and GPC3 levels. Among the 39 liver cancer samples with a cell proliferation index <5%, only 2.6% (1/39) exhibited strong positive GPC3 staining, but of the 16 cases with a high cell proliferation index >50%, 6 exhibited strong GPC3 staining (37.5%). The difference of cell proliferation indexes between cancer cells were well, moderate and poorly differentiated, and was markedly significant (χ2=26.334, P=0.002), and suggested that liver cancer cell proliferation was positively correlated with GPC3 expression (r=0.316, P=0.001). Consistently, in vitro analysis indicated that GPC3 promoted HepG2 and HLE cell growth, which was more apparent in HepG2 cells. The RT-qPCR results indicated that GPC3 promoted proliferation through the Hedgehog (Hh) pathway in HepG2 cells, but not in HLE cells. In the present study, it was demonstrated that patients with liver cancer with higher GPC3 levels exhibited poorer differentiation and higher proliferation levels. In vitro GPC3 may promote liver cancer cell lines proliferation through the Hh pathway.

Targeting human liver cancer cells with lactobionic acid-G(4)-PAMAM-FITC sorafenib loaded dendrimers

Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R.

Abstract 4421: Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver

Abstract (Background) The incidence of hepatocellular carcinoma (HCC) arising from non-alcoholic fatty liver (NAFL) has been recently increasing according to increase of patients having metabolic syndrome. HCC, which shows steatosis, cellular ballooning, Mallory-Denk bodies, inflammation and fibrosis in cancer tissue, are recognized as steatohepatitic HCC (SH-HCC). This HCC phenotype is more frequently found in NAFL patients than in viral-hepatitic patients. (Purpose) The purpose of this study is to elucidate molecular features of SH-HCC with NAFL background. (Methods) To profile SH-HCC-related genes, mRNA expression patterns in SH-HCCs were compared with those in viral hepatitis HCCs by RNA array. RNA expression of the identified SH-HCC related gene was analyzed in three human liver carcinoma cell lines (HepG2, Huh7, and HLE) in normal conditioned medium. By mimicking the lipid-rich environment, these cell lines were also cultured in the presence of saturated (palmitic or stearic acid, recognized as endoplasmic reticulum stressors) or unsaturated fatty acids (oleic acid: OA). To address the role of SH-HCC-related gene, cell proliferation and apoptosis were assessed in the presence or absence of fatty acids, by knock-down of the objective gene using siRNA. (Results) (1) Caveolin (cav)-1 and -2 were identified as SH-HCC related genes (1.63 and 1.58 fold expression of cav-1 and -2, respectively) by RNA microarray analysis. (2) HLE and Huh7 showed high and low expression of both cav-1 and -2 mRNA, whereas HepG2 did not express either on qRT-PCR. (3) In the presence of OA, HepG2 and Huh7 did not well proliferate, whereas HLE cell proliferation increased with concentration of OA and culture duration. (4) Knock-down of cav-1 and -2 canceled OA-induced increase of cell proliferation. In the same condition, expression of cleaved caspase 3 was increased, suggesting the promotion of apoptosis. (Consideration) Cav-1 and -2, the important protein for endocytosis and cellular signaling, are highly expressed in SH-HCC. These proteins would increase cell proliferation and inhibit apoptosis in the presence of unsaturated fatty acids, presumably by using these fatty acids as energy source or by caveolae-related anti-apoptotic process. Citation Format: Makoto Takeda, Takanori Sakaguchi, Ryouta Kiuchi, Takanori Hiraide, Yasushi Shibasaki, Yoshihumi Morita, Hirotoshi Kikuchi, Mitsutoshi Setou, Hiroyuki Konno. Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4421.

MicroRNA profiles in various hepatocellular carcinoma cell lines.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC.

Selenoprotein R Protects Human Lens Epithelial Cells against D-Galactose-Induced Apoptosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress.

Selenium is an essential micronutrient for humans. Much of selenium’s beneficial influence on health is attributed to its presence within 25 selenoproteins. Selenoprotein R (SelR), known as methionine sulfoxide reductase B1 (MsrB1), is a selenium-dependent enzyme that, like other Msrs, is required for lens cell viability. In order to investigate the roles of SelR in protecting human lens epithelial (hLE) cells against damage, the influences of SelR gene knockdown on d-galactose-induced apoptosis in hLE cells were studied. The results showed that both d-galactose and SelR gene knockdown by siRNA independently induced oxidative stress. When SelR-gene-silenced hLE cells were exposed to d-galactose, glucose-regulated protein 78 (GRP78) protein level was further increased, mitochondrial membrane potential was significantly decreased and accompanied by a release of mitochondrial cytochrome c. At the same time, the apoptosis cells percentage and the caspase-3 activity were visibly elevated in hLE cells. These results suggested that SelR might protect hLE cell mitochondria and mitigating apoptosis in hLE cells against oxidative stress and endoplasmic reticulum (ER) stress induced by d-galactose, implying that selenium as a micronutrient may play important roles in hLE cells.

Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma.

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.

BackgroundCancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.MethodsThe human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.ResultsPoorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.ConclusionOur novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-722) contains supplementary material, which is available to authorized users.

A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion

LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-kappaB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kappaB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kappaB signaling, and increasing stability of the LZAP protein.

LAPTM4B‐35, a novel tetratransmembrane protein and its PPRP motif play critical roles in proliferation and metastatic potential of hepatocellular carcinoma cells

Lysosomal protein transmembrane 4 beta (LAPTM4B) was originally identified as a hepatocellular carcinoma (HCC)-associated gene. This gene and its protein product LAPTM4B-35, are both overexpressed in a variety of human cancers. However, its specific role in cell transformation and malignancy has remained elusive. In the present study we investigated the effects of LAPTM4B-35 overexpression on the malignant phenotypic features in the HLE cell line. Our data show that overexpression of LAPTM4B-35 promotes cell proliferation, exogenous growth-stimulating factor-independent and anchorage-independent growth, and enhances metastatic potential, including promotion of both cell migration and invasion. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included upregulation of proliferation-promoting transcription factors such as c-Myc, c-Jun, and c-Fos, and cell cycle-promoting proteins such as cyclin D1 and cyclin E. In addition, mutagenesis study showed that the PPRP motif in the N-terminal region of LAPTM4B-35 plays a critical role in promoting proliferation, migration, and invasion, as well as in the upregulation of the oncoproteins noted above. These data offer insight into the mechanism by which this novel tetratransmembrane protein contributes to the pathogenesis of liver cancer, and suggest that it may be a potential target for cancer therapy.

Establishment and some biological characteristics of human hepatoma cell lines.

Two cell lines of human hepatoma, HLE and HLF lines, were established in vitro from the hepatocellular carcinoma of a 68-year-old patient. One clone (HLEC) was obtained from a single HLE cell. The cells of HLE and HLEC were epithelial-like and both of these cells demonstrated glycogen granules in the cytoplasm when stained with periodic acid and Schiff reagent. Although HLF cells resembled fibroblasts in morphology, they appear to have originated from hepatoma cells, judging from epithelial characteristics in aggregates reconstituted by rotation culture and heterotransplantability. HLE cells produced alpha-fetoprotein until day 187 of culture, but HLF cells did not produce alpa-fetoprotein at any period examined. Chromosome number of both cell lines was distributed near the triploid range. HLF cells were transplantable into the cheek pouch of adult hamsters treated with cortisone acetate, but not HLE cells.

Overexpression of discoidin domain receptor 1 increases the migration and invasion of hepatocellular carcinoma cells in association with matrix metalloproteinase

The discoidin domain receptor (DDR) is a class of receptor tyrosine kinases that binds to several collagens. DDR1 is widely expressed in fast-growing invasive tumors of the breast, ovary, esophagus, brain and lung. However, there is little information on the expression of DDR1 in hepatocellular carcinoma (HCC) or its function in migration and invasion. Western blot analysis was performed to determine if four HCC cell lines (HLE, Huh-7, HepG2 and SH-J1) express DDR1. The HLE and Huh-7 cell lines were transfected with two isoforms of DDR1, DDR1a and DDR1b. Immunoprecipitation for DDR1 was then performed. Migration and invasion assays were carried out and the number of migrating cells was counted in 6 randomly selected fields per well under an optical microscope. Zymography was used to determine the level of the matrix metalloproteinase (MMP)-2 and -9 expression. DDR1 was expressed in all four cell lines. In the migration assay, the number of migrating cells was significantly higher in the DDR1a- or DDR1b-overexpressing HLE and Huh-7 cells, particularly after collagen type I stimulation (P<0.001). Collagen type I stimulation activated DDR1. In the invasion assay, there was a significantly higher number of invading cells in the DDR1a- or DDR1b-overexpressing HLE cells and DDR1a-overexpressing Huh-7 cells than in the control (P<0.01). The DDR1a- and DDR1b-overexpressing HLE cells showed a remarkable increase in the MMP-9 and -2 expression, particularly the active MMP-2. The DDR1a- and DDR1b-overexpressing Huh-7 cells showed a slight increase in the MMP-9 and -2 expression. The increased invasiveness of the HCC may be associated with the overexpression of either DDR1a or DDR1b mediated by MMP-2 and -9. Although this study provided one possible mechanism for the invasion of HCC cells, more studies are needed to understand the signal through which DDR1a and DDR1b act in invasion.

A peroxisome proliferator-activated receptor γ antagonist induces vimentin cleavage and inhibits invasion in high-grade hepatocellular carcinoma

Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor metastasis. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been intensively evaluated as a potential target for the inhibition of cell growth and metastasis in cancer cells. In the present study, we examined whether PPARgamma is a possible target molecule for the prevention of cell growth and invasion by treatment with agonists (troglitazone, rosiglitazone) and antagonists (T0070907, GW9662) in four different hepatocellular carcinoma (HCC) cell lines. We also evaluated the effects of the PPARgamma agonists and antagonists on tumor cell migration and invasion. The expression level of PPARgamma protein was higher in the sarcomatoid SH-J1 and poorly differentiated HLE cell lines than that in the well-differentiated HCC cell lines (HepG2 and Huh-7). Expression of vimentin was high in the SH-J1 HCC cell line and minimally detected in the HLE cell line. Treatment with low doses of the PPARgamma antagonists inhibited cell growth and colony formation of all four of the HCC cell lines. Vimentin in the high-grade HCC cells was cleaved by the treatment with the PPARgamma antagonists. Furthermore, treatment with the PPARgamma antagonists also strongly inhibited migration and invasion of the SH-J1 and HLE cells. However, treatment with low doses of the agonists had no effect on vimentin expression, migration, and invasion of the high-grade HCC cells but cell growth was inhibited by treatment with high concentrations of the agonists. Our results indicate that treatment with a PPARgamma antagonist may prevent cell growth and invasion of high-grade HCC cells. Our findings also suggest that PPARgamma antagonists inhibit cell growth and invasion through vimentin disarrangement in high-grade HCC.

Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry

To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE. Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide. The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da. MAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.