Human Hepatoma Hep3B Cells Research Articles

Piper betle leaf extracts induced human hepatocellular carcinoma Hep3B cell death via MAPKs regulating the p73 pathway in vitro and in vivo.

Extracts of Piper betle leaf (PBLs) are rich in bioactive compounds with potential chemopreventive ability. In this study, Hep3B cells which are p53 null were used to investigate the anti-tumor effect of PBLs in the cell and in the xenograft model. The results revealed that PBLs (0.1 to 1 mg mL(-1)) induced a dose- and time-dependent increase of cell toxicity. The underlying mechanisms as evidenced by flow cytometry and western blot analysis showed that PBLs triggered ATM, cAbl, and p73 expressions and activated JNK and p38 pathways that subsequently led to cell cycle arrest and mitochondria-dependent apoptosis. PBLs also inhibited tumor growth in Hep3B-bearing mice via inducing the MAPK-p73 pathway. Our results demonstrated the in vitro and in vivo anti-tumor potential of PBLs, supporting their application as a novel chemopreventive agent for the treatment of human hepatocellular carcinoma (HCC) in the future via targeting the p73 pathway.

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

The extract of Ziziphus jujuba fruit (jujube) induces expression of erythropoietin via hypoxia-inducible factor-1α in cultured Hep3B cells.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as health supplement or herbal medicine worldwide. To study the beneficial role of jujube in enhancing hematopoietic function, we investigated its roles on the expression of erythropoietin in cultured Hep3B human hepatocellular carcinoma cells. Application of chemically standardized jujube water extract stimulated erythropoietin expression in a dose-dependent manner, with the highest response by ~ 100 % of increase. A plasmid containing hypoxia response element, a critical regulator for erythropoietin transcription, was transfected into Hep3B cells. Application of jujube water extract onto the transfected cells induced the transcriptional activity of the hypoxia response element. To account for its transcriptional activation, the expression of hypoxia-inducible factor-1α was increased after treatment with jujube water extract: the increase was in both mRNA and protein levels. These results confirmed the hematopoietic function of jujube in the regulation of erythropoietin expression in liver cells.

An acetate switch regulates stress erythropoiesis.

Endocrine erythropoietin (Epo), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor Hypoxia Inducible Factor 2 (HIF-2). We previously reported that the lysine acetyltransferase Cbp is required for HIF-2α acetylation and efficient HIF-2 dependent Epo induction during hypoxia. We now show these processes require acetate-dependent acetyl CoA synthetase 2 (Acss2). In Hep3B hepatoma cells and in Epo-generating organs of hypoxic or acutely anemic mice, acetate levels increase and Acss2 is required for HIF-2α acetylation, Cbp/HIF-2α complex formation and recruitment to the Epo enhancer, and efficient Epo induction. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an Acss2-dependent manner. In acquired and genetic chronic anemia mouse models, acetate supplementation also increases Epo expression and resting hematocrits. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and Epo induction during pathophysiological states marked by tissue hypoxia.

Role of Bax/Bcl-2 family members in green tea polyphenol induced necroptosis of p53-deficient Hep3B cells.

Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

Suppression of hepatocellular carcinoma proliferation and hepatitis B surface antigen secretion with interferon-λ1 or PEG-interferon-λ1.

Hepatocellular carcinoma (HCC) is a common cancer associated with chronic hepatitis B virus (HBV) infection. Conventional interferon-α (IFN-α) and pegylated IFNs (PEG-IFNs) approved for chronic HBV infection treatment can reduce the risk of HCC but are not suitable for the majority of patients and cause significant side effects. IFN-λ1 is a type III IFN with antiviral, antiproliferative, and immunomodulatory functions similar to type I IFNs but with fewer side effects. However, the tolerability and antitumor activity of PEG-IFN-λ1 in HCC xenograft mice are unknown. In vitro IFN-λ1 treatment of Hep3B and Huh7 human hepatoma cell lines increased MHC class I expression, activated JAK-STAT signaling pathways, induced IFN-stimulated gene expression, and inhibited hepatitis B surface antigen (HBsAg) expression. IFN-λ1 treatment also caused 23.2 and 19.9% growth inhibition of Hep3B and Huh7 cells, respectively, and promoted cellular apoptosis. PEG-IFN-λ1, but not IFN-λ1 treatment, significantly suppressed tumor growth (P=0.002) and induced tumor cell apoptosis in a Hep3B cell xenograft mouse model without significant weight loss or toxicity. PEG-IFN-λ1 also significantly inhibited (P=0.000) serum HBsAg secretion from Hep3B xenograft tumors in vivo. Thus, PEG-IFN-λ1 can suppress Hep3B xenograft tumor growth and inhibit HBsAg production and may be a potential treatment for HBV-related HCC.

Synthesis and in vitro anticancer evaluation of 2-isopropyl-5-methylphenol Loaded PLGA based iron oxide nanoparticles

For precise delivery of 2-isopropyl-5-methylphenol (IPMP), Poly (lactic-co-glycolic acid) (PLGA) based superparamagnetic nanoparticles loaded with IPMP (IMNPs) were synthesized and characterized. IMNPs with a particle size of 223±20.7nm were synthesized by solvent evaporation method. Fe3O4 nanoparticles of 33±3.63nm encapsulated in the PLGA polymer provides the superparamagnetic property to the IMNPs. The entrapment efficacy of Fe3O4 nanoparticles, 2-isopropyl-5-methylphenol and average cumulative percentage release profile of IPMP from the IMNPs were investigated. The magnetic nanocarrier enhanced the stability and activity of IPMP. Release of IPMP from IMNPs occurred in a controlled manner. The cytotoxicity of IMNPs against human hepatoma Hep3B cell line was investigated and the IC50 value was found to be 20μg/mL for IMNPs compared to 25μg/mL for free IPMP. The result showed that IMNPs had significant antitumor activity. Therefore, IMNPs may be considered as an effective anticancer drug delivery system for cancer chemotherapy.

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

BackgroundVascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.ResultsThe predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.ConclusionSilencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.

Anticancer effects of deproteinized asparagus polysaccharide on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in the world whose chemoprevention became increasingly important in HCC treatment. Although the anticancer effects of asparagus constituents have been investigated in several cancers, its effects on hepatocellular carcinoma have not been fully studied. In this study, we investigated the anticancer effects of the deproteinized asparagus polysaccharide on the hepatocellular carcinoma cells using the in vitro and in vivo experimental model. Our data showed that deproteinized asparagus polysaccharide might act as an effective inhibitor on cell growth in vitro and in vivo and exert potent selective cytotoxicity against human hepatocellular carcinoma Hep3B and HepG2 cells. Further study showed that it could potently induce cell apoptosis and G2/M cell cycle arrest in the more sensitive Hep3B and HepG2 cell lines. Moreover, deproteinized asparagus polysaccharide potentiated the effects of mitomycin both in vitro and in vivo. Mechanistic studies revealed that deproteinized asparagus polysaccharide might exert its activity through an apoptosis-associated pathway by modulating the expression of Bax, Bcl-2, and caspase-3. In conclusion, deproteinized asparagus polysaccharide exhibited significant anticancer activity against hepatocellular carcinoma cells and could sensitize the tumoricidal effects of mitomycin, indicating that it is a potential therapeutic agent (or chemosensitizer) for liver cancer therapy.

Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

BackgroundThe Wnt pathway plays an important role in Hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection.MethodsWe performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1.ResultsCell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them.ConclusionNiclosamide is a potential candidate for the treatment of hepatoma.

DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac

Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons.

Application of the DataChip/MetaChip technology for the evaluation of ajoene toxicity in vitro

The DataChip is a universal platform for three-dimensional (3D) cell cultures on a micropillar chip, which can be applicable to a variety of human cells to simulate organ-specific toxicity. In addition, the MetaChip is developed for various combinations of drug metabolizing enzymes that can be spotted into the microwell chip and incubated with 3D human cells to simulate systematic compound metabolism in the human liver on a microscale format. Ajoenes have been known for various therapeutics activities, including anticancer effects, but there was limited information available in regard to their metabolism and cytotoxicity. In the present work, the metabolism-mediated toxicity of ajoenes was evaluated on a DataChip/MetaChip platform. In detail, we tested cytotoxicity of E- and Z-ajoene on 3D cultured Hep3B human hepatoma cells coupled with mixtures of drug metabolizing enzymes. Metabolic profiles of ajoenes were assessed with 23 representative drug metabolizing enzymes on the MetaChip. As a result, cytotoxicity of E-ajoene was significantly augmented in the presence of cytochrome P450 (CYP) isoforms, such as CYP2E1 and CYP3A5. Both E- and Z-ajoene were drastically detoxified in the presence of Phase II enzymes, including major UGTs, SULTs, NATs, and GSTs. Interestingly, All Mix, an artificial human liver microsome containing representative P450 mixture and phase II enzyme mixture, attenuated P450-induced cytotoxicity of ajoenes. Conclusively, we were able to confirm the metabolism-medicated toxicity of ajoenes on the chip.

Cordycepin increases sensitivity of Hep3B human hepatocellular carcinoma cells to TRAIL-mediated apoptosis by inactivating the JNK signaling pathway

Resistance to tumor necrosis factor-related apoptosis‑inducing ligand (TRAIL)-induced apoptosis has been reported in various cancer cells. Cordycepin, a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, which possesses many pharmacological activities including antitumor and anti-inflammation. In the present study, we demonstrated that treatment of cordycepin sensitized TRAIL-resistant Hep3B human hepatocellular carcinoma cells to TRAIL-mediated apoptosis as evidenced by formation of apoptotic bodies, chromatin condensation and accumulation of cells in the sub-G1 phase. The induction of apoptosis following co-treatment with cordycepin and TRAIL in Hep3B cells appeared to be correlated with modulation of Bcl-2 family protein expression and activation of the caspase cascade, which resulted in the cleavage of poly(ADP-ribose) polymerase and β-catenin. In addition, cordycepin treatment also inhibited activation of c-Jun N-terminal kinase (JNK). Pretreatment with SP600125, a JNK inhibitor, resulted in a significantly increased sub-G1 population and caspase activity in cordycepin plus TRAIL-mediated apoptosis. Taken together, these results indicate that JNK acts as a key regulator of apoptosis in response to combined treatment with cordycepin and TRAIL in human hepatocellular carcinoma Hep3B cells.

Bufalin induces G2/M phase arrest and triggers autophagy via the TNF, JNK, BECN-1 and ATG8 pathway in human hepatoma cells

Liver cancer is the fifth most common cause of cancer death worldwide. The study of more effective anti-hepatoma drugs is urgently required. Bufalin has been isolated from a traditional Chinese medicine and possesses less toxicity to normal cells. However, it has been found to inhibit growth of cancer cells. In this study, we aimed to investigate the efficacy and mechanism of bufalin in Huh7, Hep3B and HA22T human hepatoma cells. The three cell lines were treated with bufalin, the proliferation was detected by WST-1 assay and cell cycle was detected by flow cytometry analysis. The results showed that bufalin inhibited the proliferation of hepatoma cells and regulated the hepatoma cell death program in a dose- and time-dependent manner without typical features of apoptosis. RT-PCR arrays were used to investigate the autophagy transcriptional response triggered by bufalin and 13 genes were altered and further confirmed by real-time PCR. The translation levels of selected genes were examined by western blot analysis to reveal the bufalin-induced autophagy cascade. Bufalin synergized with the JNK pathway to induce autophagy of hepatoma cells and is closely associated with the upregulation of TNF, BECN-1, MAPK and ATG8, together with the downregulation of Bcl-2 and Bid. Our study provided a multi-angle evaluation system for anti-hepatoma pharmacology for pre-clinical drug investigation. In this case, bufalin was capable of inducing hepatoma cell autophagy, suggesting a potential regimen for single or combined chemotherapy to overcome hepatoma in clinical practice.

NC‐6300, an epirubicin‐incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin-incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC-6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure of 40-80 nm in diameter in an aqueous milieu. NC-6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC-6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC-6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC-6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin-treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC-6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC-6300 in patients with hepatocellular carcinoma or other cancers.

Abstract 295: PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells.

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, a zinc finger transcription factor Krüppel-like factor 4 (KLF4) was found to be reduced in tumors of HCC patients. Additionally, results from our laboratory showed that overexpressed KLF4 repressed epithelial-mesenchymal transition (EMT) process and cell motility by induction of E-cadherin expression and reduction of EMT markers in hepatoma cells. Interestingly, it has been found that PPARγ agonists upregulated KLF4 in colorectal cancer cells. However, the mechanism of PPARγ-induced KLF4 in the EMT process of HCC remains unclear. In this study, Hep3B, PLC/PRF/5, and Mahlavu human hepatoma cells were treated with PPARγ agonists rosiglitazone, troglitazone, and PPARγ antagonist GW9662, respectively. After rosiglitazone or troglitazone treatment for various time periods, growth inhibition was observed in both Hep3B and PLC/PRF/5 cells at 48 hours by surforhodamine B assay. Mahlavu cells showed less response to rosiglitazone and troglitazone treatment as compared to the other two cell lines. However, pretreatment of GW9662, a PPARγ antagonist, prior to PPARγ agonists treatment did not block the PPARγ agonists-induced cell growth inhibition. Moreover, cotreatment of PPARγ agonist and antagonist showed additive growth inhibition effect. In addition, rosiglitazone and troglitazone inhibited the migration of Hep3B, PLC/PRF/5 and Mahlavu cells differentially in transwell assay. Western blotting analysis showed upregulation of KLF4 and E-cadherin in Hep3B and PLC/PRF/5 cells after rosiglitazone and troglitazone treatment. Interestingly, decreased KLF4 and E-cadherin were observed in Hep3B, PLC/PRF/5, and Mahlavu cells after GW9662 treatment, indicating that the regulation of KLF4 and E-cadherin were PPARγ-dependent. Moreover, GW9662 also inhibited cell migration of Hep3B, PLC/PRF/5, and Mahlavu cells, with a concomitant decrease of vimentin. These results together suggested that PPARγ agonists and antagonist not only inhibited the growth but also decreased migration of hepatoma cells. Modulation of the PPARγ agonists induced expression of KLF4 and E-cadherin played important role in migration of hepatoma cells. Citation Format: Hui-Tzu Hsu, Ming-Ta Sung, Chin-Wen Chi. PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2013-295

Identification of FN1BP1 as a Novel Cell Cycle Regulator through Modulating G1 Checkpoint in Human Hepatocarcinoma Hep3B Cells

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

Anticancer effects of O-desmethylangolensin are mediated through cell cycle arrest at the G2/M phase and mitochondrial-dependent apoptosis in Hep3B human hepatocellular carcinoma cells

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200µM) for 24, 48 or 72h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclinA andB. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an AnnexinV assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochromec release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

MicroRNA-122-dependent and -independent replication of Hepatitis C Virus in Hep3B human hepatoma cells

The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122.

Cytotoxic quassinoids from Ailanthus altissima

Two new quassinoids, altissinol A (1) and B (7), together with 12 known quassinoids, were isolated from the 95% ethanol extract of the barks of Ailanthus altissima. The structures of the new compounds (1 and 7) were determined on the basis of the spectroscopic methods including UV, IR, HR-ESI-MS, 1D and 2D NMR. The cytotoxic potential of all isolates were evaluated in vitro against three human hepatoma cell lines. Quassinoids 1–7 displayed potent cytotoxic activities against human hepatoma Hep3B and HepG2 cell lines. Interestingly, compounds 2, 3, and 5 exhibited cytotoxic activity against multidrug resistance HepG2/ADM cell line with IC50 value 4.3-fold more sensitive to Doxorubicin (DOX).