Human Hepatoblastoma Cell Line Research Articles

Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism in a TPA-treated human hepatocellular carcinoma cell line, HuH-7.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes. Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. The expression of IL-6 mRNA was measured using quantitative real-time PCR, and intracellular signaling molecules were detected using western blotting. When the HuH-7 human hepatocellular carcinoma cell line and HepG2 human hepatoblastoma cell line were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose-dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.

Focal adhesion kinase and its epigenetic interactors as diagnostic and therapeutic hints for pediatric hepatoblastoma.

Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients. Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The in vitro experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226. Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, AFP, EPCAM, OCT4, and SOX2, in association with anti-proliferative and pro-apoptotic effects on HB cells. Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability.

The importance of cell uptake in photothermal treatments mediated by biomimetic magnetic nanoparticles

Biomimetic magnetic nanoparticles (BMNPs) mediated by MamC have proven to be photothermal agents able to allow an optimized cytotoxicity against tumoral cells when used simultaneously as drug nanotransporters and as hyperthermia agents. However, it remains unclear whether BMNPs need to be internalized by the cells and/or if there is a threshold for internal Fe concentration for the photothermal therapy to be effective. In this study, three different situations for photothermal treatments have been simulated to disentangle the effect of BMNPs cell uptake on cell viability after photothermal treatments. Human hepatoblastoma (HepG2) cell line was treated with suspensions of BMNPs, and protocols were developed to have only intracellular BMNPs, only extracellular BMNPs or both, followed by photothermal exposure of the treated cell cultures. Our data demonstrate that: (1) Although the heating efficiency of the photothermal agent is not altered by its location (intra/extracellular), the intracellular location of BMNPs is crucial to ensure the cytotoxic effect of photothermal treatments, especially at low Fe concentration. In fact, the concentration of BMNPs needed to reach the same cytotoxic effect following upon laser irradiation of 0.2 W/cm2 is three times larger if BMNPs are located extracellularly compared to that needed if BMNPs are located intracellularly; (2) For a given location of the BMNPs, cell death increases with BMNPs (or Fe) concentration. When BMNPs are located intracellularly, there is a threshold for Fe concentration (∼ 0.5 mM at laser power intensities of 0.1 W/cm2) needed to affect cell viability following upon cell exposure to photothermia. (3) Bulk temperature rise is not the only factor accounting for cell death. Actually, temperature increases inside the cells cause more damage to cell structures and trigger cell death more efficiently than an increase in the temperature outside the cell.

Everolimus inhibits hepatoblastoma by inducing autophagy-dependent ferroptosis.

Everolimus, a known inhibitor of the mammalian target of rapamycin (mTOR), has shown uncertain efficacy in treating hepatoblastoma. This study delves into the potential anti-hepatoblastoma properties of everolimus and its intricate relationship with autophagy and ferroptosis, both in vitro and in vivo. In vivo, tumor tissue from hepatoblastoma patient and human hepatoblastoma cell line HuH-6 were xenografted into nude mice to establish xenograft models for observing the effect of everolimus on tumor growth. In vitro, HuH-6 cells were cultured to evaluate the anti-hepatoblastoma activity of everolimus. Transmission electron microscopy and microtubule-associated proteins 1 light chain 3 (LC3), beclin 1, and p62 protein expressions were employed to investigate autophagy. Additionally, indicators of cell apoptosis, reactive oxygen species (ROS) and proteins associated with ferroptosis were measured to evaluate ferroptosis. The results demonstrate that everolimus treatment effectively induced the formation of autophagosomes in hepatoblastoma cells, upregulated the LC3II/I ratio and beclin 1 expression, and downregulated p62 expression, indicating an enhanced autophagy level both in vitro and in vivo. Furthermore, everolimus treatment induced cell apoptosis, increased ROS level, elevated concentrations of malondialdehyde, 4-hydroxynonenal, and iron content, while reducing the ratio of glutathione/oxidized glutathione, and downregulating the protein expression of glutathione peroxidase 4 and solute carrier family 7 member 11, suggesting its ability to induce ferroptosis in hepatoblastoma cells. Importantly, the induction of ferroptosis by everolimus was significantly reversed in the presence of autophinib, an autophagy inhibitor, indicating the autophagy-dependent of everolimus-induced ferroptosis. Taken together, these findings suggest that everolimus holds promise as an effective anti-hepatoblastoma drug, with its mechanism of action potentially involving the induction of autophagy-dependent ferroptosis in hepatoblastoma cells.

Sedanolide Activates KEAP1-NRF2 Pathway and Ameliorates Hydrogen Peroxide-Induced Apoptotic Cell Death.

Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death probably due to preventing the decrease in the mitochondrial membrane potential and the increase in caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway.

Investigation of TGF-α-overexpressing mouse hepatocytes (TAMH) cultured as spheroids for use in hepatotoxicity screening of drug candidates.

The immortalized mouse liver cell line TAMH has been described as a valuable tool for studying hepatotoxic mechanisms, but until now, it has only been reported to grow as a monolayer in culture. However, culturing hepatocytes as three-dimensional (3D) spheroids has been shown to result in improved liver-specific functions (e.g., metabolic capacity) by better mimicking the in vivo environment. This approach may lead to more reliable detection of drug-induced liver injury (DILI) in the early phase of drug discovery, preventing post-marketing drug withdrawals. Here, we investigated the cultivation of TAMH as 3D spheroids, characterizing them with optical and transmission electron microscopy as well as analyzing their gene expression at mRNA level (especially drug-metabolizing enzymes) compared to TAMH monolayer. In addition, comparisons were made with spheroids grown from the human hepatoblastoma cell line HepG2, another current spheroid model. The results indicate that TAMH spheroids express hepatic structures and show elevated levels of some of the key phase I and II drug-metabolizing enzymes, in contrast to TAMH monolayer. The in vitro hepatotoxic potencies of the drugs acetaminophen and flupirtine maleate were found to be very similar between TAMH spheroidal and the monolayer cultures. Both the advantages and disadvantages of TAMH spheroids as an in vitro hepatotoxicity model compared to monolayer model are discussed.

Exploring thyroxine binding globulin structural changes and its release from human hepatoblastoma cells upon interaction with silica particles: A prelude to unrevealing the mechanism of thyroid hormone dysregulation

Endocrine dysregulation in the presence of environmental chemical risk factors is a global adverse health concern. The aim of this investigation was to explore the structural changes and binding affinity of thyroxine (T4) binding protein (TBG) upon interaction with SiO2 particles as the second largest mineral in the Earth's crust and one of the most important constituents of rock, soil, and dust. Therefore, the interaction of TBG with SiO2 particles was assessed by fluorescence quenching, molecular docking, ANS and synchronous fluorescence, and far-UV CD analyses. Also, the release of TBG from human hepatoblastoma cell line, Hep G2, was assessed by ELISA assay. The results displayed that the value of stoichiometry of binding site (n) of TBG for T4 was approximately equal to one, which was reduced to 0.36 in the presence of SiO2 particles. Also, the binding affinity (Kb) values revealed that the binding affinity between T4 and TBG was strong (97.90 × 105 L/mol), while the presence of SiO2 particles resulted in the calculation of a Kb around 0.00159 × 105 L/mol, which was significantly lower than that of the absence of SiO2 particles. This data was also verified by molecular docking analyses which indicated that SiO2 particles interacted with the T4 binding pocket of TBG. Moreover, further studies exhibited that although the equimolar concentration of T4 to TBG resulted in the superior stability of TBG-T4 complex relative to free TBG, the presence of SiO2 particles with the same concentration led to denaturation of the secondary structure of TBG. Furthermore, it was seen that the amount of released TBG in the cell culture medium of Hep G2 was about 2.21 ng/mL protein, whereas this amount in SiO2 particles-treated cell group was significantly reduced to 1.71 ng/mL protein (*P < 0.05). In conclusion, this study implies that SiO2 particles show the potential to result in inhibition of TBG release, TBG denaturation, and interfere with TBG binding affinity which may lead to dysregulation of the thyroid hormone transport and associated signaling pathways.

New Anthranilic Acid Hydrazones as Fenamate Isosteres: Synthesis, Characterization, Molecular Docking, Dynamics & in silico ADME, in vitro Anti-inflammatory and Anti-cancer Activity Studies.

In this study, twenty new anthranilic acid hydrazones 6-9 (a-e) were synthesizedand their structures were characterized by Fourier-transform Infrared (FTIR), Nuclear Magnetic Resonance (1H NMR - 13C NMR), and High-resolution Mass Spectroscopy (HRMS). The inhibitory effects of thecompounds against COX-II were evaluated. IC50 values of the compounds were found in the range of >200 - 0.32 µM and compounds 6e, 8d, 8e, 9b, 9c, and 9e were determined to be the most effective inhibitors. Cytotoxic effects of the most potent compounds were investigated against human hepatoblastoma (Hep-G2) and human healthy embryonic kidney (Hek-293) cell lines. Doxorubicin (IC50: 8.68 ± 0.16 µM for Hep-G2, 55.29 ± 0.56 µM for Hek-293) was used as standard.8e is the most active compound, with low IC50against Hep-G2 (4.80 ± 0.04 µM), high against Hek-293 (159.30 ± 3.12), and high selectivity (33.15). Finally, molecular docking and dynamics studies were performed to understand ligand-protein interactions between the most potent compounds and COX II, Epidermal Growth Factor Receptor (EGFR), and Transforming Growth Factor beta II (TGF-βII). The docking scores were calculated in the range of -10.609 - -6.705 kcal/mol for COX-II, -8.652 - -7.743 kcal/mol for EGFR and -10.708 - -8.596 kcal/mol for TGF-βII.

Legal Cannabis sativa L. Dried Inflorescences: Cannabinoids Content and Cytotoxic Activity against Human HepG2 Cell Line

Cannabis sativa L. has health benefits, principally due to the levels and ratios of two important cannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC:CBD ratio affects their pharmacological interaction for the treatment of different diseases as well as its modulation allows for a custom-made product that utilizes the distinguishing effects of CBD, THC, or both, for a peculiar patient or clinical effect. This study aims to investigate the total content of THC, CBD, and their ratio in 34 dried inflorescence legally sold in physical and online stores, by using a validated liquid chromatography-ultraviolet (HPLC-UV) method, after cannabinoids identification performed through MSn studies. Cannabinol (CBN) content was also monitored to evaluate hemp age or conservation status. CBN content always resulted lower than limit of quantification, thus confirming well-stored fresh hemp. All investigated samples showed a total THC amount below 0.59% w/w, thus responding to legal requirements.. The total CBD amount ranged from 2.62 to 20.27% w/w and it was not related to THC level. THC:CBD ranged among 1:3 and 1:26, thus ascertaining their suitability for different target pharmacological uses. In vitro studies using human hepatoblastoma cell line HepG2 suggested that hemp extracts with THC:CBD ratios of 1:9 exhibited higher toxicity than pure cannabinoids.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

The Effects of Protein Phosphatase 2A Activation with Novel Tricyclic Sulfonamides on Hepatoblastoma

BackgroundThe tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. We aimed to examine the effects of two novel compounds of the tricyclic sulfonamide class, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression, on human hepatoblastoma. MethodsAn established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were treated with increasing doses of 3364 or 8385, and viability, proliferation, cell cycle and motility were investigated. Cancer cell stemness was evaluated by real-time PCR and tumorsphere forming ability. Effects on tumor growth were examined using a murine model. ResultsTreatment with 3364 or 8385 significantly decreased viability, proliferation, cell cycle progression and motility in HuH6 and COA67 cells. Both compounds significantly decreased stemness as demonstrated by decreased abundance of OCT4, NANOG, and SOX2 mRNA. The ability of COA67 to form tumorspheres, another sign of cancer cell stemness, was significantly diminished by 3364 and 8385. Treatment with 3364 resulted in decreased tumor growth in vivo. ConclusionNovel PP2A activators, 3364 and 8385, decreased hepatoblastoma proliferation, viability, and cancer cell stemness in vitro. Animals treated with 3364 had decreased tumor growth. These data provide evidence for further investigation of PP2A activating compounds as hepatoblastoma therapeutics.

SLC-0111, an inhibitor of carbonic anhydrase IX, attenuates hepatoblastoma cell viability and migration.

In response to hypoxia, tumor cells undergo transcriptional reprogramming including upregulation of carbonic anhydrase (CA) IX, a metalloenzyme that maintains acid-base balance. CAIX overexpression has been shown to correlate with poor prognosis in various cancers, but the role of this CA isoform in hepatoblastoma (HB) has not been examined. We surveyed the expression of CAIX in HB specimens and assessed the impact of SLC-0111, a CAIX inhibitor, on cultured HB cells in normoxic and hypoxic conditions. CAIX immunoreactivity was detected in 15 out of 21 archival pathology HB specimens. The CAIX-positive cells clustered in the middle of viable tumor tissue or next to necrotic areas. Tissue expression of CAIX mRNA was associated with metastasis and poor clinical outcome of HB. Hypoxia induced a striking upregulation of CAIX mRNA and protein in three HB cell models: the immortalized human HB cell line HUH6 and patient xenograft-derived lines HB-295 and HB-303. Administration of SLC-0111 abrogated the hypoxia-induced upregulation of CAIX and decreased HB cell viability, both in monolayer and spheroid cultures. In addition, SLC-0111 reduced HB cell motility in a wound healing assay. Transcriptomic changes triggered by SLC-0111 administration differed under normoxic vs. hypoxic conditions, although SLC-0111 elicited upregulation of several tumor suppressor genes under both conditions. Hypoxia induces CAIX expression in HB cells, and the CAIX inhibitor SLC-0111 has in vitro activity against these malignant cells.

Coinfection of Clonorchis sinensis and hepatitis B virus: clinical liver indices and interaction in hepatic cell models

BackgroundIn China, people infected with hepatitis B virus (HBV) are commonly found in areas with a high prevalence of Clonorchis sinensis, a trematode worm. Published studies have reported that the progression of hepatitis B is affected by coinfection C. sinensis.MethodsClinical data from a total of 72 patients with C. sinensis and HBV (as sole infection or with coinfections) and 29 healthy individuals were analysed. We also incubated the hepatic stellate cell line LX-2 with total proteins from C. sinensis adult worms (CsTPs) and HBV-positive sera. In addition, the human hepatoblastoma cell line HepG2.2.15 was treated with the antiviral drug entecavir (ETV), CsTPs and the anti-C. sinensis drug praziquantel (PZQ).ResultsOur clinical data indicated that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB) and hyaluronic acid (HA) were significantly higher in patients with coinfection than in those infected with HBV only. In cell models, compared with the model in which LX-2 cells were incubated with HBV-positive sera (HBV group), transcripts of alpha-smooth muscle actin and types I and III collagen were significantly elevated in the models of LX-2 cells treated with CsTPs and HBV-positive sera (CsTP+HBV group), while the messenger RNA levels of tumour necrosis factor-α, interleukin (IL)-1β and IL-6 in the CsTP+HBV group were clearly lower. The HBV surface antigen and hepatitis B e-antigen levels were higher in the HepG2.2.15 cells treated with ETV and CsTPs than in those in the ETV group and in the cells administered a mixture of ETV, CsTPs and PZQ.ConclusionsThese results confirmed that C. sinensis and HBV coinfection could aggravate the progression of liver fibrosis. CsTPs might promote chronic inflammation of the liver in individuals with HBV infection, resulting in the development of hepatic fibrosis.Graphic abstract

Preclinical Evidence Supporting Novel Protein Phosphatase 2A Activators as Therapeutics in Hepatoblastoma

INTRODUCTION: The tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. Our laboratory has shown that PP2A activation using the sphingosine analog, FTY720, decreased hepatoblastoma viability in vitro and tumor growth in vivo. Because FTY720 has significant immunosuppressive properties, we wished to examine the effects 2 novel compounds, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression in hepatoblastoma. METHODS: The established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, COA67, were used. Cells were treated with increasing doses of 3364 or 8385 for 24 hours. Proliferation and viability were investigated using CellTiter96 and alamarBlue assays, respectively. The ability of the COA67 cells to form tumor spheres, a marker of cancer cell stemness, was assessed following treatment with 3364 or 8385 (2 μM). RESULTS: Viability was significantly decreased following doses as low as 5 μM treatment in HuH6 (Fig. 1A, upper panel) and 0.5 μM treatment in COA67 (Fig. 1A, lower panel). Proliferation was significantly decreased in HuH6 after treatment with 3364 (7.5 μM, 57 ± 0.1%; p ≤ 0.01) and 8385 (12.5 μM, 54 ± 0.1%; p ≤ 0.01) vs 0 μM and COA67 cells at 4 μM (3364: 64 ± 0.7%; 8385: 93 ± 0.3%, vs 0 μM; p ≤ 0.05) after treatment. Both compounds significantly affected the ability of COA67 cells to form tumor spheres (Fig. 1B).CONCLUSION: The novel PP2A activators, 3364 and 8385, decreased proliferation, viability, and stemness in hepatoblastoma. These findings provide evidence for further investigation of these novel PP2A activators as potential therapeutics for hepatoblastoma.

Multiomics characterization of mouse hepatoblastoma identifies yes-associated protein 1 target genes.

Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1) S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis -regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

Inhibitory effects of LOXL2 knockdown on cellular functions of liver cancer stem cells.

BackgroundLysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.MethodsCD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knockdown in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, immunofluorescence and reverse transcription-PCR (RT-PCR).ResultsKnockdown of LOXL2 decreased spheroid formation, migration and invasion (P<0.05), also induced apoptosis (P<0.05) and cell cycle arrest (P<0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (P<0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3B) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (P<0.01).ConclusionsThe results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

Chemical investigations and cytotoxic effects of metabolites from Antrodia camphorata against human hepatocellular carcinoma cells

Antrodia camphorata is used as a medicinal fungus in Taiwan to treat fatigue, food intoxication, and enhance liver function. Here we identified fermented metabolic components from the mycelium of A. camphorata KH37 and explored their anti-hepatoma potentials with study models of human hepatoblastoma cell lines. Bioassay-guided fractionation of the solid fermentation powder of A. camphorata KH37 led to the isolation of one new quinonol, antroquinonol Z (1), and nine known compounds (2–10). Treatment with 10 μM antrocamols LT1 (2) or LT3 (3) reduced cell viability of HepG2 and Huh-7 cells to about 60% in 48 hours. Antroquinonol Z (1) exhibited mild cytotoxicity against Huh-7 cells in 48 and 72 hours. Interestingly, two fractions showed cytotoxicity in HepG2 and Huh-7 cells, even better than compounds isolated from these fractions. The significant cytotoxicity of partially purified samples from A. camphorata KH37 exhibited a potential for developing alternative or complementary therapeutics against hepatoma.

Inhibition of cIAP1 in the effective suppression of chemotherapy‑resistant hepatoblastoma.

Cellular inhibitor of apoptosis protein‑1 (cIAP1) is a key regulator of programmed cell death and is known to be associated with chemotherapeutic resistance. The present study aimed to investigate the antitumor efficacy of birinapant, a novel selective inhibitor of cIAP1, against cisplatin (CDDP)‑resistant hepatoblastoma (HB) cells. Western blot analysis was used to investigate the antitumor effect of birinapant on cIAP1 expression in Huh6 cells at the protein level. A WST‑8 assay was performed to evaluate the tumor growth inhibitory effect of birinapant on the human HB cell lines, Huh6 and HepG2. Huh6 cells were exposed to CDDP and/or birinapant in order to confirm tumor growth inhibition. The antitumor efficacy of birinapant plus CDDP combination therapy was significantly higher than that of CDDP monotherapy in a dose‑dependent manner (P=0.035). The study also investigated the antitumor efficacy of birinapant plus CDDP combination therapy in an established xenograft model of SCID mice. Compared with CDDP monotherapy, birinapant combined with CDDP showed better inhibition of tumor growth (P=0.121). It was observed that the mRNA expression of cIAP1 in tumors was significantly enriched in the CDDP monotherapy group compared with that in the untreated group. Furthermore, immunohistochemical staining was performed to compare cIAP1 expression in pre‑ and post‑chemotherapy specimens in patients with HB, and a significant increase was observed in the post‑chemotherapy specimens (P<0.001). CDDP‑resistant Huh6 (Huh6‑CDDPR) cells were also established following repeated exposure to CDDP. Birinapant was substantially more effective against the Huh6‑CDDPR cells than against the Huh6 wild‑type cells. Taken together, these findings suggest that repeated exposure to CDDP enhances cIAP1 expression in HB cells and that birinapant is a promising therapeutic drug for CDDP‑resistant HB.

Metastatic human hepatoblastoma cells exhibit enhanced tumorigenicity, invasiveness and a stem cell-like phenotype

Background/PurposeMetastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. Materials/MethodsUsing a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. ResultsThe HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. ConclusionThis newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

Isolation of the hemeoxygenase-1 inducer from rice-derived peptide.

Bioactive peptides with various health benefits have been reported from rice protein hydrolysates. We previously showed that rice-derived peptides (RP) increased intracellular glutathione levels and induced the expression of γ-glutamylcysteine synthetase, which is regulated by nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is an important Nrf2 downstream antioxidant enzyme that protects against oxidative stress. This study aimed to investigate the protective effects of RP on hydrogen peroxide (H2O2)-induced oxidative stress in human hepatoblastoma cell line HepG2 and identified HO-1 induced peptides from RP. Pretreatment of cells with RP reduced the cytotoxicity caused by H2O2 in a dose-dependent manner. Moreover, RP induced HO-1 expression in a concentration- and time-dependent manner. Next, we attempted to isolate the HO-1 inducer from RP by bioactivity-guided fractionation. Purification of the active peptides using a Sep-Pak C18 cartridge and reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of the three peptides. These peptides effectively reduced H2O2-induced oxidative stress. Among them, only P3 (peptide sequence: RSAVLLSH) increased HO-1 protein expression. Additionally, the knockdown of Nrf2 suppressed the induction of HO-1 expression by P3. Our results indicated that P3 identified from RP induced HO-1 by activating the Nrf2 signaling pathway.