Human Hepatoma Hep3B Cells Research Articles

Hep3B 간암세포에서 개똥쑥추출물로부터 Akt-mTOR-GSK3β 신호경로에 의한 apoptosis 효과

Hep3B 간암세포에서 개똥쑥추출물로부터 Akt-mTOR-GSK3β 신호경로에 의한 apoptosis 효과

Combined therapy with oncolytic adenoviruses encoding TRAIL and IL-12 genes markedly suppressed human hepatocellular carcinoma both in vitro and in an orthotopic transplanted mouse model.

BackgroundGene-based virotherapy mediated by oncolytic viruses is currently experiencing a renaissance in cancer therapy. However, relatively little attention has been given to the potentiality of dual gene virotherapy strategy as a novel therapeutic approach to mediate triplex anticancer combination effects, particularly if the two suitable genes are well chosen. Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interleukin-12 (IL-12) have been emerged as promising pharmacological candidates in cancer therapy; however, the combined efficacy of TRAIL and IL-12 genes for treatment of human hepatocellular carcinoma (HCC) remains to be determined.MethodsHerein, we investigated the therapeutic efficacy of concurrent therapy with two armed oncolytic adenoviruses encoding human TRAIL gene (Ad-ΔB/TRAIL) and IL-12 gene (Ad-ΔB/IL-12), respectively, on preclinical models of human HCC, and also elucidated the possible underlying mechanisms. The effects of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy were assessed both in vitro on Hep3B and HuH7 human HCC cell lines and in vivo on HCC-orthotopic model established in the livers of athymic nude mice by intrahepatic implantation of human Hep3B cells.ResultsCompared to therapy with non-armed control Ad-ΔB, combined therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 elicited profound anti-HCC killing effects on Hep3B and HuH7 cells and on the transplanted Hep3B-orthotopic model. Efficient viral replication and TRAIL and IL-12 expression were also confirmed in HCC cells and the harvested tumor tissues treated with this combination therapy. Mechanistically, co-therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 exhibited an enhanced effect on apoptosis promotion, activation of caspase-3 and-8, generation of anti-tumor immune response evidenced by upregulation of interferon gamma (IFN-γ) production and infiltration of natural killer-and antigen presenting cells, and remarkable repression of intratumor vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) expression and tumor microvessel density.ConclusionsOverall, our data showed a favorable therapeutic effect of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy against human HCC, and may therefore constitute a promising and effective therapeutic strategy for treating human HCC. However, further studies are warranted for its reliable clinical translation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0353-8) contains supplementary material, which is available to authorized users.

The protective effects of epigallocatechin gallate on lipopolysa ccharide-induced hepatotoxicity: an in vitro study on Hep3B cells.

In the present study, our aim was to investigate the possible protective effects of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced hepatotoxicity by using Hep3B human hepatoma cells. Specifically, the study examines the role of some proinflammatory markers and oxidative damage as possible mechanisms of LPS-associated cytotoxicity. Consequently, the hepatocellular carcinoma cell line Hep3B was chosen as a model for investigation of LPS toxicity and the effect of EGCG on LPS-exposed cells. The Hep3B human hepatoma cells were used for this study. The cytotoxic effects of chemicals (EGCG and LPS), AST and ALT levels, SOD and CAT activities, GSH-Px level and TNF-alpha and IL-6 levels were detected by using different biochemical and molecular methods. LPS and EGCG were applied to cells at various times and doses. The highest treatment dose of EGCG (400 µM) led to a dramatic decrease in SOD level and increase in CAT and GSH levels. Additionally, the highest dose of EGCG also led to a dramatic increase in TNF-alpha and IL-6 levels. On the other hand, effective doses of EGCG (200 and 100 µM) normalized all related parameters levels. LPS caused hepatotoxicity, but interestingly, a high dose of EGCG was found to be a cytotoxic agent in this study. However, other two doses of EGCG led to a decrease in both inflammatory cytokine levels and antioxidant enzyme levels. Further studies should examine the effect of EGCG on secondary cellular signaling pathways.

Anti‐inflammatory, Anti‐proliferative Activities from the Skin of the Catfish Arius Bilineatus, Valenciennes

The catfish secretes a gelatinous substance composed of biochemically active lipids and proteins from its skin upon stress or injury. Preparations from the skin have previously been shown to affect blood clotting and accelerates healing of non‐healing diabetic foot ulcers in man. Here catfish skin preparation (CSP) and its lipid fraction were investigated with the focus on its ability to alter key inflammatory mediators and to inhibit the proliferation of human non‐small cell lung cancer (NSCLC) A549 cells and human hepatocellular carcinoma Hep3B cells. The anti‐inflammatory activity was determined by its effect on arachidonate metabolism in cells in vitro or in mouse ear tissues by LC/MS/MS method, and on the inflammatory genes analyzed by inflammation array kits (PCR based). The antiproliferative activity of CSP was evaluated by MTT assay. When A549 cells were treated with CSP or different lipid fraction of CSP (200 μg/ml), total lipid fraction noticeably reduced the cellular level of PGE2 by 64.2%, whereas the petroleum either fraction of CSP decreased the production of PGI2, TEXB2, PGF2α, PGE2 and PGE1 by 42%, 24%, 76%, 55% and 68%, respectively compared to that of control vehicle treated cells. Similarly, chloroform fraction of CSP also reduced the cellular level of PGI2 and PGF2α by 67% and 74%, respectively in A549 cells. To further identify the anti‐inflammatory properties of CSP, we then tested the anti‐inflammatory effect of CSP in arachidonic acid induced mouse ear model. Intriguingly, the cream prepared from the total lipid CSP (10 mg) reduced the redness of ears caused by arachidonic acid, and significantly reduced mouse ear weight (p < 0.05) suggesting anti‐inflammatory activity of the CSP. Analyzing mouse ear eicosanoid levels revealed that PGE2 was reduced by 28% as well as 5‐lipoxygenase metabolite 5‐HETE by 30% in the CSP cream treated group compared to that of control group. The inflammatory genes were further tested in the control and CSP cream treated mouse ear tissues. Intriguingly, the LTA4 hydrolase gene which is downstream of 5‐LOX and responsible for formation of pro‐inflammatory mediator LTB4 was reduced by 50%. Given inflammation is one of the key factors associated with tumor development, we then tested the anti‐proliferative activities of the total lipid extract and its lipid fractions in A549 cells and Hep3B cells. The growth of both A549 and Hep3B cells was significantly inhibited by chloroform fraction of CSP with IC50 around 25 mg/ml. Together, these data suggest that CSP and its lipid soluble fraction have both anti‐inflammatory and anti‐proliferative activities which may be mediated through alteration of arachidonate metabolism and warrants further investigation.Support or Funding InformationThis study was support by a grant from Kuwait Foundation for the Advancement of Science No. KFAS 201320701A‐D, and Kuwait University Research Grant No. SL03/14.

RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo.

This study aimed to investigate the anti-tumor activity of RY10-4, a small molecular that was designed and synthesized based on the structure of protoapigenone. A previous screening study showed that RY10-4 possessed anti-proliferative effects against HepG2 human hepatocellular carcinoma cells. However, the full range of RY10-4 anti-cancer effects on liver tumors and the underlying mechanisms have not been identified. Herein, employing flow cytometry, and Western blot analysis, we demonstrate that RY10-4 can induce cell cycle arrest, intracellular reactive oxygen species (ROS) production and apoptosis in HepG2 cells. In HepG2 cell xenograft tumor model, RY10-4 significantly inhibited the growth of tumors and induced apoptosis in tumor cells, with little side effects. Moreover, RY10-4 caused the suppression of STAT3 activation, which may be involved the apoptosis induction. In addition, RY10-4 inhibited the proliferation of Hep3B and HuH-7 human hepatocellular carcinoma cells in a concentration-dependent manner. Taken together, our results suggest that RY10-4 has a great potential to develop as chemotherapeutic agent for liver cancer.

The antitumor effect of TIG3 in liver cancer cells is involved in ERK1/2 inhibition.

Tazarotene-induced gene 3 (TIG3) was first characterized in tazarotene-treated human keratinocytes and identified as a retinoic acid responder gene, an important mediator of antitumor effects by retinoids. In this study, we aim to investigate the inhibitory effect of TIG3 on the growth of liver cancer and explore its underlying mechanism. Human hepatocellular carcinoma (HCC) Hep3B cells were transfected with plasmid GV141 carrying full-length TIG3 complementary DNA (cDNA). The effects of TIG3 on cell proliferation, apoptosis, and migration were determined in vitro. The suppressor effect of TIG3 on tumor growth was evaluated in vivo in a nude mouse HCC model. We observed that TIG3 expression is decreased in the Hep3B cell line as well as primary HCC tumors, and TIG3 expression inversely correlates with Ki-67 expression. Overexpression of TIG3 suppresses tumor growth in HCC both in vitro and in vivo via ERK1/2 inhibition by promoting apoptosis and inhibiting proliferation and migration. These findings identify TIG3 as an attractive therapeutic target for HCC.

Antimetastatic effect of fluvastatin on breast and hepatocellular carcinoma cells in relation to SGK1 and NDRG1 genes.

Metastasis occurs due to migration of the cells from primary tumor toward other tissues by gaining invasive properties. Since metastatic invasion shows a strong resistance against conventional cancer treatments, the studies on this issue have been focused. Within this context, inhibition of migration and determination of the relationships at the gene level will contribute to treatment of metastatic cancer cases. We have aimed to demonstrate the impact of TGF-β1 and fluvastatin on human breast cancer (MCF-7) and human hepatocellular carcinoma (Hep3B) cell cultures via Real-Time Cell Analyzer (RTCA) and to test the expression levels of some genes (NDRG1, SGK1, TWIST1, AMPKA2) and to compare their gene expression levels according to RTCA results. Both of cell series were applied TGF-β1 and combinations of TGF-β1/fluvastatin. Primer and probes were synthesized using Universal Probe Library (UPL, Roche) software, and expression levels of genes were tested via qPCR using the device LightCycler 480 II (Roche). Consequently, fluvastatin dose-dependently inhibited migration induced by TGF-β1 in both groups. This inhibition was accompanied by low level of SGK1 messenger RNA (mRNA) and high levels of NDRG1 and AMPKA2 mRNA. Thus, we conclude that fluvastatin plays an important role in reducing resistance to chemotherapeutics and preventing metastasis.

Abstract 3547: Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model

Abstract Human telomerase reverse transcriptase (hTERT) RNA and p53 tumour suppressor were previously suggested as useful targets for cancer gene therapy. In this study, we investigated the effects of systemically delivered adenoviruses harboring trans-splicing ribozyme that could target hTERT and induce p53 transcript in targeted cancer cells in vivo using mice with Hep3B human hepatocellular carcinoma cells that metastasized to the liver. We observed that intravenous injection of this virus (0.5 X1011 Virus) produced efficient regression of Hep 3B tumor metastasized nodules. Next we performed in vitro study to know whether it has a synergistic effects with CDDP, we observed that CDDP (2.5 ug/ml) increased cytotoxic effects in vitro given with the virus (10 MOI), but in vivo study revealed that addition of CDDP or arsenic hexoxide (As4O6) did not improve the therapeutic efficacy of the virus treatment. Next we performed toxicity test with 4 doses (0.5 X1011, 1 X1011, 2 X1011, and 5 X1011, Virus), which revealed that there were no toxic death, but 2-4 fold increased in AST and ALT with no changes in bilirubin, BUN, Creatinine levels. In conclusion, this study demonstrates that the adenoviruses harboring trans-splicing ribozyme targeting hTERT and inducing p53 transcript represents a safe and promising cancer treatment for hepatocellular carcinoma. [This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (0820050), and National Research Foundation of Korea (NRF) grant funded by the Korea government(MEST) (20120002631).] Keywords: Adenovirus, trans-splicing ribozyme, p53 tumour suppressor, Human Telomerase Reverse Transcriptase, cancer Citation Format: Won Sup Lee, Hye Lim Kang, Arulkumar Nagappan, Jeong Won Yun, GonSup Kim, Soon Chan Hong, Sang-Jin Lee, In-Hoo Kim. Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2015-3547

Abstract 5501: Antagonism of sorafenib and regorafenib actions by epidermal growth factor in hepatoma cells

Abstract Background. Platelet numbers are related to growth and aggressiveness of several tumor types, including hepatoma (hepatocellular carcinoma). We previously found that platelet lysates also stimulated growth and migration of hepatoma lines in vitro and antagonized the growth-inhibitory effects on hepatoma cells of both multikinase inhibitors sorafenib and regorafenib (BMC Cancer 2014; 14:351). Aims. To investigate the mechanisms for the platelet-mediated multikinase drug resistance, we examined the actions of several mitogens that are known to be contained by platelets (PDGF, VEGF, TGF beta, FGF, IGF-1, EGF). Results. We found that epidermal growth factor (EGF) alone antagonized the growth inhibitory actions of Sorafenib or Regorafenib on HepG2, PLC/PRF/5 and Hep3B human hepatoma cells, using an MTT assay. Detailed studies revealed that EGF also antagonized Regorafenib-mediated inhibition of cell migration and invasion. Furthermore, EGF also blocked regorafenib-induced apoptosis, measured by Annexin V using a MUSE cell analyzer. These effects of EGF were antagonized by concomitant addition to the cultures of EGF receptor antagonist Erlotinib, showing that the EGF receptor was involved in the mechanisms of EGF-mediated blocking of Regorafenib effects. In addition, Erlotinib also partially blocked the effects of platelet lysates in antagonizing regorafenib-mediated growth inhibition. Similar antagonism was found using IGF-1, and those effects were blocked by GSK IGF-1 receptor antagonist. Other platelet factors had only weak actions in these assays. Conclusions. EGF antagonized regorafenib-mediated growth and migration inhibition and apoptosis induction in hepatoma cells. These EGF actions were mediated via the EGF receptor, as were the similar platelet lysate actions. Citation Format: Brian I. Carr, Rosalba D'Alessandro, Maria Grazia Refolo, Catia Lippolis, Caterina Messa, Aldo Cavallini. Antagonism of sorafenib and regorafenib actions by epidermal growth factor in hepatoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5501. doi:10.1158/1538-7445.AM2015-5501

Differential effects of tetrahydropyridinol derivatives on β-catenin signaling and invasion in human hepatocellular and breast carcinoma cells.

In continuation of previous efforts to investigate the biological potency of tetrahydropyridinol derivatives, the present study synthesized three target compounds: N-(bromoacetyl)-3-carboxyethyl-2,6-diphenyl-4-O-(pentafluorobenzoyl)-Δ3-tetra-hydropyridine (5a), N-(chloroacetyl)-3-carboxyethyl-2,6-diphenyl-4-O-(pentafluorobenzoyl)-Δ3-tetrahydropyridine (5b) and N-(2-bromopropanoyl)-3-carboxyethyl-2,6-diphenyl-4-O-(pentafluorobenzoyl)-Δ3-tetrahydropyridine (5c), and examined their anticancer potency. Experiments were performed using the Sk-Hep1 and Hep3B human hepatocellular carcinoma cell lines and MDA-MB-231 breast adenocarcinoma cell line. Among the three compounds, 5a and 5b were comparably and significantly cytotoxic to the Sk-Hep1, Hep3B and MDA-MB-231 cells. The highest level of cytotoxicity was detected in theSk-Hep1 cells with half maximal inhibitory concentrations for compounds 5a and 5b at 12 and 6 µM, respectively. These two compounds induced cell cycle arrest in the Sk-Hep1 and MDA-MB-231 cells through the downregulation of β-catenin and upregulation of glycogen synthase kinase-3β and E-cadherin. By contrast, 5a and 5b induced G1 arrest in the Hep3B cells by modulating the p21 and p27 cell cycle regulatory molecules and cyclin-dependent kinase 2. In addition, 5a and 5b significantly inhibited the invasion of Sk-Hep1 and MDA-MB-231 cells. These results suggested that the 5a and 5b compounds induce cell cycle arrest by suppressing Wnt/β-catenin signaling in highly invasive Sk-Hep1 and MDA-MB-231 cells, and by inducing p53 independent cell cycle arrest in Hep3B cells.

Preclinical Study of Locoregional Therapy of Hepatocellular Carcinoma by Bioelectric Ablation with Microsecond Pulsed Electric Fields (μsPEFs).

Unresectable hepatocellular carcinoma (HCC) needs locoregional ablation as a curative or downstage therapy. Microsecond Pulsed Electric Fields (μsPEFs) is an option. A xenograft tumor model was set up on 48 nude mice by injecting human hepatocellular carcinoma Hep3B cells subcutaneously. The tumor-bearing mice were randomly divided into 3 groups: μsPEFs treated, sham and control group. μsPEFs group was treated by μsPEFs twice in 5 days. Tumor volume, survival, pathology, mitochondria function and cytokines were followed up. μsPEFs was also conducted on 3 swine to determine impact on organ functions. The tumors treated by μsPEFs were completely eradicated while tumors in control and sham groups grew up to 2 cm3 in 3 weeks. The μsPEFs-treated group indicated mitochondrial damage and tumor necrosis as shown in JC-1 test, flow cytometry, H&E staining and TEM. μsPEFs activates CD56+ and CD68+ cells and inhibits tumor proliferating cell nuclear antigen. μsPEFs inhibits HCC growth in the nude mice by causing mitochondria damage, tumor necrosis and non-specific inflammation. μsPEFs treats porcine livers without damaging vital organs. μsPEFs is a feasible minimally invasive locoregional ablation option.

Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B

Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

Fibroblast Growth Factor (Fgf) 21 is a Novel Target Gene of Glucocorticoid Receptor

Fibroblast growth factor (Fgf) 21 is a metabolic regulator that plays an important role in the maintenance of glucose, lipid, and energy homeostasis. Recent studies also suggest that Fgf21 induction can attenuate chemical‐induced toxicities, such as of acetaminophen, alcohol and dioxin. Glucocorticoid receptor (GR) is indispensable for cell growth and development, drug metabolism as well as immune response. However, the impact of GR activation on regulation of Fgf21 is unknown. The present study was designed to investigate whether activation of GR by the synthetic glucocorticoid Dexamethasone (DEX), can alter Fgf21 expression. Our results showed that in mouse liver, both low (2mg/kg) and high (50mg/kg) doses of DEX up‐regulated Fgf21 mRNA and protein expression. In addition, DEX induced Fgf21 mRNA and protein expression in both mouse (Hepa1c1c7) and human (Hep3B) hepatoma cells in a concentration‐ and time‐dependent manner. Generally, DEX produces its effects via activation of GR and/or pregnane X receptor (PXR). By using PXR‐null mouse model, we showed that 50mg/kg of DEX induced Fgf21 mRNA expression similarly in both wild‐type and PXR‐null mouse liver, indicating that the induction of Fgf21 by DEX is PXR‐independent. The in silico DNA sequence analysis revealed the presence of several putative GR response elements (GREs) in the 3kb promoter of mouse and human Fgf21/FGF21 genes. Further, GR silencing using GR‐specific siRNA, attenuated DEX‐induced Fgf21 expression in both mouse (Hepa1c1c7) and human (Hep3B) hepatoma cells. In summary, activation of GR induced Fgf21 expression in mouse liver as well as in cultured mouse and human hepatocytes.

Leucine-rich α-2-glycoprotein promotes TGFβ1-mediated growth suppression in the Lewis lung carcinoma cell lines.

Leucine-rich α2-glycoprotein (LRG) is an approximately 50-kDa glycoprotein that has been found to be elevated in the sera of patients with several types of cancer. LRG directly binds to transforming growth factor beta 1 (TGFβ1) and modulates TGFβ1 signaling in endothelial cells; however, the precise function of LRG in cancer remains unclear. This study aimed to investigate the role of LRG in cancer. Lewis lung carcinoma (LLC) cells hardly expressed LRG. The growth of LLC tumors allografted in the LRG knockout (KO) mice was significantly increased compared with wild-type (WT) mice. Conversely, overexpression of LRG significantly inhibited the growth of LLC tumors in WT mice. In the presence of LRG, TGFβ1 significantly inhibited the proliferation of LLC cells and human hepatocellular carcinoma Hep3B cells in vitro by inducing apoptosis via the potent activation of smad2 and its downstream signaling pathway. Furthermore, administration of a TGFβR1 inhibitor (SB431542) significantly enhanced the growth of LLC tumors in WT mice compared with LRG KO mice via inhibition of apoptosis. We propose that LRG potentiates the effect of TGFβ1 in cancer cells whose growth is suppressed in the presence of TGFβ1.

Antiproliferative activity and phenotypic modification induced by selected Peruvian medicinal plants on human hepatocellular carcinoma Hep3B cells

Ethnopharmacological relevanceThe high incidence of human hepatocellular carcinoma (HCC) in Peru and the wide use of medicinal plants in this country led us to study the activity against HCC cells in vitro of somes species used locally against liver and digestive disorders. Materials and methodsEthnopharmacological survey: Medicinal plant species with a strong convergence of use for liver and digestive diseases were collected fresh in the wild or on markets, in two places of Peru: Chiclayo (Lambayeque department, Chiclayo province) and Huaraz (Ancash department, Huaraz province). Altogether 51 species were collected and 61 ethanol extracts were prepared to be tested.Biological assessment: All extracts were first assessed against the HCC cell line Hep3B according a 3-step multi-parametric phenotypic assay. It included 1) the evaluation of phenotypic changes on cells by light microscopy, 2) the measurement of the antiproliferative activity and 3) the analysis of the cytoskeleton and mitosis by immunofluorescence. Best extracts were further assessed against other HCC cell lines HepG2, PLC/PRF/5 and SNU-182 and their toxicity measured in vitro on primary human hepatocytes. ResultsEthnopharmacological survey: Some of the species collected had a high reputation spreading over the surveyed locations for treating liver problems, i.e. Baccharis genistelloides, Bejaria aestuans, Centaurium pulchellum, Desmodium molliculum, Dipsacus fullonum, Equisetum bogotense, Gentianella spp., Krameria lapacea, Otholobium spp., Schkuhria pinnata, Taraxacum officinale.Hep3B evaluation: Fourteen extracts from 13 species (Achyrocline alata, Ambrosia arborescens, Baccharis latifolia, Hypericum laricifolium, Krameria lappacea, Niphidium crassifolium, Ophryosporus chilca, Orthrosanthus chimboracensis, Otholobium pubescens, Passiflora ligularis, Perezia coerulescens, Perezia multiflora and Schkuhria pinnata) showed a significant antiproliferative activity against Hep3B cells (IC50≤ 50µg/mL). This was associated with a lack of toxicity on primary human hepatocytes in vitro. Immunofluorescence experiments on Hep3B cells showed that crude extracts of Schkuhria pinnata and Orthrosanthus chimboracensis could block Hep3B cells in mitosis with an original phenotype. Crude extracts of Perezia coerulescens, Perezia multiflora, Achyrocline alata, Ophryosporus chilca, Otholobium pubescens and Hypericum laricifolium could modify the overall microtubule cytoskeletal dynamics of Hep3B cells in interphase by an original mechanism. ConclusionsOur method allowed us to select 9 extracts which displayed antiproliferative activities associated with original cellular phenotypes on Hep3B cells, regarding known microtubule-targeting drugs. Both chemical and cellular studies are ongoing in order to elucidate natural compounds and cellular mechanisms responsible of the activities described.

Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells

Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Perylenetetracarboxylic anhydride as a precursor of fluorescent carbon nanoonion rings.

Thermal annealing at 400 °C of perylenetetracarboxylic anhydride in low molecular mass PEG gives rise to the formation of well defined nanoobjects of 2.5 nm height and size distribution from 10 to 65 nm (average 40 nm) after purification of the raw mixture with silicagel chromatography. TEM reveals that the flat nanoobjects are constituted of concentric graphenic rings (0.34 nm interlayer distance). The morphology of the nanoparticles resembles onion rings of nanometric dimensions (nanoonion rings C-NOR). C-NOR particles have an excitation dependent emission with λem from 430 to 570 nm and a maximum emission quantum yield of 0.49. C-NOR particles can be internalized into Hep3B human hepatoma cells as determined by confocal fluorescence microscopy and are remarkably biocompatible affecting slightly cell viability according to the MTT test.

Purification and cloning of lectin that induce cell apoptosis from Allium chinense

A 8.7 kDa lectin with high agglutin activity was isolated by affinity chromatography and cloned from Allium chinense in this study. For the MTT assay, approximately 60 µg/ml A. chinense lectin (ACL) inhibited 50% of the human hepatoma Hep-3B cells grown after 48 h. In addition, no antiproliferative effect was observed on normal human umbilical vein endothelial cells (HUVEC) even at 100 µg/ml concentration. After treatments with ACL on Hep-3B cells, morphologic changes in the nucleus and cytoskeleton were observed under laser scanning confocal microscopy with 4′,6-diamidino-2-phenylindole and tubulin Alexa Fluor 488 staining; whereas, the mitochondrial membrane potential was observed through Mito Tracker Red CMXRos staining. The results showed that ACL led to cell morphology and structure change (e.g., round cell shrinkage). Moreover, ACL resulted in significant change in the shape of the nucleus, damaged the cytoskeleton when tubulin was degraded, and reduced the mitochondrial transmembrane potential. By contrast, no changes were observed on HUVEC cells under the same treatment conditions. DNA fragmentation analysis was used to detect DNA damage. Western blot showed that ACL upregulated caspase-3 and Bax expression during apoptosis and cloned the structural gene of ACL with an open reading frame of 456 bp encoding 151 amino acid residues. The results showed that ACL is a potential anticancer drug.

Camptothecin sensitizes human hepatoma Hep3B cells to TRAIL-mediated apoptosis via ROS-dependent death receptor 5 upregulation with the involvement of MAPKs

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of malignant cancer cells, but several cancers have acquired potent resistance to TRAIL-induced cell death by unknown mechanisms. Camptothecin (CPT) is a quinolone alkaloid that induces cytotoxicity in a variety of cancer cell lines. However, it is not known whether CPT triggers TRAIL-induced cell death. In this study, we found that combined treatment with subtoxic doses of CPT and TRAIL (CPT-TRAIL) potentially enhanced apoptosis in a caspase-dependent manner. CPT-TRAIL effectively induced the expression of death receptor (DR) 5, which is a specific receptor of TRAIL, and treatment with a chimeric blocking antibody for DR5 reduced CPT-TRAIL-induced cell death, indicating that CPT functionally triggers DR5-mediated cell death in response to TRAIL. CPT-induced generation of reactive oxygen species (ROS) also preceded the upregulation of DR5 in response to TRAIL. The involvement of ROS in DR5 upregulation confirmed that pretreatment with antioxidants, including N-acetyl-L-cysteine and glutathione, significantly inhibits CPT-TRAIL-induced cell death by suppressing DR5 expression. The specific inhibitors of ERK and p38 also decreased CPT-TRAIL-induced cell death by blocking DR5 expression. In conclusion, our results suggest that CPT sensitizes cancer cells to TRAIL-mediated apoptosis via ROS and ERK/p38-dependent DR5 upregulation.