Metastatic Human Hepatocellular Carcinoma Cell Research Articles

MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2

BackgroundThis study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines.MethodsA decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration.ResultsmiR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo.ConclusionsIn summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

A multi-parallel N-glycopeptide enrichment strategy for high-throughput and in-depth mapping of the N-glycoproteome in metastatic human hepatocellular carcinoma cell lines

N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high-throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 μL as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.

Oncolytic vaccinia virus inhibits human hepatocellular carcinoma MHCC97-H cell proliferation via endoplasmic reticulum stress, autophagy and Wnt pathways.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy. Vaccinia virus (VV) possessed many inherent advantages with respect to being engineered as a vector for cancer gene therapy, although the mechanism of action remains to be explored further. We constructed a thymidine kinase gene insertional inactivated VV, named VV-Onco, and then tested its effects on cell viability, apoptosis and colony formation ability in a highly metastatic human hepatocellular carcinoma cell line MHCC97-H, and also investigated the potential cell signal pathways involved in this action. VV-Onco induced strong cytotoxicity and apoptosis and also inhibited the colony formation of MHCC97-H cells. The tumor cell apoptosis induced by VV-Onco is likely mediated via endoplasmic reticulum stress, autophagy and Wnt signaling pathways. The downregulation of survivin and c-Myc may also play a role in VV-Onco induced cell death. The results of the present study provide new insights into the mechanisms of VV-induced tumor cell death. The engineered recombinant VV containing optimized therapeutic transgenes may represent a new avenue for cancer gene therapy. Copyright © 2016 John Wiley & Sons, Ltd.

New isoindolinones from the fruiting bodies of Hericium erinaceum

Hericium erinaceus is a well-known medicinal and edible mushroom, which is considered as a potential source to obtain antitumor candidates. In this work, five new isoindolinones, named erinaceolactams A-E (1–5), along with five known compounds (6–10), were isolated from 70% ethanol extract of the fruiting bodies of H. erinaceus. The structures of new compounds were validated by HRESIMS and 1D, 2D NMR. It's worth mentioning that there are two pairs of isomers included in the new compounds. Moreover, their cytotoxicity against metastatic human hepatocellular carcinoma cell lines SMMC-7221 and MHCC-97H were evaluated. The results showed that compounds 6 and 7 exhibited promising inhibitory potency against the growth of two cell lines.

Glutamine Transporter Expression Profiling Reveal Major Role for ASCT2 and LAT1 in Primary and Metastatic Human Hepatocellular Carcinoma Cells

A concerted effort is underway to identify metabolic or physiological derangements in hepatocellular carcinoma (HCC) that may offer targets for therapy. While past work from our lab has documented ASCT2 as the major glutamine transporter in human HCC cells, little is known regarding differences in the array of transporters that operate in primary and metastatic HCC. To address this issue, fourteen human HCC cell lines (7 epithelial and 7 mesenchymal) were evaluated for glutamine transporter expression via reverse‐transcriptase – quantitative polymerase chain reaction (RT‐qPCR) and functional glutamine uptake analyses. The transporters screened via RT‐qPCR were: ASCT2 and LAT1, and six members of the SLC38 glutamine transporter family (System N and A transporters (SNATs)), including SNAT3 and SNAT5 – the dominant System N glutamine transporters in normal hepatocytes. All cell lines universally expressed ASCT2 and LAT1 as well as the SLC38 transporters SNAT2 and SNAT7. The System N transporters SNAT3 and SNAT5 were differentially expressed in some epithelial HCC, while the System A transporters SNAT1 and SNAT4 were variably expressed in both HCC cell types. Functional analysis revealed that the majority of glutamine in all HCC cell lines was transported by a mechanism consistent with ASCT2, independent of transporter mRNA expression signatures. Importantly, ASCT2 and LAT1 transporter activities and cognate mRNA and protein expression levels were discordant, highlighting the complex and layered regulation between gene expression and physiological activity. The results also support continued focus on ASCT2 and LAT1 as a primary therapeutic targets for both primary and metastatic HCC. Support: NIH Grant# R15CA108519 (BPB)

Transcriptome and proteome of human hepatocellular carcinoma reveal shared metastatic pathways with significant genes.

Previously isolated pathways screened from individual genes were investigated at either the transcriptional or translational level; however, the consistency between the pathways screened at the gene expression levels was obscure in metastatic human hepatocellular carcinoma (HCC). To elucidate this question, we performed a transcriptomic (16,353 genes) and proteomic (7861 proteins) analysis simultaneously on six metastatic HCC cell lines against two nonmetastatic HCC cell lines, with all HBV traceable and close genetic-backgrounds for a comparative study. The quantitative and integrated results showed that significant genes were screened differentially with 351 transcripts from the transcriptome and 304 proteins from the proteome, with limited overlapping genes (7%). However, we discovered that these discrete 351 transcripts and 304 proteins screened share extrusive significant-pathways/networks with a 77% overlap, including active TGF-β, RAS, NFκB, and Wnt, and inactive HNF4A, which are responsible for HCC metastasis. We conclude that the discrete, but significant genes predicted by either ome play intrinsically important roles in the linkage of responsible pathways shared by both omes in HCC metastasis.

The transcriptional profiling of glycogenes associated with hepatocellular carcinoma metastasis.

Background and objectiveMetastasis is one of the important reasons for the poor prognosis of hepatocellular carcinoma (HCC), abnormal glycosylation plays a pivotal role in HCC metastasis. The goal of this study was to screen and validate the transcriptional profiling of glycogenes associated with HCC metastasis.MethodologyThe differentially transcribed glycogenes were screened out by the Human Glycosylation RT2 Profiler PCR Array, and were identified by qRT-PCR in human HCC cell lines and their orthotopic xenograft tumors. Further analyses were performed with K-mean clustering, Gene Ontology (GO) and ingenuity pathways analysis (IPA). Four differentially transcribed glycogenes were validated in clinical cancer specimens by qRT-PCR.ResultsA total of thirty-three differentially transcribed glycogenes were obtained by comparison the transcription in the metastatic human HCC cell lines (MHCC97L, MHCC97H and HCCLM3) with the transcription in the non-metastatic HCC cell line Hep3B. Seven differentially transcribed glycogenes were selected to further identification in human HCC cell lines and their orthotopic xenograft tumors. According to their trends by K-mean clustering, all of the differentially transcribed glycogenes were classified in six clusters. GO analysis of the differentially transcribed glycogenes described them in biological process, subcellular location and molecular function. Furthermore, the partial regulatory network of the differentially transcribed glycogenes was acquired through the IPA. The transcription levels of galnt3, gcnt3, man1a1, mgat5b in non-metastatic and metastatic HCC clinical cancer specimens showed the same changing trends with the results in human HCC cell lines and their orthotopic xenograft tumors, and the divergent transcription levels of gcnt3 and mgat5b were statistically significant.ConclusionsThe transcriptional profiling of glycogenes associated with HCC metastasis was obtained and validated in this study and it might provide novel drug targets and potential biological markers for HCC metastasis.

MiR-503 regulates metastatic function through Rho guanine nucleotide exchanger factor 19 in hepatocellular carcinoma

BackgroundOur previous work described a metastasis-related microRNAs expression profiling and revealed miR-503 regulating metastatic function in hepatocellular carcinoma (HCC) cells. Here, we investigate to define the mechanism of miR-503 regulating metastasis in HCC. Materials and methodsThe expressions of miR-503 in HCC cell lines and clinical tissues with different metastatic potential were investigated. Meanwhile, a metastatic human HCC cell BALB/c nude mice model was used to investigate whether miR-503 regulates metastasis of HCC in vivo. Furthermore, luciferase activity of reporter gene, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescence-activated cell sorting analysis (FACS), and invasion assay were carried out to characterize the mechanism of miR-503 regulating metastasis in HCC. ResultsWe confirmed the negative correlation between miR-503 expression and metastatic potential of HCC in cell lines and in clinical HCC tissues. We also showed that overexpression of miR-503 resulted in inhibition of proliferation and metastasis of HCC in vivo. Furthermore, we demonstrated that ARHGEF19 is a direct target gene of miR-503. Finally, our results indicated that ARHGEF19 overcomes the suppressive influence of miR-503 in HCC cells. ConclusionsOur results suggest an important role of miR-503 in inhibiting metastasis of HCC through deregulating ARHGEF19.

Oridonin induces the apoptosis of metastatic hepatocellular carcinoma cells via a mitochondrial pathway

The selective induction of apoptosis is a promising strategy for cancer therapy. The antitumor effects of oridonin have been reported in several types of malignant tumors. However, the effects of oridonin on MHCC97-H cells, a highly metastatic human hepatocellular carcinoma cell line, have not been reported. The present study aimed to determine the effect of oridonin on the apoptosis of MHCC97-H cells and to identify the underlying molecular mechanisms that are involved. Compared with the untreated control cells, oridonin significantly decreased (P<0.05) cell proliferation in a concentration- and time-dependent manner. Oridonin at concentrations of 12.5, 25, 50 and 100 μM resulted in increased apoptotic Annexin V-positive and propidium iodide-negative cells by 9.5, 15.6, 22.2 and 31.7%, respectively, compared with the control groups (P<0.05). The mitochondrial membrane potential was significantly decreased by 6.0, 12.9, 18.9 and 27.1% in the MHCC97-H cells that were treated with oridonin at concentrations of 12.5, 25, 50 and 100 μM, respectively, for 24 h compared with the control groups (P<0.05). Oridonin increased the activity of caspase-3 and the expression of cleaved caspase-9 and cytochrome c in the cytoplasm and decreased the Bcl-2:Bax ratio in a concentration-dependent manner. The data indicate that oridonin inhibited the proliferation of the MHCC97-H cells by inducing apoptosis via a mitochondrial pathway. This mitochondrial pathway of apoptosis involved a reduction in the mitochondrial membrane potential and the subsequent release of cytochrome c and activation of caspase-3 and -9.

Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis.

Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells

Human hepatocellular carcinoma (HCC) ranks second in cancer mortality in China; recurrence and metastasis have been the cause of the high mortality. Glycans on the cell surface play a pivotal role in tumor metastasis. The global alteration in the structure and composition of N-glycans during HCC metastasis remains unknown. To understand glycan alterations of glycoproteins by correlating the glycosyltransferase expression profile with glycan structure, we systematically used glycan profiling tools: glycogene microarray analyses of 115 genes, including glycotransferases, glycosidases and nuclear sugar transporters and lectin chips to investigate the glycan-related gene expression signatures in the high metastatic potential HCC cell line, HCCLM3, in comparison to the HCC cell line, Hep3B, with low metastatic potential. Of the 115 genes, 18 genes were up-regulated in high metastatic potential HCCLM3 cells in comparison to Hep3B cells, while 11 genes were down-regulated. The differentially expressed genes, such as ST3GalI, FUT8, β3GalT5, MGAT3 and MGAT5, were mainly involved in the synthesis of N-glycan and glycolipids, particularly the sialyl Lewis antigen. The results of the glycogene microarray analysis were further validated by quantitative real-time PCR analysis and lectin-based analysis. The differentially expressed glycogenes identified in this study may provide new insights and represent novel factors for investigating the functional role of cell surface carbohydrate-mediated HCC metastasis.

Notch1‐Snail1‐E‐cadherin pathway in metastatic hepatocellular carcinoma

Notch signaling, a critical pathway for tissue development, also contributes to tumorigenesis in many cancers, but its pathological function in liver cancer is not well defined. In our study, Notch1 expression and its clinicopathological parameters were evaluated in 82 human hepatocellular carcinoma (HCC) patients. Plasmid-based siNotch1 shRNA was transiently or stably transfected into metastatic HCC cells and subsequently evaluated for the effects on orthotopic liver tumor metastasis in a mouse model as well as the effects on downstream pathways. Aberrant high expression of Notch1 was significantly associated with metastatic disease parameters in HCC patients, such as tumor-node-metastasis Stages III-IV and tumor venous invasion. Knocking-down Notch1 reduced cell motility in vitro and orthotopic tumor metastasis from the liver to the lung in vivo in a mouse model. In metastatic HCC cells, abnormal expression of Notch1 was associated with increased expression of Snail1 and repressed expression of E-cadherin; the Notch1-Snail1-E-cadherin association can also be found in HCC patient tumors. Inhibition of Notch1 by shRNA abolished Snail1 expression, which further resulted in the re-establishment of repressed E-cadherin in metastatic HCC cells. Thus, abnormal Notch1 expression was strongly associated with HCC metastatic disease, which might be mediated through the Notch1-Snail1-E-cadherin pathway. Knock-down of Notch1 reversed HCC tumor metastasis in a mouse model. Therefore, these data suggest that effective targeting of Notch signaling might also inhibit tumor metastasis.

Inhibition of glypican-3 expression via RNA interference influences the growth and invasive ability of the MHCC97-H human hepatocellular carcinoma cell line

Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is found to be overexpressed in hepatocellular carcinoma (HCC). The purpose of the present study was to investigate the possible role of GPC3 in the development of HCC. In this study, RNA interference (RNAi) with a GPC3 small hairpin RNA (GPC3 shRNA) was used to identify the effects of GPC3 on the regulation of malignant behaviors of HCC. MHCC97-H, a highly metastatic human HCC cell line in which GPC3 mRNA and protein levels were detected as the highest among the 4 HCC cell lines assessed in this study, and was thus selected as a cell model for in vitro and in vivo experiments. The results showed that down-regulation of GPC3 can significantly inhibit the proliferative and invasive ability of MHCC97-H. Compared with the parental HCC cells, GPC3-silenced cells exhibited attenuated capacities in developing tumors in nude mice, while the growth of tumor xenografts derived from these cells dramatically regressed. In conclusion, our results suggest that GPC3 contributes to the proliferation and metastasis of HCC and that it may be a potential molecular target for HCC treatment.

Clinicopathological Significance of ZEB1 Protein in Patients with Hepatocellular Carcinoma

ZEB1, a member of the ZFH family of proteins (zinc-finger E-box binding homeobox), plays a central role in epithelial-mesenchymal transition (EMT) during carcinogenesis. In this study, we investigated the expression of ZEB1 in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. Expression levels of ZEB1 were assessed by Western blot in 5 HCC cell lines and in paired cancerous and noncancerous tissues from 110 patients with HCC. Short-hairpin RNA (shRNA) interference for ZEB1 was performed in MHCC-97H cell line. ZEB1 protein was detected at a relatively high level in metastatic human HCC cell lines (MHCC-97L and MHCC-97H) when compared with that in nonmetastatic HCC cell lines (Hep3B, PLC and Huh-7). ZEB1 was expressed at high levels in 72 of 110 HCC patients (65.4%) and correlated with advanced TNM stage, tumor size >5 cm, intrahepatic metastasis, vascular invasion, and frequent early recurrence. The results of multivariate analysis revealed that ZEB1 high expression was a significant prognostic factor for poor overall and disease-free survivals. Silencing ZEB1 resulted in significant suppression of motility of MHCC-97H cell line, which was accompanied with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal markers N-cadherin and vimentin. Furthermore, silencing ZEB1 prevented the spread of intrahepatic metastasis and increased overall survival in mouse orthotopic tumor models. This study shows that ZEB1 high expression was correlated with HCC malignant progression and subsequent poor patient survival by induction of EMT changes.

Abstract 4236: Moscatilin inhibits invasion by suppressing urokinase plasminogen activator expression through Akt inactivation in human hepatocellular carcinoma cells

Abstract Purpose: Hepatocellular carcinoma (HCC) frequently shows early invasion into blood vessels as well as intrahepatic metastasis. The innovations of novel small-molecule agents to block HCC invasion and subsequent metastasis are urgently needed. Moscatilin is a bibenzyl derivative extracted from the stems of a traditional Chinese medicine, Orchid Dendrobrium loddigesii. Previous studies demonstrated that moscatilin exerted potent cytotoxic effects against numerous lines of cancer cells. Recently, moscatilin has been reported to suppress tumor angiogenesis. However, the anti-metastatic property of moscatilin has not been elucidated. Methods: Multiple molecular and pharmacological approaches such as invasion assay, MTT assay, ELISA assay, zymography, real-time PCR, Western blot, luciferase reporter gene assay, and chorioallantoic membrane (CAM) intravasation assay were used to determine the anti-metastatic effects of moscatilin in SK-Hep-1 cells, a highly metastatic human HCC cell line. Results: We found moscatilin inhibited cell invasion in a concentration-dependent manner in SK-Hep-1 cells in the absence of cytotoxicity. Moscatilin did not affect proteolytic activities of matrix metalloproteinase (MMP) -2 and MMP-9, but profoundly suppressed urokinase plasminogen activator (uPA) activity. We next found that moscatilin substantially inhibited protein expression of uPA but did not obviously impair uPAR and PAI-1 expression. To examine the upstream signaling of uPA suppression, the ERK1/2, Akt, mTOR, and NF-κB signaling pathways were evaluated in moscatilin-treated cells. We demonstrated that moscatilin dramatically inhibited Akt phosphorylation, but not ERK1/2 phosphorylation. Moscatilin did not reduce the phosphorylation of mTOR as well as of components of the translational machinery including p70S6K, 4E-BP1 and eIF4E. Additionally, IKKα/β phosphorylation, IκBα phosphorylation, p65 phosphorylation at Ser536, and κB-luciferase activity were not inhibited by moscatilin. However, Moscatilin repressed the expression of uPA mRNA, suggesting that moscatilin regulated the uPA production in transcriptional level. Transfection of constitutively active Akt (Myr-Akt) partly restored the moscatilin induced Akt inactivation. The inhibition of cell invasion, uPA activity and gene expression in response to moscatilin were also attenuated. Moreover, we found that moscatilin significantly inhibited cell metastasis using CAM intravasation assay, a well established in vivo model for metastasis. Conclusions: This study provides evidence that moscatilin induces Akt inactivation and uPA suppression, which leads to inhibition of invasion and metastasis in human HCC cells. Our results suggest that moscatilin is a novel anti-metastatic agent, which has great potential for further development for treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2011-4236

Lack of association between stretch‐activated and volume‐activated Cl− currents in hepatocellular carcinoma cells

Stretch-activated chloride currents (I(Cl,SA) ) have been considered to be a component of volume-activated chloride currents (I(Cl,vol) ) for some time. This is due to a similarity in biophysical and pharmacological properties that involve a membrane curvature-induced mechanism and rearrangement of the cytoskeleton induced by cell swelling or membrane stretch. In the present study, we demonstrated that current density, along with the time taken from the activation of currents to the peak, were significantly different between the two currents, in highly metastatic human hepatocellular carcinoma cells. In addition, the activation of I(Cl,vol) or I(Cl,SA), induced maximally by hypotonic solutions or membrane stretch, respectively, did not affect the following activation of the other one. Moreover, neither inhibition of I(Cl,vol) by sh-ClC-3 transfection, nor functional blocking of I(Cl,vol) by intracellular dialysis of anti-ClC-3 antibody had an effect on the activation and properties of I(Cl,SA). Collectively, our results suggest that I(Cl,SA) is different from I(Cl,vol) in activation mechanism and/or in molecular entity responsible for formation of the currents. ClC-3 is involved in the activation of I(Cl,vol), but not of I(Cl,SA).

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

BackgroundFibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved.MethodsThe MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively.ResultsBoth of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity.ConclusionsPolypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Effects of high metastatic potential hepatocellular carcinoma cell-binding peptide on the invasion and metastasis of liver cancer

To evaluate the effects of specific peptide (AWYPLPP peptide) binding to high metastatic potential human hepatocellular carcinoma (HCC) cells on the invasion and metastasis of liver cancer. The effects of AWYPLPP peptide on the invasion, migration, proliferation and adhesion of high metastatic potential human HCC cell line (HCCLM3) were evaluated in vitro by Matrigel invasion assay, migration assay, MTT assay and adhesion assay. The effect of AWYPLPP peptide on lung metastasis of HCC in vivo was evaluated in male nude mice with subcutaneously implanted HCCLM3 cells. Incubation with the AWYPLPP peptide, but not the control peptide, resulted in a concentration-dependent increase of invasion ability in HCCLM3 cells at the concentration of 0.1 to 100 micromol/L. At any concentration used for the invasion assay, the peptide had no effect on cell migration, proliferation and adhesion. After 30 days of transplantation, eight of nine (88.9%) mice in the AWYPLPP peptide group showed obvious lung metastasis. The metastatic rate of lung metastasis was significantly increased in the AWYPLPP peptide group compared with that in the control group. There was no significant difference among the weights of primary tumor in the PBS, control peptide and AWYPLPP peptide groups. AWYPLPP peptide can promote in vitro invasion and in vivo lung metastasis of high metastatic potential human HCC cells. Identification of the receptor for AWYPLPP peptide binding may provide new insights into the molecular mechanism underlying HCC invasion and metastasis as well as new targets for intervention.

Cytokeratin 10 and Cytokeratin 19: Predictive Markers for Poor Prognosis in Hepatocellular Carcinoma Patients after Curative Resection

Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of alpha-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10-/CK19- patients (56.1% and 60.0%, respectively; P < 0.001). CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.

A novel peptide that selectively binds highly metastatic hepatocellular carcinoma cell surface is related to invasion and metastasis

Using a phage display approach, we identified AWYPLPP peptide as a specific peptide ligand that binds to the cell surface of highly metastatic human hepatocellular carcinoma (HCC). Moreover, the peptide was able to promote in vitro invasion of highly metastatic HCC cells by activating matrix metalloproteinase-9 and in vivo lung metastasis of HCC tumors. These results indicate that AWYPLPP peptide likely recognizes a novel receptor that is selectively expressed on the cell surface of highly metastatic HCC and mediates cellular activities associated with the invasive phenotype. Identification of the receptor for the AWYPLPP peptide may provide new insights into the molecular mechanism of HCC metastasis.