Potential Of Hepatocellular Carcinoma Cells Research Articles

B-Cell Receptor-Associated Protein 31 Promotes Metastasis via AKT/β-Catenin/Snail Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal cancer worldwide, characterized with high heterogeneity and inclination to metastasize. Emerging evidence suggests that BAP31 gets involved in cancer progression with different kinds. It still remains unknown whether and how BAP31 plays a role in HCC metastasis. Epithelial–mesenchymal transition (EMT) has been a common feature in tumor micro-environment, whose inducer TGF-β increased BAP31 expression in this research. Elevated expression of BAP31 was positively correlated with tumor size, vascular invasion and poor prognosis in human HCC. Ectopic expression of BAP31 promoted cell migration and invasion while BAP31 knockdown markedly attenuated metastatic potential in HCC cells and mice orthotopic xenografts. BAP31 induced EMT process, and enhanced the expression level of EMT-related factor Snail and decreased contents and membrane distribution of E-cadherin. BAP31 also activated AKT/β-catenin pathway, which mediated its promotional effects on HCC metastasis. AKT inhibitor further counteracted the activated AKT/β-catenin/Snail upon BAP31 over-expression. Moreover, silencing Snail in BAP31-overexpressed cells impaired enhanced migratory and invasive abilities of HCC cells. In HCC tissues, BAP31 expression was positively associated with Snail. In conclusion, BAP31 promotes HCC metastasis by activating AKT/β-catenin/Snail pathway. Thus, our study implicates BAP31 as potential prognostic biomarker, and provides valuable information for HCC prognosis and treatment.

Annexin A7 and JNK knockdown suppress the lymphatic metastasis potential of hepatocellular carcinoma cells: Possible contributions of ATF2 and sequence-related lncRNA NONMMUT114121.1.

BackgroundOur previous studies identified the calcium-dependent phospholipid binding protein Annexin A7 (ANXA7) as a critical mediator of hepatocellular carcinoma (HCC) lymph node metastasis (LNM) in mice, possibly through c-Jun N-terminal kinase (JNK) signaling. Activating transcription factor-2 (ATF2) is a downstream target of JNK, so we examined if modulation of LNM capacity by ANXA7 and JNK is associated with changes in ATF2 activity and its sequence-related long non-coding (lnc)RNA NONMMUT114121.1.MethodsThe effects of shRNA-induced ANXA7 and JNK knockdown on HCC cell transwell invasion, HCC cell lymphatic tissue adherence, ATF2 mRNA and protein expression, and ATF2 subcellular distribution were examined in the murine HCC cell line Hca-P.ResultsInvasive capacity, lymphoid adhesion, and ATF2 expression at both mRNA and protein levels were significantly reduced by ANXA7 or JNK knockdown (all P<0.05). Immunofluorescence revealed that ATF2 was mainly localized to the nucleus of control Hca-P cells, but this nuclear expression was markedly reduced by ANXA7 or JNK knockdown. LncRNA NONMMUT114121.1 was associated with ATF2 by sequencing and its expression level was significantly increased by ANXA7 knockdown (P<0.05).ConclusionsJNK and ANXA7 promote the invasive capacity and lymphatic adherence of Hca-P cells, possibly through ATF2 activation and its related lncRNA NONMMUT114121.1. Nuclear ATF2 may thus be a potential diagnostic and/or prognostic biomarker for HCC.

Sublethal irradiation promotes the metastatic potential of hepatocellular carcinoma cells.

Radiotherapy (RT) represents one of the major treatment methods for cancers. However, many studies have observed that in descendant surviving tumor cells, sublethal irradiation can promote metastatic ability, which is closely related to the tumor microenvironment. We therefore investigated the functions and mechanisms of sublethal irradiated liver nonparenchymal cells (NPCs) in hepatocellular carcinoma (HCC). In this study, primary rat NPCs and McA‐RH7777 hepatoma cells were irradiated with 6 Gy X‐ray. Conditioned media (CM) from nonirradiated (SnonR), irradiated (SR), or irradiated plus radiosensitizer celecoxib‐treated (S[R + D]) NPCs were collected and added to sublethal irradiated McA‐RH7777 cells. We showed that CM from sublethal irradiated NPCs significantly promoted the migration and invasion ability of sublethal irradiated McA‐RH7777 cells, which was reversed by celecoxib. The differentially expressed genes in differently treated McA‐RH7777 cells were enriched mostly in the AMP‐activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. SR increased the migration and invasion ability of HCC cells by inhibiting AMPK/mTOR signaling, which was enhanced by the AMPK inhibitor compound C and blocked by the AMPK activator GSK‐621. Analyses of HCC tissues after neoadjuvant radiotherapy confirmed the effects of radiation on the AMPK/mTOR pathway. Cytokine antibody arrays and further functional investigations showed that matrix metalloproteinase‐8 (MMP‐8) partly mediates the promotion effects of SR on the migration and invasion ability of HCC cells by regulating AMPK/mTOR signaling. In summary, our data indicate that MMP‐8 secreted by irradiated NPCs enhanced the migration and invasion of HCC by regulating AMPK/mTOR signaling, revealing a novel mechanism mediating sublethal irradiation–induced HCC metastasis at the level of the tumor microenvironment.

Mongolian Chelidonium majus Suppresses Metastatic Potential of Hepatocellular Carcinoma Cells through TIMP Up-regulation and MMP Down-regulation

In this study, the antimetastatic effect of Chelidonium majus ethanol extract on hepatocellular carcinoma in vitro was investigated. The viability of SK-Hep1 and normal liver cells was determined using a cell counting kit-8 assay. Wound healing assays were performed to investigate SK-Hep1 cell migration and various metastatic characteristics including adhesion, aggregation and invasion were also measured using these cells. Furthermore, the proteolytic activity of extracellular matrix metalloproteinase-9 was measured using gelatin zymography. Expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, membrane type 1-matrix metalloproteinase and tissue inhibitor of metalloproteinase-1 were measured using reverse transcription-polymerase chain reaction and Western blotting. Chelidonium majus ethanol extract significantly inhibited the proliferation of SK-Hep1 hepatocellular carcinoma cells in a dose-dependent manner. Moreover, metastatic characteristics including adhesion, migration, aggregation and invasion were significantly suppressed by Chelidonium majus ethanol extract treatment. Further, this preparation downregulated the expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and membrane type 1-matrix metalloproteinase, but upregulated tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2, in a dose-dependent manner. Additionally, the proteolytic activity of matrix metalloproteinase-9 was greatly diminished with 400 µg/ml of Chelidonium majus ethanol extract. Taken together, it was suggested that Chelidonium majus ethanol extract might exert an antimetastatic effect on hepatocellular carcinoma cells by inhibiting proliferation, adhesion, migration, aggregation and invasion through the downregulation of matrix metalloproteinases and upregulation of tissue inhibitor of metalloproteinases. Thus, this extract could represent a promising therapeutic agent for this disease.

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing. Key words: Whole exome sequencing; Hepatocellular carcinoma; Mutations; Metastasis

Mining featured biomarkers associated with vascular invasion in HCC by bioinformatics analysis with TCGA RNA sequencing data

This study aims to identify the feature genes associated with vascular invasion in hepatocellular carcinoma (HCC). Here, the RNA sequencing data related to vascular invasion in The Cancer Genome Atlas (TCGA) database, including 292 HCC patients with complete clinical data were included in our study as the training dataset for construction and E-TABM-36, including 41 HCC patients with complete clinical data was used as the validation dataset. Following data normalization, differentially expressed mRNA and copy number (CN) were selected between with and without vascular invasion samples. A support vector machine (SVM) classifier was constructed and validated in GSE9828 and GSE20017 datasets. Total 59 feature genes were found by the SVM classifier. Using Cox regression analysis, three clinical features, including Patholigic T, Stage and vascular invasion and 6 optimal prognostic genes, including ANO1, EPHX2, GFRA1, OLFM2, SERPINA10 and TKT were significantly correlated with prognosis. A risk score formula was developed to assess the prognostic value of 6 optimal prognostic genes, which were identified to possess the most remarkable correlation with overall survival in HCC patients. By performing in vitro experiments, we observed TKT was significantly increased, but OLFM2 was decreased in high metastatic potential HCC cell lines (SK-HEP-1 and MHCC-97 H) compared with low metastatic potential cell line Huh7 and normal human liver cell line LO2 using western blotting analysis. Knockdown of TKT in MHCC-97H or overexpression of OLFM2 in SK-HEP-1 significantly suppressed cell migration and invasion using transwell assays. Our results demonstrated that TKT and OLFM2 might be novel independent biomarkers for predicting survival based on the presence of vascular invasion in patients with HCC.

Long non-coding RNA SNHG1 functions as a competitive endogenous RNA to regulate PDCD4 expression by sponging miR-195-5p in hepatocellular carcinoma

Long non-coding RNA (lncRNA) potentially regulates tumorigenesis. LncRNA small nucleolar RNA host gene 1 (SNHG1) expression remains high in hepatocellular carcinoma cells; however, its biological mechanism in hepatocellular carcinoma remains unknown. In this study, SNHG1 expression in hepatocellular carcinoma cells was detected by qRT-PCR. Proliferative and migratory potentials of hepatocellular carcinoma cells were determined by CCK-8 and Transwell assay, respectively. Then, the nude mice model of xenograft was employed to verify the effect of SNHG1 on tumor formation in vivo. We identified the potential target of SNHG1 through bioinformatics and dual-luciferase reporter gene. Furthermore, Western blot and RIP assay was used for clarifying their interaction and functions in regulating the development of hepatocellular carcinoma. Our results indicated a high expression of SNHG1 in hepatocellular carcinoma cells. Downregulation of SNHG1 inhibited proliferative and migratory potentials of hepatocellular carcinoma cells in vitro and in vivo. Moreover, the expression of programmed cell death 4 (PDCD4) was positively regulated by SNHG1 through competing with miR-195-5p. These results indicated that SNHG1 participated in the development of hepatocellular carcinoma as a ceRNA to competitively bind to miR-195-5p and thus mediate PDCD4 expression.

Plasminogen activator inhibitor 2 (PAI2) inhibits invasive potential of hepatocellular carcinoma cells in vitro via uPA- and RB/E2F1-related mechanisms

Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues. First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining. The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival. Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

SET7/9 promotes hepatocellular carcinoma progression through regulation of E2F1.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide. Histone-lysine N-methyltransferase SET7/9 is a protein lysine monomethylase that methylates histone H3K4 as well as various non-histone proteins. Deregulation of SET7/9 is frequently detected in human cancers. However, the role of SET7/9 in HCC development remains unclear. In the present study, upregulation of SET7/9 and E2F transcription factor 1 (E2F1) expression was detected in 68 samples of HCC tissues compared with these levels noted in the paired healthy liver samples. The expression levels of SET7/9 and E2F1 were significantly correlated with pathological stage and tumor size. Subcellular fractionation and co-immunoprecipitation analyses revealed protein-protein interaction between SET7/9 and E2F1 in the cytoplasm of HCC cells. Silencing of SET7/9, as well as treatment with 5′-deoxy-5′-methylthioadenosine (MTA), a protein methylation inhibitor, led to reduced E2F1 protein abundance in HCC cells. Using Cell Counting Kit-8 (CCK-8) assay, Transwell migration assay and wound healing assay, significantly decreased cell proliferation, migration and invasion were observed in cells exhibiting downregulation of SET7/9 and E2F1 expression, as well as in wild-type HCC cells treated with MTA. Furthermore, SET7/9 downregulation and MTA treatment resulted in reduced expression of downstream targets of E2F1, including cyclin A2, cyclin E1 and CDK2. In conclusion, the present study revealed an oncogenic function of SET7/9 in HCC and demonstrated that SET7/9 may be responsible for alterations in the proliferative ability, aggressiveness and invasive/metastatic potential of HCC cells through post-translational regulation of E2F1.

Abstract 4479: Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma

Abstract Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently down-regulated in hepatocellular carcinoma (HCC) cells and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis and therapy resistance potential of HCC cells. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a DEN+CCL4 HCC induced PRMT6-/- mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and that PRMT6 interacted with CRAF, and likely other RAF family members, and their methylation at conserved arginine 100, negatively regulating its activity, and as a consequence resulting in enhanced MEK/ERK signaling. Our work uncovered a critical repressive function for PRMT6 in maintenance of HCC cells by regulating the MEK/ERK pathway via arginine methylation of RAF, providing a new avenue of molecular mechanism by which ERK mediated stemness in HCC cells are developed. Citation Format: LH Chan, L Zhou, Kai Yu Ng, TL Wong, TK Lee, YP Ching, YF Yuan, D Xie, S Richard, MS Huen, XY Guan, S Ma. Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4479.

T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

Recurrence and metastasis are major challenges in the management of hepatocellular carcinoma (HCC) patients after resection. To identify a metastasis-associated gene signature, we performed comparative gene expression analysis with recurrent HCC tissues from HCC patients who underwent partial or total hepatectomy and from non-metastatic primary HCC tissues. From this, we were able to identify genes associated with HCC recurrence. TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed genes in patients with recurrent HCC who had undergone total hepatectomy. The significant overexpression of TCIRG1 was confirmed using the Liver Hepatocellular Carcinoma dataset from The Cancer Genome Atlas. High expression of TCIRG1 was significantly associated with poor 5-year disease-free and recurrence-free survival of HCC patients. TCIRG1 knockdown suppressed tumor cell growth and proliferation in HCC cell lines; caused a significant increase in the proportion of cells in the G1/S phase of cell cycle; induced cell death; suppressed the metastatic potential of HCC cells by selectively regulating the epithelial–mesenchymal transition (EMT) regulatory proteins E-cadherin, N-cadherin, Fibronectin, Snail and Slug; and significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. These findings suggest that TCIRG1 functions as a metastatic enhancer by modulating growth, death and EMT in HCC cells. TCIRG1 could be a therapeutic target for the treatment of liver malignancy and metastasis.

Up-regulation of brain-expressed X-linked 2 is critical for hepatitis B virus X protein-induced hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Chronic hepatitis B virus (HBV) infection is a major cause for HCC. Hepatitis B virus X (HBx), one of four proteins encoded by HBV genome, plays a vital role in the pathogenesis of HBV-induced HCC. However, the molecular mechanisms of HBx-triggered HCC remain largely undetermined. Here we revealed that the expression of Brain-expressed X-linked 2 (BEX2) and Osteopontin (OPN) were elevated in liver tissues of HBV transgenic mice and human HCC specimens. Moreover, a positive correlation between BEX2 and OPN was exhibited in samples from HCC patients with HBV infection. The protein levels of BEX2 and OPN were both higher in HBV-positive HCC specimens compared to that of HBV-negative HCC specimens. HBx potentiated OPN expression through up-regulation of BEX2. Importantly, the depletion of BEX2 suppressed tumorigenic potential of HCC cells with highly expressed HBx. We demonstrated the important role of BEX2 in HCC pathogenesis, and BEX2 may be a novel therapeutic target for HCC patients with HBV infection. The newly identified HBx/BEX2/OPN signaling cassette is implicated in the pathogenesis of HBV-induced HCC.

Abstract 3829: TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis

Abstract Recurrence and metastasis are a major challenge in the management of hepatocellular carcinoma (HCC) patients after HCC resection. In order to identify metastatic molecular signature, we performed comparative gene expression profiling analysis with recurred HCC tissues from HCC patients who underwent partial or total hepatectomy and non-metastatic primary HCC. From this, we were able to recapitulate molecular signatures associate with HCC recurrence, and found that TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed gene elements in recurred HCC patients with total hepatectomy. We then observed that TCIRG1 is significantly overexpressed in TCGA-LIHC (Liver Hepatocellular Carcinoma Dataset of The Cancer Genome Atlas), and high expression of TCIRG1 was significantly associated with poor prognosis of HCC patients in 5-year disease-free. TCIRG1 knockdown suppressed tumor cell growth and proliferation in Huh7 and SNU475, liver cancer cell lines. From flow cytometric analysis for cell cycle analysis and cell death, we observed that TCIRG1 knockdown caused a significant increase of G1/S phase, and also induced programmed cell death processing in liver cancer cells. TCIRG1 knockdown also reduced the metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins, E-cadherin, N-cadherin, Vimentin, Fibronectin, Snail and Slug, in liver cancer cells. In addition, sustained expression of TCIRG1 in liver cancer cell line reduced tumor growth rate in mouse xenograft model. Moreover, targeted-disruption of TCIRG1 significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. In conclusion, our findings suggest that TCIRG1 functions as metastatic enhancing gene by modulating cellular growth, death, and EMT in liver cancer cells providing TCIRG1 as a therapeutic target for the treatment of liver malignancy and metastasis. Citation Format: Hee Doo Yang, Jung Woo Eun, Qingyu Shen, Hyung Seok Kim, Sang Yean Kim, Suk Woo Nam. TCIRG1, T-cell immune regulator 1, functions as a metastatic enhancing gene in liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2017-3829

Huaier Restrains Proliferative and Migratory Potential of Hepatocellular Carcinoma Cells Partially Through Decreased Yes-Associated Protein 1.

In China, Trametes robiniophila Murr (Huaier), a traditional Chinese herbal medicine, has been widely used in adjuvant therapies of hepatocellular carcinoma (HCC). However, the molecular mechanisms have not been fully understood. The aims of this study are to investigate the functions and mechanisms of Huaier on inhibiting proliferation and migration of HCC cells. Firstly, cell counting kit-8 (CCK-8) and colony formation shown Huaier inhibited proliferation of HCC Bel-7404, Bel-7402 and SMMC-7721 cells in a dose-dependent manner, and this inhibition might be due to Huaier decreased the expressions of the proliferating cell nuclear antigen (PCNA), the nuclear proliferation related antigen (Ki-67) and CyclinD1 detected by western blotting analysis. Notably, we also found Huaier treatment did not cause any cytotoxicity to normal human hepatocyte L-02 cells. Next, we found Huaier dose-dependently decreased Bcl-2 expression and increased Bax expression in Bel-7404 cells. The activities of cleaved caspase substrates had also been enhanced after Huaier treatment, suggesting Huaier treatment could induce HCC cell apoptosis. Then, the inhibitory effects of Huaier on migration of Bel-7404, Bel-7402 and SMMC-7721 cells via inhibiting Epithelial mesenchymal transition (EMT) had also been proved. Moreover, we confirmed yes-associated protein 1 (YAP1) was up-regulated in HCC cells and tissues, and overexpression of YAP1 promoted HCC cell proliferation and migration. Then, western blot and immunefluorescence shown Huaier had the inhibitory effects on YAP1 in HCC cells. On the other hand, human liver cancer tissue microarray (TMA) shown YAP1 expression was closely to clinic. Our study also confirmed Huaier had the inhibitory effects on YAP1 in xenograft mice models, it could be because Huaier treatment translocated YAP1 from nucleus to cytoplasm, and further promoted phosphorylation of YAP1 to be degraded by ubiquitination. Hence, we conclude that Huaier may restrain the proliferation and migration of HCC cells via down-regulation of YAP1. In summary, our study reveals the potential mechanisms of Huaier on inhibiting proliferation and migration of HCC cells. Importantly, for the first time, we found that Huaier can inhibit YAP1 expression in this anti-tumor process. We believe this finding is beneficial for the clinical applications of Huaier and the targeted therapies for HCC.

The Chinese medicine Sini-San inhibits HBx-induced migration and invasiveness of human hepatocellular carcinoma cells.

BackgroundSini-San (SNS) is a formulation of four Traditional Chinese Drugs that exhibits beneficial therapeutic effects in liver injury and hepatitis. However, there are no reports describing its effects on the hepatitis B X-protein (HBx)-induced invasion and metastasis in hepatoma cells, and the detailed molecular mechanisms of its actions are still unclear.MethodsIn this study, we investigated the mechanisms underlying SNS-mediated inhibition of HBx-induced cell invasion and the inhibition of secreted and cytosolic MMP-9 production, using gelatin zymography and Western blot analysis in a human hepatoma cell line (HepG2). Relative luciferase activity was assessed for MMP-9, NF-κB, or AP-1 reporter plasmid-transfected cells.ResultsSNS suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-κ B (NF-κB) activity. SNS suppressed HBx-induced AP-1 activity through inhibition of phosphorylation in the extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. SNS also suppressed HBx-induced inhibition of NF-κB nuclear translocation through IκB and suppressed HBx-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-κB and AP-1.ConclusionsSNS suppresses the invasiveness and metastatic potential of hepatocellular carcinoma cells by inhibiting multiple signal transduction pathways.

Long non-coding RNAs are differentially expressed in hepatocellular carcinoma cell lines with differing metastatic potential.

Metastasis is a major reason for poor prognosis in patients with cancer, including hepatocellular carcinoma (HCC). A salient feature is the ability of cancer cells to colonize different organs. Long non-coding RNAs (lncRNAs) play important roles in numerous cellular processes, including metastasis. In this study, the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2) were assessed using the Arraystar Human LncRNA Array v2.0, which contains 33,045 lncRNAs and 30,215 mRNAs. Coding-non-coding gene co-expression (CNC) networks were constructed and gene set enrichment analysis (GSEA) was performed to identify lncRNAs with potential functions in organ-specific metastasis. Levels of two representative lncRNAs and one representative mRNA, RP5-1014O16.1, lincRNA-TSPAN8 and TSPAN8, were further detected in HCC cell lines with differing metastasis potential by qRT-PCR. Using microarray data, we identified 1,482 lncRNAs and 1,629 mRNAs that were differentially expressed (≥1.5 fold-change) between the two HCC cell lines. The most upregulated lncRNAs in H2 were RP11-672F9.1, RP5-1014O16.1, and RP11-501G6.1, while the most downregulated ones were lincRNA-TSPAN8, lincRNA-CALCA, C14orf132, NCRNA00173, and CR613944. The most upregulated mRNAs in H2 were C15orf48, PSG2, and PSG8, while the most downregulated ones were CALCB, CD81, CD24, TSPAN8, and SOST. Among them, lincRNA-TSPAN8 and TSPAN8 were found highly expressed in high lung metastatic potential HCC cells, while lowly expressed in no or low lung metastatic potential HCC cells. RP5- 1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines, while lowly expressed in no lymphatic metastatic potential HCC cell lines. We provide the first detailed description of lncRNA expression profiles related to organ-specific metastasis in HCC. We demonstrated that a large number of lncRNAs may play important roles in driving HCC cells to metastasize to different sites; these lncRNAs may provide novel molecular biomarkers and offer a new basis for combating metastasis in HCC cases.

Incomplete radiofrequency ablation enhances invasiveness and metastasis of residual cancer of hepatocellular carcinoma cell HCCLM3 via activating β-catenin signaling.

BackgroundRadiofrequency ablation (RFA) is one of the curative therapies for hepatocellular carcinoma (HCC), however, accelerated progression of residual HCC after incomplete RFA has been reported more frequently. The underlying molecular mechanism of this phenomenon remains to be elucidated. In this study, we used an incomplete RFA orthotopic HCC nude mouse model to study the invasive and metastatic potential of residual cancer as well as the correlated mechanism.MethodsThe incomplete RFA orthotopic nude mouse models were established using high metastatic potential HCC cell line HCCLM3 and low metastatic potential HCC cell line HepG2, respectively. The changes in cellular morphology, motility, metastasis and epithelial–mesenchymal transition (EMT), and HCC cell molecular markers after in vitro and in vivo incomplete RFA intervention were observed.ResultsPulmonary and intraperitoneal metastasis were observed in an in vivo study. The underlying pro-invasive mechanism of incomplete RFA appeared to be associated with promoting EMT, including down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. These results were in accordance with the in vitro response of HCC cells to heat intervention. Further studies demonstrated that β-catenin was a pivotal factor during this course and blocking β-catenin reduced metastasis and EMT phenotype changes in heat-treated HCCLM3 cells in vitro.ConclusionIncomplete RFA enhanced the invasive and metastatic potential of residual cancer, accompanying with EMT-like phenotype changes by activating β-catenin signaling in HCCLM3 cells.

Inhibition of TGF-β/SMAD3/NF-κB signaling by microRNA-491 is involved in arsenic trioxide-induced anti-angiogenesis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of metastasis and recurrence, which are primarily attributed to the angiogenesis. So, the anti-angiogenesis treatment has become the new approach for HCC therapy. In addition to treating leukemia, arsenic trioxide (As2O3) also suppresses other solid tumors, including HCC. However, the roles of As2O3 in the angiogenesis potential of HCC cells remain unclear. In our present study, As2O3 attenuated the angiogenic ability by the microRNA-491 (miR-491)-mediated inhibition of TGF-β/SMAD3/NF-κB signal pathway in MHCC97H and MHCC97L cells. Briefly, in these cells, As2O3 improved the expression of miR-491 via DNA-demethylation; miR-491, which targeted the SMAD3-3′-UTR, decreased the expression/function of SMAD3, leading to the inactivation of NF-κB/IL-6/STAT-3 signaling; knockdown of miR-491 abolished the As2O3-induced inhibitions of the TGF-β/SMAD3/NF-κB pathway, the VEGF secretion, and the angiogenesis. By understanding a novel mechanism whereby As2O3 inhibits the angiogenic potential in HCC cells, our study would help in the design of future strategies of developing As2O3 as a potential chemopreventive agent when used alone or in combination with other current anticancer drugs.

The effects of the CCR6/CCL20 biological axis on the invasion and metastasis of hepatocellular carcinoma.

Chemokines and their receptors have recently been shown to play major roles in cancer metastasis. Chemokine receptor 6 (CCR6) and its ligand, CCL20, were highly expressed in a variety of human cancers. In our present study, we aimed to clarify whether CCR6/CCL20 was correlated with the migration of hepatocellular carcinoma (HCC). RT-PCR and Western blot results showed that CCR6 was overexpressed in different invasive potential HCC cell lines (p < 0.05), while the expression of CCL20 had no obvious difference (p > 0.05). CCR6 was suppressed by siRNA in HCCLM6, and then the biological behaviors of HCCLM6 cells were observed. The results showed that the CCR6/CCL20 biological axis increased the capacity of proliferation and adhesion, as well as the chemotactic migration and the level of cytokines related to degraded extracellular matrix. In conclusion, these findings indicate that CCR6 indeed participates in regulating the migration and invasion of HCC, and it might become a prognostic factor of HCC.

Platycodin D Induces Apoptosis, and Inhibits Adhesion, Migration and Invasion in HepG2 Hepatocellular Carcinoma Cells

Platycodin D (PD), a triterpenoid saponin isolated from the Chinese medicinal herb Platycodonis radix, possesses anti-cancer effects in several cancer cell lines. The aim of this study was to evaluate its anti- cancer activities in hepatocellular carcinoma cells. MTT and colony formation assays were performed to evaluate cell proliferation, along with flow cytometry and Western blotting for apoptosis. Cell adhesion was tested by observing cellular morphology under a microscope, while the transwell assay was employed to investigate the cell migration and invasion. PD concentration-dependently inhibited cell proliferation in both HepG2 and Hep3B cells, and significantly suppressed colony formation and induced apoptosis in HepG2 cells. The protein levels of cleaved poly ADP-ribose polymerase (PARP) and Bax were up-regulated while that of survivin was down-regulated after treatment with PD. Moreover, PD not only obviously suppressed the adhesion of HepG2 cells to Matrigel, but also remarkably depressed their migration and invasion induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). PD presents anti-cancer potential in hepatocellular carcinoma cells via inducing apoptosis, and inhibiting cell adhesion, migration and invasion, indicating promising features as a lead compound for anti-cancer agent development.