Migration Of Hep3B Cells Research Articles

ASTAXANTHIN INHIBITS CELL PROLIFERATION, MIGRATION, INVASION AND INDUCED APOPTOSIS VIA AMPK-mTOR SIGNALING PATHWAY IN HEPATOCELLULAR CARCINOMA HEP 3B CELLS

"Hepatocellular carcinoma (HCC) is a fatal malignancy with a poor prognosis. There is an urgent need to study the molecular mechanisms of HCC development and explore potential drugs to improve survival. This study aims to investigate the antitumor effects of astaxanthin on HCC proliferation, migration, invasion and apoptosis through regulation of adenosine-activated protein kinase (AMPK). CCK8, wound healing, transwell and flow cytometry assays were used to evaluate Hep 3B cell viability, migration, invasion and apoptosis after astaxanthin treatment. Protein expression was determined by Western blot. CCK8 assays showed that all concentrations (200 μM, 400 μM, 500 μM) of astaxanthin used in this study significantly inhibited the proliferation of Hep 3B cells (P < 0.05). Wound healing, transwell and flow cytometry showed that astaxanthin inhibited Hep 3B cell migration and invasion and induced apoptosis. Western blot showed that astaxanthin increased the expression of p-AMPK (P <0.05) and inhibited its downstream p-mTOR protein expression (P < 0.05). The effects of astaxanthin on cell proliferation, migration, invasion and apoptosis was attenuated after addition of the AMPK blocker Compound C. To conclude, astaxanthin inhibits the proliferation, migration and invasion of HCC by regulating AMPK, and promotes tumor cell apoptosis in a dose-dependent manner."

Transcriptome analysis revealed the role of mTOR and MAPK signaling pathways in the white strain of Hypsizygus marmoreus extracts-induced cell death of human hepatoma Hep3B cells.

The aim of this study was to investigate the anticancer mechanisms of white genius mushroom (WGM). WGM is a popular edible mushroom in Taiwan and has been demonstrated to mediate potent antiproliferation effects against human Hep3B liver cancer cells in our previous study. According to next generation sequencing technology and KEGG pathway enrichment analysis, mTOR and MAPK signaling pathways were markedly changed during treatment with WGM extracts in Hep3B cells. Therefore, this study examined the effects of WGM extracts on the expression of mTOR and MAPK signaling pathway-related proteins, such as PI3K, Akt, mTOR, Ras, Raf, MEK, ERK, p38 and JNK in Hep3B cells. According to the results of immunoblotting, we demonstrated that the protein expression of the members of PI3K/Akt/mTOR and MAPK signaling pathways were involved in WGM extracts-induced cell death. Furthermore, the inhibitors of PI3K/Akt/mTOR and MAPK signaling pathways such as rapamycin, MK2206, LY3214996 and SB202190, blocked the induction of cell death and vacuoles formation induced by WGM extracts. This study also demonstrated that WGM extracts is able to inhibit Hep3B cell migration and colony formation in a dose-dependent manner. In addition to being a very popular food, WGM should be a pharmacologically safe natural agent for cancer treatment. Therefore, WGM might be designed to develop into a dietary chemopreventive agent for the cancer treatment.

Low concentration triphenyl phosphate fuels proliferation and migration of hepatocellular carcinoma cells.

Organophosphate flame retardants (OPFRs) have been widely used due to their unique properties. The OPFRs are mainly metabolized in the liver. However, whether the plasma level of OPFRs was involved in the progression of liver cancer remains unclear. Triphenyl phosphate (TPP) is one of the OPFRs that are mostly detected in environment. In this study, we performed CCK8, ATP, and EdU analyses to evaluate the effect of TPP at the concentrations at 0.025-12.8μM on the proliferation, invasion, and migration of Hep3B, a hepatocellular carcinoma (HCC) cell line. Tumor-bearing mouse model was used for in vivo validation. The results showed that low concentrations of TPP at (0.025-0.1μM), which are obtained in the plasma of patients with cancers, remarkably promoted cell invasion and migration of Hep3B cells. Animal experiments confirmed that TPP treatment significantly enhanced tumor growth in the xenograft HCC model. To explore the possible molecular mechanisms that might mediate the actions of TPP on Hep3B cells, we profiled gene expression in groups treated with or without TPP at the concentrations of 0.05 and 0.1μM using transcriptional sequencing. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Protein-protein interaction (PPI) analyses demonstrated that pathways affected by differentially expressed genes (DEGs) were mainly in nuclear-transcribed mRNA catabolic processes, cytosolic ribosome, and ATPase activity. A 0.05 and 0.1μM TPP led to up-regulation of a series of genes including EREG, DNPH1, SAMD9, DUSP5, PFN1, CKB, MICAL2, SCUBE3, and CXCL8, but suppressed the expression of MCC. These genes have been shown to be associated with proliferation and movement of cells. Taken together, our findings suggest that low concentration of TPP could fuel the proliferation, invasion, and migration of HCC cells. Thus, TPP is a risk factor in the progression of HCC in human beings.

Effect and Mechanism of LncRNA HOTAIR on Migration, Apoptosis and Proliferation of Hepatocellular Carcinoma Cells

<sec> <title>Objective:</title> This study was designed to probe the influence and mechanism of lncRNA HOTAIR on migration, apoptosis and proliferation of hepatocellular carcinoma (HCC) cells. </sec> <sec> <title>Methods:</title> We evaluated LncRNA HOTAIR expression in HCC tissues and adjacent tissues, and serum of HCC patients and healthy controls. Later, we knocked down lncRNA HOTAIR, and utilized CCK-8 to determine Hep3B cell proliferation, flow cytometry for prospecting Hep3B cell apoptosis, and cell scratch assay for observing Hep3B cell migration.We anticipated the direct target of lncRNA HOTAIR, and adopted luciferase reporter assay to verify. Moreover, we inhibitedmiR-126-5p expression, and rescue experiment for evaluating the influence of si-HOTAIR+miR-126-5p inhibitors on Hep3B cell migration, apoptosis as well as proliferation. </sec> <sec> <title>Results:</title> Our results showed that lncRNA HOTAIR expression in tumor tissues and serum was significantly increased. Moreover, lncRNA HOTAIR inhibition significantly decreased the Hep3B cell proliferation rate, elevated Hep3B cell apoptosis rate, and inhibited Hep3B cell migration. Luciferase reporter assay suggested that miR-126-5p was the direct target of lncRNA HOTAIR. Furthermore, co-transfection of si-HOTAIR+miR-126-5p inhibitor could diminishthe effects of HOTAIR silencing on apoptosis, proliferation and migration. </sec> <sec> <title>Conclusion:</title> Silencing of lncRNA-HOTAIR can inhibit the HCC cell migration and proliferation, and increase the apoptosis by up-regulating miR-126-5p expression. </sec>

Anticancer and Antiangiogenic Activities of Novel α-Mangostin Glycosides in Human Hepatocellular Carcinoma Cells via Downregulation of c-Met and HIF-1α.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In the present study, for the first time, we evaluated the anticancer and antiangiogenic activities of α-mangostin-3-O-β-D-2-deoxyglucopyranoside (Man-3DG) and α-mangostin 6-O-β-D-2-deoxyglucopyranoside (Man-6DG), glycosides of α-mangostin, against human HCC cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed the growth of three different HCC cells (Hep3B, Huh7, and HepG2) as well as the migration of Hep3B cells. Furthermore, they induced cell cycle arrest in the G0/G1 phases and apoptotic cell death by regulating apoptosis-related proteins of mitochondria in Hep3B cells. Noticeably, Man-3DG and Man-6DG also caused autophagy, while co-treatment of the α-mangostin glycosides with an autophagy inhibitor 3-MA enhanced the inhibitory effect on Hep3B cell growth in comparison to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway that plays a critical role in the pathogenesis of HCC. Furthermore, the α-mangostin glycosides decreased Hep3B cell-induced angiogenesis in vitro through the downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Notably, Man-6DG more effectively inhibited the growth, tumorsphere formation, and expression of cancer stemness regulators compared to α-mangostin and Man-3DG in 3D spheroid-cultured Hep3B cells. These findings suggest that the α-mangostin glycosides might be promising anticancer agents for HCC treatment with superior pharmacological properties than the parent molecule α-mangostin.

Characterization and Inception of a Triterpenoid Astrakurkurol, as a Cytotoxic Molecule on Human Hepatocellular Carcinoma Cells, Hep3B.

Mushrooms are customary influential sources of pharmaceutically active metabolites. Usually lanostane-type triterpenoids from mushrooms had prospective for cancer disease treatments. Recently, a triterpenoid, astrakurkurol obtained from the fresh basidiocarps of the edible mushroom Astraeus hygrometricus, drew attention as a new cytotoxic therapeutic. The structural stability of this triterpenoid had been established with the amalgamation of density functional theory (DFT) calculations and study of single-crystal X-ray diffraction. To successfully manifest astrakurkurol as a potent cytotoxic therapeutics, a wide apprehension on the molecular and cellular mechanisms underlying their action is prerequisite. On this account, our study was directed to scrutinize the influence of this triterpenoid on human hepatocellular cancer cell model Hep3B. Encapsulating all experimental facts revealed that astrakurkurol had significantly decreased cell viability in a concentration-dependent manner. This effect was unveiled to be apoptosis, documented by DNA fragmentation, chromatin condensation, nuclear shrinkage, membrane blebing, and imbalance of cell cycle distribution. Astrakurkurol persuaded the expression of death receptor associated proteins (Fas), which triggered caspase-8 activation following tBid cleavage. Moreover, tBid mediated ROS generation, which triggered mitochondrial dysfunction and activated the mitochondrial apoptotic events. Astrakurkurol cytotoxicity was based on caspase-8-mediated intrinsic apoptotic pathway and was associated with inhibition at Akt and NF-κB pathway. Astrakurkurol had also inhibited the migration of Hep3B cells, indicating its antimigratory potential. These findings led us to introduce astrakurkurol as a feasible and natural source for a safer cytotoxic drug against hepatocellular carcinoma.

BAY 87‑2243 sensitizes hepatocellular carcinoma Hep3B cells to histone deacetylase inhibitors treatment via GSK‑3β activation

Hepatocellular carcinoma (HCC) is associated with some of the highest cancer-associated mortality rates. Histone deacetylase (HDAC) inhibitors anti-HCC activities have been shown to promote Snail-induced metastasis. In the present study, it was shown that BAY 87-2243, a hypoxia-inducible transcription factor-1α inhibitor, could enhance the anti-HCC effects of HDAC inhibitors, including trichostatin A and vorinostat. In addition, BAY 87-2243 plus HDAC inhibitors exhibited synergistic cytotoxicity and induced significant cell death in Hep3B cells. Additionally, BAY 87-2243 combined with HDAC inhibitors-treated Hep3B cells formed fewer and smaller colonies as compared with either the control or single agent-treated cells. Furthermore, glycogen synthase kinase-3β might be involved in the enhanced cell death induced by BAY 87-2243 plus HDAC inhibitors. The present data also indicated that BAY 87-2243 combined with HDAC inhibitors could suppress the migration of Hep3B cells, and BAY 87-2243 could reverse the HDAC inhibitor-induced Snail activation in Hep3B cells. In conclusion, BAY 87-2243 combined with HDAC inhibitors might be an attractive chemotherapy strategy for HCC therapy.

Rffect of salt-induced kinase 1 on proliferation, invasion and migration of human hepatoma cell lines MHCC97-L and HEP3B

Objective To investigate the effect of salt-induced kinase 1 (SIK1) on the proliferation, invasion and migration of hepatoma cells. Methods Inhibitors were transfected into MHCC97-L and HEP3B cells to construct SIK1 knockdown group. The expression of SIK1 was detected by quantitative polymerase chain reaction (qPCR). The effect of SIK1 on cell proliferation at 1, 2, 3, 4, 5 days was measured by MTT assay. Cell cycle assay was used to detect the effect of SIK1 on G1, S and G2 phases. Apoptosis assay was used to detect cell invasion. The migration of osteosarcoma cells at 6, 24, 48 h was determined by wound healing test. Results The expression of SIK1 in MHCC97-L and HEP3B cells was lower than that in normal liver cells (0.51±0.01, 0.33±0.02, tMHCC97-L=19.630, P=0.000; tHEP3B=24.940, P=0.000). After knocking down SIK1, the expression of SIK1 in MHCC97-L and HEP3B cells was significantly decreased(0.39±0.02, 0.51±0.03, tMHCC97-L=11.479, P=0.000; tHEP3B=9.543, P=0.001). MTT assay showed that the proliferation of MHCC97-L and HEP3B cells was significantly improved (1.20±0.06 vs. 0.90±0.07, tMHCC97-L=5.630, P=0.005; 1.09±0.11 vs. 0.63±0.05, tHEP3B=6.460, P=0.003); Cell cycle experiments showed that the ratio of S phase cells was higher than that of the control group [(24.77±0.99)% vs. (17.22±1.01)%, tMHCC97-L=9.280, P=0.001; (21.63±0.94)% vs. (15.32±0.88)%, tHEP3B=8.460, P=0.001], and the ratio of G2 cells was higher than that of the control group [(16.75±0.50)% vs. (14.94±0.64)%, tMHCC97-L=3.860, P=0.018; (15.99±0.51)% vs. (13.97±0.44)%, tHEP3B=5.170, P=0.007]. The apoptosis rate of MHCC97-L and HEP3B cells was decreased [(1.91±0.10)% vs. (3.27±0.31)%, tMHCC97-L=7.180, P=0.002; (2.01±0.14)% vs. (3.81±0.13)%, tHEP3B=16.600, P=0.000]. The results of wound healing test showed that the migration of MHCC97-L and HEP3B cells was promoted [(202.00±15.61) vs. (285.33±10.07) μm; (229.33±13.61) vs. (321.33±16.04) μm, tMHCC97-L=7.770, P=0.001; tHEP3B=7.570, P=0.002]. Conclusion Decreasing the expression of SIK1 promotes the proliferation, invasion and migration of MHCC97-L and HEP3B cells. Key words: Salt-inducible kinase 1; Hepatic carcinoma; Proliferation; Invasion; Migration

Epigenetic analysis of FHL1 tumor suppressor gene in human liver cancer.

Liver cancer is one of the most common types of cancer among human malignancies. Four and a half LIM domains 1 (FHL1), as a tumor suppressor gene, is frequently downregulated in multiple types of human cancer. However, the role and specific mechanisms of FHL1 as a tumor suppressor in liver cancer are poorly understood. The present study aimed to investigate the role and associated mechanisms of FHL1 in human liver cancer. The level of FHL1 mRNA in hepatocellular carcinoma (HCC) tissue specimens and cell lines derived from the human liver was determined using reverse transcription polymerase chain reaction and western blot analysis. The association between FHL1 expression and clinicopathological characteristics of patients with liver cancer was analyzed. Western blotting, small interfering RNA (siRNA) and chromatin immunoprecipitation were used to study the expression association of FHL1 and enhancer of zeste homolog 2 (EZH2) in human liver cancer and to explore the regulatory mechanism of FHL1 downregulation. Colony formation and migration assays were performed while FHL1 was overexpressed in Hep3B cells. The results showed that the expression of FHL1 mRNA in tumor tissue decreased, exhibiting a significant difference compared with the adjacent non-cancerous tissue (P<0.05). However, the downregulation of FHL1 was not significantly associated with the sex, age, hepatitis B virus infection status, tumor size, distant metastasis status or level of tumor differentiation of the patients. FHL1 was synergistically silenced by DNA methylation and histone modification, and 3-deanzaneplanocin A (DZNep), an inhibitor of EZH2, which is a histone methyltransferase of the polycomb repressive complex 2, which catalyzes histone H3 lysine 27 tri-methylation (H3K27me3). A significant association between FHL1 and EZH2 expression was identified in the female hepatocellular carcinoma (HCC) samples, but was not in the male HCC samples. FHL1 overexpression and DZNep treatment significantly suppressed the growth and migration of Hep3B cells by restoring FHL1 expression. H3K27me3 was significantly enriched at the FHL1 promoter region, as indicated by a chromatin immunoprecipitation assay, and associated with the epigenetic repression of the FHL1 tumor suppressor gene in HCC cell lines. In conclusion, the present study provides an insight into DNA methylation and EZH2-H3K27me3 epigenetic repression of FHL1 in human liver cancer.

Targeting Pyk2 gene on the proliferation, invasion and migration induction of hepatocelluar cancer Hep3B cells

Objective To investigate the influence of proline-rich tyrosine kinase 2 (Pyk2) gene RNA interference on proliferation, invasion and migration of Hep3B hepatocellular carcinoma cells. Methods The Pyk2 gene RNA interference vector was transfected in Hep3B hepatocellular carcinoma cells by lipofectamine. The Hep3B cells divided into three groups: siRNA group (the vector with Pyk2 RNAi gene was transfected), negative control group (the vector without Pyk2 RNAi gene was transfected), and blank control group (no vectors was transfected). Pyk2 mRNA and protein were detected using reverse transcription reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The biological behavior including cell proliferation, invasion and migration were detected by 3-(4, 5-dimethyl-2-thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), transwell and wound healing assay, respectively. Results The expression of Pyk2 mRNA of Hep3B cell line in siRNA group (0.16±0.03) was significantly decreased than those in negative group (0.74±0.13) and blank control group (0.77±0.16), with statistically significant differences (t=51.46, P=0.000; t=53.21, P=0.000). The expression of Pyk2 protein of Hep3B cell line in siRNA group (0.24±0.06) was significantly decreased than those in negative group (0.83±0.05) and blank control group (0.91±0.06), with statisti-cally significant differences (t=57.29, P=0.000; t=68.53, P=0.000). The cell proliferation inhibition rate at 48 hours in siRNA group (26.17%±0.28%) was significantly raised than those in negative group (9.28%±0.22%) and blank control group (6.47%±0.31%), with statistically significant differences (t=31.45, P=0.004; t=34.64, P=0.002). The number of transmembrane cells in siRNA group (32.5±8.5)/10 HP was significantly declined than those in negative group (98.4±12.3)/10 HP and blank control group (112.6±11.3)/10 HP, with statistically significant differences (t=95.64, P=0.000; t=105.17, P=0.000). The wound healing assay in siRNA group (28.17%±1.46%) was significantly lower than those in negative group (77.38%±2.24%) and blank control group (79.41%±3.17%), with statistically significant (t=85.86, P=0.000; t=89.37, P=0.000). Conclusion Pyk2 gene involves the proliferation, invasion and migration of Hep3B cells, which has close correction with development and metastasis of hepatocellular carcinoma. Pyk2 gene is very helpful to become a molecular target for the diagnosis and treatment of hepatocellular carcinoma. Key words: Liver neoplasms; Cell proliferation; Neoplasm invasiveness; RNA interference; Proline-rich tyrosine kinase 2

Korean Byungkyul - Citrus platymamma Hort.et Tanaka flavonoids induces cell cycle arrest and apoptosis, regulating MMP protein expression in Hep3B hepatocellular carcinoma cells.

Citrus platymamma Hort.et Tanaka is an indigenous fruit of Jeju island in Korea. In this study the bioactivity of C.platymamma flavonoids were evaluated on human hepatoma Hep3B cell lines. Eleven flavonoids were identified from the peels of C.platymamma Hort.et Tanaka through high-performance liquid chromatography-Tandem mass spectrometry and the anticancer effect of these C.platymamma flavonoids on human hepatoma Hep3B were studied. Chromatin condensation was observed in Hep3B cells treated with C.platymamma flavonoids. DNA fragmentation was confirmed through agarose gel electrophoresis and TUNEL assay. An increase in the total apoptotic cells and G2/M cell cycle arrest with decreased protein expression of CDC25C, CDK1, cyclinB1 and p21 were observed in Hep3B cells treated with flavonoids of C.platymamma. Further, protein expression of Bcl-XL, Bax, caspase-3 and -9 were also modulated by C.platymamma flavonoids treatment indicating that cell death is through intrinsic apoptotic pathway. Moreover, C.platymamma flavonoids also regulated the phosphorylation of MAPKs, PI3K, and Akt in Hep3B cells. Relevant to inhibiting metastasis, C.platymamma treatment reduced wound closure of Hep3B cells and the protein expression of matrix metalloproteinase-2 and -9 were reduced in C.platymamma treated cells. The results show that C.platymamma flavonoids induce cell cycle arrest and apoptosis following activation of MAPKs and suppression of PI3K/Akt pathway which eventually inhibits cell migration in Hep3B cells. The finding provides evidence on biochemical activities of C.platymamma Hort.et Tanaka, which would be an essential agent for hepatocellular carcinoma (HCC) treatment.

Na+/K+-ATPase α1 subunit, a novel therapeutic target for hepatocellular carcinoma.

We aimed to identify the expression patterns of Na+/K+-ATPase (NKA) α subunits in human hepatocellular carcinoma (HCC) samples and evaluate these subunits as potential targets for HCC treatment. The mRNA expression profiles of NKA α subunits in human HCC samples were analyzed. We found that the mRNA expression for NKA α1 subunit (ATP1A1) was higher than that for other NKA α subunits. Also, ATP1A1 gene expression was markedly higher in HCC samples than in adjacent nontumor tissue samples. Western blotting data suggested that 6 of 14 (43%) HCC samples had elevated ATP1A1 protein expression. Furthermore, knockdown of ATP1A1 expression in human HCC HepG2 and MHCC97H cells markedly reduced their proliferation in vitro and suppressed the tumorigenicity of MHCC97H cells in vivo. Downregulation of ATP1A1 expression resulted in cell-cycle arrest at G2/M phase and apoptosis in HepG2 cells as well as decreased migration in Hep3B cells. We further validated that ATP1A1 downregulation caused intracellular accumulation of reactive oxygen species. Pretreatment with N-acetyl cysteine blocked cell-growth inhibition induced by ATP1A1 downregulation. Collectively, these data suggested that targeting ATP1A1 is a novel approach to the treatment of HCC.

MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is a highly invasive tumor with frequent distant metastasis, which is the main cause for the poor prognosis. However, the mechanisms for metastasis remain poorly investigated. MicroRNAs (miRNAs) have been implicated in HCC progression. MicroRNA-122 (miR-122) is considered as a tumor suppressor in human cancer. In the present study, miR-122 expression was found to be significantly lower in HCC than the level in normal tumor-adjacent tissues. miR-122 was clearly silenced or downregulated in five HCC cell lines (HepG2, Hep3B, MHCC97H, Huh7 and SMMC-7721) compared with normal hepatocytes (LO2). HCC patients with low expression of miR-122 had a poor 3-year survival. Univariate analysis and multivariate Cox regression analysis indicated that miR-122 is an independent prognostic factor in HCC. Downregulation of miR-122 promoted proliferation and inhibited apoptosis in Hep3B cells. We found that the public miRNA database (TargetScan) predicted that PKM2 may be a target for miR-122, and the 3'-untranslated region (3'-UTR) of PKM2 contains a highly conserved binding site for miR-122. To identify this, pre-miR-122/anti-miR-122 were respectively transfected into the Hep3B cell line. We found that miR-122 overexpression significantly reduced the level of PKM2. Moreover, knockdown of PKM2 significantly increased miR-122 inhibitor-mediated Hep3B cell apoptosis and reduced miR-122 inhibitor-mediated Hep3B cell migration and invasion. Moreover, re-expression of PKM2 partially abrogated miR-122-induced HCC cell growth arrest and apoptosis in vivo. In conclusion, miR-122 serves as a prognostic biomarker and induces apoptosis and growth arrest by downregulating PKM2 in HCC.

Raf/ERK/Nrf2 signaling pathway and MMP-7 expression involvement in the trigonelline-mediated inhibition of hepatocarcinoma cell migration

BackgroundTrigonelline occurs in many dietary food plants and has been found to have anti-carcinogenic activity. Trigonelline is also found in coffee which is one of the most widely consumed beverages. Many epidemiological studies have reported that coffee consumption has an inverse relationship with the risk of cirrhosis or hepatocellular carcinoma. It would be interesting to investigate whether trigonelline is an ideal chemoprevent agent to prevent cancer progression.MethodsThe protein expression was performed by western blotting. The trigonelline content in snow pea (Pisum sativum) was analyzed by high-performance liquid chromatography (HPLC). The migratory activity of human hepatocarcinoma cells (Hep3B) was assessed by using a wound migration assay. The percentage of each phase in the cell cycle was analyzed on a FACScan flow cytometer. Gene expression was detected by real-time reverse transcriptase-polymerase chain reaction techniques. Native gel analysis was performed to analyze the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase.ResultsAccording to the data of HPLC analysis, P. sativum, which is a popular vegetable, has relatively high content of trigonelline. Our findings suggest that trigonelline is an efficient compound for inhibiting Hep3B cell migration. Trigonelline inhibited the migration of hepatoma cells at concentrations of 75–100 µM without affecting proliferation. Raf/ERK/Nrf2 protein levels and further downstream antioxidative enzymes activity, such as SOD, catalase, and glutathione peroxidase, significantly decreased after treatment with 100 µM of trigonelline for 24 h. The migration inhibition of trigonelline is also related to its ability to regulate the matrix metalloproteinases 7 (MMP-7) gene expression.ConclusionsIn this study, protein kinase Cα (PKCα) and Raf/ERK/Nrf2 signaling pathway and MMP-7 gene expression were involved in the trigonelline-mediated migration inhibition of Hep3B cells. We also demonstrated that trigonelline inhibits Hep3B cell migration through downregulation of nuclear factor E2-related factor 2–dependent antioxidant enzymes activity. This study analyzed the trigonelline content in a popular vegetable, snow pea, as a representative proof to prove that trigonelline is often found in the daily intake of food. Our finding suggested that trigonelline should be a useful chemopreventive agent derived from the daily intake of food to prevent cancer progression.

Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation.

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR1 and PAR4 are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms. Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H2DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate. Thrombin, the PAR1 selective agonist peptide TFLLRN-NH2 (PAR1-AP), and the PAR4 selective agonist peptide, AYPGKF-NH2 (PAR4-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR1-AP and the PAR4-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR1- and PAR4-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-L-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR1-AP and the PAR4-AP, but had no effect on PAR1/4-mediated activation of Met and PDGFR in Hep3B cells. Collectively, our data indicate that PAR1 and PAR4 activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.

Effects of endothelial cells conditioned medium on the proliferation and invasion of hepatocellular carcinoma cells

Objective To investigate the effects of vascular endothelial cells on the proliferation and invasion of hepatocellular carcinoma cells,and better understand the role of endothelial cells in the development of hepatocellular carcinoma (HCC).Methods The biological characteristics of Hep3B cells,such as proliferation,migration and invasion,were comparatively analyzed under HUVECs-derived conditioned medium (CM) stimulation and endothelial cell basal medium (EBM) stimulation,and its related signal molecules were detected by Western blotting.Data analysis was performed using SPSS 16.0 software.Results Cell Counting Kit-8 (CCK-8) test showed that the proliferation of Hep3 B cells treated with CM was significantly increased as compared with that of the control cells (P ＜ 0.01).The wound healing assay and cell migration assay demonstrated that the migration of Hep3B cells was obviously enhanced underCM stimulation [(33.65±1.22)％ vs.(57.85±2.20)％,P＜0.01 and 51.2±3.1 vs.28.4± 4.6,P ＜0.01],and invasion ability of Hep3B cells induced by CM was also improved (16.2 ± 2.3 vs.5.4 ± 1.5).The expressions of invasion/metastasis associated genes matrix metalloproteinase-2 (MMP-2),matrix metalloproteinase-9 (MMP-9),osteopontin (OPN) and cluster of differentiation (CD44) and the serine-threonine kinase (Akt) phosphorylation and Extracellular Regulated Protein kinases (ERK) phosphorylation level were all upregulated in the treated Hep3B with CM.Conclusion Vascular endothelial cell conditioned mediun containing some soluble cytokines and/or chemokines which may facilitate the proliferation,migration and invasion of HCC cells via paracrine pathway. Key words: Hepatocellular carcinoma ; Endothelial cell ; Paracrine ; Invasiveness and metastasis

Abstract 5494: The effect of honokiol combined with PPARγ agonists in regulation of cell growth and migration in human hepatoma cells

Abstract Hepatocellular carcinoma (HCC) is one of the malignant cancers worldwide. At present, surgical resection is the best treatment for HCC. Despite some other options such as chemotherapy and radiotherapy, recurrence and metastasis are the major causes of increased mortality. It has been reported honokiol, a natural biphenolic compound, exerts inhibitory effects on the growth and migration of hepatoma cells. Most recently, it was found that honokiol functions as not only an RXR agonist but also a PPARγ agonist, and is capable of potentiating the activation of PPARγ in the presence of PPARγ agonist rosiglitazone in 3T3-L1 adipocytes and HLE human hepatoma cells. Moreover, PPARγ agonists exhibited inhibitory role in cell growth and migration in various cancers through PPARγ-dependent and -independent pathway. However, the combination effect and the molecular mechanism of cotreatment with honokiol and PPARγ agonists are still unclear in hepatoma cells. In this study, Hep3B, Mahlavu, and SK-Hep1 human hepatoma cells were treated with honokiol and/or PPARγ agonists. Honokiol and PPARγ agonists rosiglitazone and troglitazone inhibited cell growth in hepatoma cells by surforhodamine B assay, respectively. Moreover, cotreatment with honokiol and PPARγ agonist showed additive effect in cell growth inhibition. Furthermore, retrovirus-mediated PPARγ overexpression in SK-Hep1 cells resulted in higher sensitivity to honokiol treatment in cell growth inhibition compared to vector control. Additionally, PPARγ agonists rosiglitazone and troglitazone inhibited cell migration in Hep3B cells by wound healing and transwell assays. These results together suggest that honokiol combined with PPARγ agonist had additive effect in cell growth inhibition and migration of hepatoma cells. The molecular mechanism and potential application of this combination for treatment of hepatoma will be presented. Citation Format: Chin-Wen Chi, Hui-Tzu Hsu. The effect of honokiol combined with PPARγ agonists in regulation of cell growth and migration in human hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2014-5494

Bone morphogenetic protein 2 inhibits hepatocellular carcinoma growth and migration through downregulation of the PI3K/AKT pathway

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Previous studies have suggested that abnormal expression of BMP-4, BMP-7, and BMP-9 is correlated with tumor progression in HCC, but the role played by BMP-2 in HCC has not yet been reported. To determine the role of BMP-2 in HCC, we first investigated the effect of exogenous BMP-2 on the growth of the cell lines HCC SK-Hep-1, Hep G2, and Hep 3B. Next, we studied the function of BMP-2 in SK-Hep-1 HCC cell line using a recombinant lentivirus vector to deliver BMP-2. We also used siRNA to silence endogenous BMP-2 expression in the HCC Hep 3B cell line. Then, cell growth and migration were assayed in vitro using WST-8, wound-healing, and transwell invasion assays. Cellular apoptosis and cell-cycle distribution were assessed using flow cytometry. We also investigated the effects of BMP-2 overexpression and knockdown on the expression of proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-2 (MMP-2), phosphorylated AKT (p-AKT), phosphoinositide 3-kinase p85α (PI3Kp85α), Bax, Bcl-2, caspase-3, cleaved caspase-3, p21, and cyclin E. As a result, we observed that BMP-2 inhibited the proliferation of HCC cells. Furthermore, HCC cell proliferation and migration were significantly diminished by BMP-2 overexpression, as was indicated by WST-8, would healing, and transwell assays, while knockdown of BMP-2 led to an increase in proliferation and migration of Hep 3B cells. BMP-2 overexpression significantly increased the susceptibility of SK-Hep-1 cells to low-serum-induced apoptosis, while BMP-2 knockdown reduced the susceptibility of Hep 3B cells. Overexpression of BMP-2 induced G1 phase arrest through upregulation of p21. When BMP-2 expression was elevated in SK-Hep-1 cells, the expression of PI3Kp85α, p-AKT, PCNA, and MMP-2 declined. These results suggest that BMP-2 exerts an inhibitory effect on the growth and migration of HCC cells, possibly via a blockade of PI3K/AKT signaling.

Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: Molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes.

Role of GSK-3β in hepatocellular carcinoma cell migration

Objective To study the effects of GSK-3β on the migration ability of hepatocellular carcinoma cells. Methods Inhibition tests with specific inhibitors of GSK -3 ( LiCl and SB216763) and transfection with different plasmids( GSK-3β WT,GSK-3β S9A,and GID5 -6) were used to alter the activity of GSK -3β. The migration ability of SMMC-7221 and Hep3B cells was observed using wound healing assay,transwell assay and the microtubule-organizing center ( MTOC) test before and after treatment. Immunocytochemistry was used to investigate the distribution of phospho-GSK-3β. Results Wound healing assay showed that LiCl and SB216763 decreased the migration of SMMC-7721 cells by 36. 44% and 41. 78% ,respectively; and decreased the migration of Hep3B cells by both 26. 66%. Transfection with GSK-3β WT increased the expression of GSK-3β and pGSK-3β,and the migration rate of SMMC-7721 cells decreased to 61. 27%. Transfection with GSK-3β S9A increased GSK-3β expression and had no noticeable influence on pGSK -3β expression,and the migration rate of SMMC-7721 cells decreased to 38. 61%. Transfection with GID5-6 had no noticeable influence on GSK-3β and increased pGSK-3β expression,and the migration rate decreased to 36. 49%. Immunocytochemical staining showed that LiCl blocked pGSK-3β accumulation on cell edge as that in normally cultured cells. MTOC test showed that the MTOC positive rate was above 60% in Hep3B cells before adding inhibitors of GSK-3β,and it decreased after exposure to the inhibitors. Conclusion GSK-3β plays an important role in migration of hepatocellular carcinoma cells. Inhibitors of GSK-3β can affect the polarization and decrease the migration ability of hepatocellular carcinoma cells.