Hepatoblastoma Cell Line HepG2 Research Articles

Alsterpaullone induces apoptosis of HepG2 cells via a p38 mitogen-activated protein kinase signaling pathway.

Alsterpaullone (Alp) is a small-molecule inhibitor that targets cyclin-dependent kinases to inhibit tumor cell activity. However, to the best of our knowledge, the effect of Alp on hepatoblastoma has not been investigated. Therefore, the function of Alp in apoptotic induction of hepatoblastoma cells and a potential mechanism of action were investigated. Results indicated that low doses of Alp (1 µM) significantly induced apoptosis in the HepG2 hepatoblastoma cell line. In vivo experiments of tumor suppression further indicated that Alp (3 mg/kg) exerted an inhibitory effect on HepG2 ×enograft tumor growth. Following Alp treatment, the expression level of B-cell lymphoma 2 (Bcl-2)-associated X protein, and cleaved caspase-3 and −9 in HepG2 cells was significantly increased; however, the expression of Bcl-2 was significantly decreased. In addition, phosphorylation of p38 mitogen-activated protein kinase (MAPK) significantly decreased Alp-induced caspase-3 and −9 activation. These results suggested that Alp induces apoptosis and inhibited proliferation via the p38MAPK signaling pathway. Therefore, Alp may be a therapeutic agent for treating hepatoblastoma.

Evaluation of Aflatoxin M1 Effects on the Metabolomic and Cytokinomic Profiling of a Hepatoblastoma Cell Line.

Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in mothers’ milk and in marketed milk products, in this study we decided to test the effects of AFM1 on a hepatoblastoma cell line (HepG2). Firstly, we evaluated the effects of AFM1 on the cell viability, apoptosis, cell cycle, and metabolomic and cytokinomic profile of HepG2 cells after treatment. AFM1 induced: (1) a decrease of HepG2 cell viability, reaching IC50 at 9 µM; (2) the blocking of the cell cycle in the G0/G1 phase; (3) the decrease of formiate levels and incremented level of some amino acids and metabolites in HepG2 cells after treatment; and (4) the increase of the concentration of three pro-inflammatory cytokines, IL-6, IL-8, and TNF-α, and the decrease of the anti-inflammatory interleukin, IL-4. Our results show that AFM1 inhibited the growth of HepG2 cells, inducing both a modulation of the lipidic, glycolytic, and amino acid metabolism and an increase of the inflammatory status of these cells.

Curcumin and allopurinol ameliorate fructose-induced hepatic inflammation in rats via miR-200a-mediated TXNIP/NLRP3 inflammasome inhibition.

Excess fructose consumption causes high prevalence of metabolic syndrome and inflammatory liver diseases. The aim of the current study was to investigate the therapeutic effects and underlying molecular mechanisms of curcumin and allopurinol in high fructose-induced hepatic inflammation. Male Sprague-Dawley rats were supplied with standard rat chow and drinking water containing 10% (w/v) fructose for consecutive 12 weeks. Curcumin (15, 30 and 60 mg/kg) and allopurinol (5 mg/kg) were administered to rats via oral gavage daily from Week 7 to 12. For in vitro experiments, curcumin (2.5 μM) and allopurinol (100 μM) were treated to 5 mM fructose-exposed Buffalo rat liver cell line (BRL-3 A) and human hepatoblastoma cell line (HepG2), respectively. The data from these animal and hepatocyte models showed that curcumin and allopurinol ameliorated fructose-induced metabolic symptom, especially hepatic inflammation in rats. Interestingly, down-regulation of microRNA-200a (miR-200a) was screened out in livers of fructose-fed rats and then validated in fructose-exposed BRL-3 A and HepG2 cells. Fructose-induced miR-200a low-expression was identified as a negative mediator of thioredoxin interacting protein (TXNIP) by direct targeting of 3'UTR-rTXNIP, subsequently activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in BRL-3 A cells. Curcumin, as well as allopurinol, notably up-regulated miR-200a expression, accordingly, down-regulated TXNIP and inhibited NLRP3 inflammasome activation in fructose-fed rat livers and fructose-exposed BRL-3 A and HepG2 cells. Taken together, this study firstly identified miR-200a as a biomarker of fructose-induced hepatic inflammation, and revealed the hepatoprotection of curcumin and allopurinol via up-regulating miR-200a-mediated TXNIP/NLRP3 inflammasome pathway.

In vitro toxicity assessment of rivaroxaban degradation products and kinetic evaluation to decay process

Degradation kinetics of oral anticoagulant rivaroxaban (RIV) was assessed in acid and alkaline media and while exposed to UVC radiation. Among all stress conditions tested, kinetic degradation process was better described by a zero-order model. A stability indicating method was validated for the analysis of the anticoagulant RIV in tablets by high-performance liquid chromatography. Robustness was evaluated with a two-level Plackett–Burman experimental design. The effect of acute exposition of the human hepatoblastoma HepG2 cell line to RIV stressed samples (100 and 500 µM) was assessed through in vitro toxicity tests. MTT reduction, neutral red uptake, mitochondrial membrane potential, and low molecular weight DNA diffusion assays were employed for cytotoxicity evaluation (5×104 cells/well). The genotoxic potential was assessed by comet assay (2×104 cells/well). Acute toxicity to HepG2 cells was assessed after 24 h incubation with sample solutions, for each test. A direct relationship between the increased amount of alkaline degradation products and higher cytotoxic potential was found. Results obtained by viability assay investigations support the concerns on risks associated with acute toxicity and genotoxicity of pharmaceutical samples containing degradation products as impurities.

Inhibition of CREB binding protein-beta-catenin signaling down regulates CD133 expression and activates PP2A-PTEN signaling in tumor initiating liver cancer cells

BackgroundThe WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells.MethodsHuman hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitro. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation.ResultsCBP inhibitor ICG001 and CBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phosphoSer380/Tyr382/383PTEN, phosphoSer473-AKT, Phospho-Ser552beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid.ConclusionsCBP-beta-catenin signaling promotes stemness via CD133 induction and cell proliferation in TICs. We found a novel functional link between CBP-beta-catenin and PP2A-PTEN-AKT pathway in liver TICs. Therefore, CBP-beta-catenin-PP2A-PTEN-AKT signaling axis could be a novel therapeutic target to prevent liver tumor initiation and cancer recurrence.

Effect of a third-generation oncolytic herpes simplex virus on the growth of human hepatocellular carcinoma.

321 Background: Multimodality therapies are used to manage patients with hepatocellular carcinoma (HCC), although advanced HCC is incurable. The third-generation oncolytic herpes simplex virus type 1 (HSV-1) T-01 replicates efficiently and selectively in tumor cells, kills tumor cells without damaging the surrounding normal tissues, and induces host immune responses specific to tumor cells. Here we investigated the antitumor effects of T-01 on HCC. Methods: The cytopathic activities of T-01 were tested at different multiplicities of infection in 14 human and one murine hepatoma cell lines in vitro. In mouse various xenograft models, HuH-7, KYN-2, PLC/PRF/5 and HepG2 human cells and Hepa1-6 murine cells were used to investigate the in vivo efficacy of T-01. Results: T-01 was cytotoxic to 13 cell lines (in vitro), including 10 human HCCs, two human hepatoblastomas, and one murine hepatoma. In mouse xenograft models of subcutaneous, orthotopic, and peritoneal tumor metastasis in athymic mice (BALB/c nu/nu), the growth of tumors formed by the HCC cell lines HuH-7, KYN-2, and PLC/PRF/5 as well as those formed by the hepatoblastoma cell line HepG2 was inhibited by T-01 compared with that of mock-inoculated tumors. In a bilateral Hepa1-6 subcutaneous tumor model in C57BL/6 mice, the growth of tumors inoculated with T-01 was inhibited and, in the contralateral tumors without T-01, T-01 also significantly reduced tumor growth compared with mock-infected tumors. T-01 infection significantly enhanced antitumor efficacy via T cell-mediated immune responses. Conclusions: These results demonstrate that a third-generation oncolytic HSV-1 may serve as a novel treatment for patients with HCC.

Sigma-1 receptor (σ1R) is downregulated in hepatic malignant tumors and regulates HepG2 cell proliferation, migration and apoptosis.

Sigma-1 receptor (σ1R), an important transmembrane structural protein, has been demonstrated to be overexpressed in various types of human cancer, and has been confirmed to be involved in many biological behaviors during tumorigenesis and tumor progression. The aim of the present study was to explore the essential role of σ1R in hepatic malignant tumors (HMTs), which, to the best of our knowledge, has not been reported to date. We assessed σ1R expression in hepatocellular carcinoma (HCC) tissues and found that σ1R was significantly decreased in HCC when compared with that in benign liver tissues (P<0.01). Additionally, the expression of σ1R was shown to be inversely correlated with HCC grade (r=-0.424, P=0.021, Kendall's τ-b-test). We further used a FLAG‑SV40‑neomycin‑plasmid strategy to increase σ1R expression in the HepG2 hepatoblastoma cell line. Overexpression of σ1R impaired cell proliferation, inhibited cell migration, induced cell cycle arrest at G1phase, and increased cell apoptosis invitro. Furthermore, overexpression of σ1R decreased the expression levels of STAT-3 and NF-κB, which provided insight into the underlying mechanisms of σ1R-associated HMT development and progression. These findings suggest that the decreased expression of σ1R plays an essential role in hepatic tumorigenesis, and that it may serve as a potential predictive factor and therapeutic target for the treatment of HMTs.

MiR‑378a enhances the sensitivity of liver cancer to sorafenib by targeting VEGFR, PDGFRβ and c‑Raf.

Liver cancer is a globally prevalent cancer with poor prognosis. The present study investigated the link between microRNA-378a (miR‑378a) expression and the sensitivity of hepatocellular carcinoma (HCC) and hepatoblastoma (HB) cancers to sorafenib therapy. miR‑378a expression was determined in liver tissue samples from healthy candidates and patients with liver cancer using the reverse transcription‑quantitative polymerase chain reaction. The antitumor effects of miR‑378a alone and in combination with sorafenib were investigated in the HB cell line HepG2 and the HCC cell line SMMC‑7721 with methyl thiazoyl tetrazolium, colony formation, flow cytometry and Transwell migration assays. The underlying mechanisms were investigated using western blot analysis. miR‑378a expression was decreased in tissue samples from patients with liver cancer. HCC and HB cell line proliferation and invasion ability was inhibited by miR‑378a. The combination of miR‑378a and sorafenib provided the greatest inhibition. Western blot indicated that mitogen activated protein kinase signaling pathway proteins, vascular endothelial growth factor receptor, platelet derived growth factor receptorβ, Raf‑1 proto‑oncogene, serine/threonine kinase and matrix metallopeptidase2 were regulated by miR‑378a alone and to a greater extent when combined with sorafenib. Results suggest that miR‑378a can inhibit liver cancer cell growth and enhance the sensitivity of liver cancer cells to sorafenib‑based chemotherapies.

Heme Oxygenase‐1 May Affect Cell Signalling via Modulation of Ganglioside Composition

Heme oxygenase 1 (Hmox1), a ubiquitous enzyme degrading heme to carbon monoxide, iron, and biliverdin, is one of the cytoprotective enzymes induced in response to a variety of stimuli, including cellular oxidative stress. Gangliosides, sialic acid-containing glycosphingolipids expressed in all cells, are involved in cell recognition, signalling, and membrane stabilization. Their expression is often altered under many pathological and physiological conditions including cell death, proliferation, and differentiation. The aim of this study was to assess the possible role of Hmox1 in ganglioside metabolism in relation to oxidative stress. The content of liver and brain gangliosides, their cellular distribution, and mRNA as well as protein expression of key glycosyltransferases were determined in Hmox1 knockout mice as well as their wild-type littermates. To elucidate the possible underlying mechanisms between Hmox1 and ganglioside metabolism, hepatoblastoma HepG2 and neuroblastoma SH-SY5Y cell lines were used for in vitro experiments. Mice lacking Hmox1 exhibited a significant increase in concentrations of liver and brain gangliosides and in mRNA expression of the key enzymes of ganglioside metabolism. A marked shift of GM1 ganglioside from the subsinusoidal part of the intracellular compartment into sinusoidal membranes of hepatocytes was shown in Hmox1 knockout mice. Induction of oxidative stress by chenodeoxycholic acid in vitro resulted in a significant increase in GM3, GM2, and GD1a gangliosides in SH-SY5Y cells and GM3 and GM2 in the HepG2 cell line. These changes were abolished with administration of bilirubin, a potent antioxidant agent. These observations were closely related to oxidative stress-mediated changes in sialyltransferase expression regulated at least partially through the protein kinase C pathway. We conclude that oxidative stress is an important factor modulating synthesis and distribution of gangliosides in vivo and in vitro which might affect ganglioside signalling in higher organisms.

Deoxynivalenol and Nivalenol Toxicities in Cultured Cells: a Review of Comparative Studies.

The in vitro studies of the toxicities of trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) including cell proliferation, cytokine secretion, and the involvement of heat shock protein 90 (Hsp90) in their toxicities were reviewed. Trichothecene mycotoxins are extremely toxic to leukocytes and leukopenia is one of the leading signs of trichothecene toxicosis, implying that trichothecenes hinder cell proliferation. Both toxins retarded proliferation of all four cell lines tested. NIV was more potent than DON in human promyelocytic leukemia cell line HL60, human lymphoblastic leukemia cell line MOLT-4, and rat aortic myoblast cell line A-10. In contrast, both toxins exhibited almost the same potencies in human hepatoblastoma cell line HepG2. While exposure to 0.3 μg/mL DON greatly induced the secretion of anti-hematopoietic cytokines CCL3 and CCL4, treatment with NIV decreased the secretion of these cytokines in HL60 cells, indicating that the toxicity mechanisms of these mycotoxins differ. Because molecular chaperone Hsp90 occupies a pivotal position in a wide range of pathological processes, the effects of an Hsp90-specific inhibitor radicicol on cytokine secretions were investigated. Radicicol counteracted the effect of DON on cytokine secretion, indicating that Hsp90 plays a crucial role in DON-induced cytokine secretion in HL60 cells. Conversely, the results of co-treatment with NIV and radicicol indicate that radicicol does not mitigate the effect of NIV. Regarding CCL3 and CCL4 secretions, DON and NIV have Hsp90-related and -unrelated mechanisms of toxicities, respectively. Taken together the results suggest that, although these toxins share similar chemical structure, there are differences in their toxic mechanisms.

Effect of 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47) and its metabolites on cell viability, oxidative stress, and apoptosis of HepG2

2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47), an extensively used brominated flame retardant (BFR), is frequently detected in biotic environments. To date, studies have reported that BDE-47 induces hepatotoxicity, reproductive toxicity, and neurotoxicity in vitro. However, little is known regarding BDE-47 metabolites-mediated cell toxicity in relevant human cell models. The cytotoxic effects of BDE-47 and its eight metabolites on hepatoblastoma cell line-HepG2 cells were investigated in this study. We found that BDE-47 and all its metabolites inhibited cell viability in both a dose- and time-dependent manner. For BDE-47 and its debromination products (BDE-28 and BDE-7), they had less severe effects on cell viability when the cells were pretreated with lower dose of the same compound, however, no significant difference was observed in control, suggesting that low concentrations have an adaptation effect on HepG2 cells. BDE-47 and its metabolites also induce changes in ROS generation, SOD and GSH activity, cell cycle regulation, DNA damage and cell apoptosis, indicating that the toxicity mechanisms of BDE-47 and its degradation products are mediated by oxidative stress, DNA damage and cell cycle dysregulation. Moreover, brominated phenol products (2,4-DBP and 4-BP) posed the highest toxic effects on HepG2, followed by hydroxylated products (6-OH-BDE-47, 5-OH-BDE-47, 2-OH-BDE-28, and 4-OH-BDE-17), and BDE-47 and its debromination products were comparatively less toxic to HepG2 cells. Taken together, these results demonstrate the hepatotoxic potential of BDE-47 and its metabolites.

Use of HepG2 cell line for evaluation of toxic and metabolic antipsychotic action

The toxic and metabolic effects of antipsychotics on cells of hepatic origin have been studied in vitro using human hepatoblastoma cell line HepG2. The cells were cultured in the presence of two first- and second-generation antipsychotics, haloperidol and olanzapine, respectively, at concentrations that can exist in the blood, liver and other tissues with high lipid content when the drugs are used for therapeutic purposes. During cultivation in several periods, the products of carbohydrate and lipid metabolism were quantified, the activities of several enzymes in the culture medium were measured, and viability (proliferation) of cells was assessed with MTS assay. Both drugs were toxic to HepG2 cells, which was manifested in inhibition of proliferation and an increased alkaline phosphatase activity in the culture medium. The toxic action of olanzapine in the doses used was less significant than that of haloperidol. According to literature data, antipsychotics increase the expression of lipogenesis genes in cells of the central nervous system, adipose tissue and liver, which can lead to hyperlipidemia. However, we have not observed increased levels of total cholesterol, cholesterol of low and high density lipoproteins, and triglycerides in the culture medium of HepG2 cells in the presence of haloperidol and olanzapine. This may be associated with the fact that both drugs, which are cationic amphiphiles, are able to inhibit intracellular lipid trafficking. Moreover, both drugs had no effect on the activity of aspartate aminotransferase and gamma-glutamyltransferase in the culture medium, but contributed to a decrease in the activity of alanine aminotransferase. Overall, our work has confirmed that HepG2 cells may serve as a useful model to obtain new data on drug effects on the metabolism of cells of hepatic origin and to assess the risk of hepatotoxicity in preclinical studies.

Multiple-endpoints gene alteration-based (MEGA) assay: A toxicogenomics approach for water quality assessment of wastewater effluents

Wastewater effluents contain a significant number of toxic contaminants, which, even at low concentrations, display a wide variety of toxic actions. In this study, we developed a multiple-endpoints gene alteration-based (MEGA) assay, a real-time PCR-based transcriptomic analysis, to assess the water quality of wastewater effluents for human health risk assessment and management. Twenty-one genes from the human hepatoblastoma cell line (HepG2), covering the basic health-relevant stress responses such as response to xenobiotics, genotoxicity, and cytotoxicity, were selected and incorporated into the MEGA assay. The genes related to the p53-mediated DNA damage response and cytochrome P450 were selected as markers for genotoxicity and response to xenobiotics, respectively. Additionally, the genes that were dose-dependently regulated by exposure to the wastewater effluents were chosen as markers for cytotoxicity. The alterations in the expression of an individual gene, induced by exposure to the wastewater effluents, were evaluated by real-time PCR and the results were validated by genotoxicity (e.g., comet assay) and cell-based cytotoxicity tests. In summary, the MEGA assay is a real-time PCR-based assay that targets cellular responses to contaminants present in wastewater effluents at the transcriptional level; it is rapid, cost-effective, and high-throughput and can thus complement any chemical analysis for water quality assessment and management.

Low-Dose Dose-Response for In Vitro Nrf2-ARE Activation in Human HepG2 Cells.

Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-like factor 2-related factor 2 (Nrf2), and the antioxidant response element (ARE) are interacting components of a master regulatory signaling pathway that coordinates redox homeostasis, cytoprotective responses, and shifts in stem cell state. This study reexamined detailed dose–response (DR) data reported for in vitro Nrf2-ARE activation in human hepatoblastoma HepG2 cell lines containing either a ARE-bla or ARE-luc reporter at 12 different concentrations of each of 15 chemicals. The normalized study data were combined among chemicals exhibiting a positive response, yielding n = 531 (179) DR data for 9 (7) chemicals using the ARE-bla (ARE-luc) assay. Three-parameter linear/kth-power regression fits obtained to each combined set of ARE-bla- or ARE-luc-assay response data provided good fits (R 2 = .99 or .91, respectively, P fit > .99) that each incorporate a highly significant negative initial linear slope (P = 4 × 10−5 or .00025) and an overall J-shaped DR pattern. Results from this reanalysis of high-resolution ARE response data support the hypothesis that nonlinear ARE-mediated adaptive cellular responses to oxidative stress are governed by an ultrasensitive molecular switch.

Association of TIP30 expression and prognosis of hepatocellular carcinoma in patients with HBV infection.

Altered expression of TIP30, a tumor suppressor, has been observed in many cancers. In this study, we have evaluated the expression of TIP30 in the tissues of 209 hepatocellular carcinomas (HCC) and their adjacent tissues by using a high‐density tissue microarray, and analyzed its correlation with the clinical pathological parameters of the patients. The results revealed negative or weak expression of TIP30 in 43.5% (91/209) of the HCC tissues, and in only 27% (56/209) of the adjacent tissues. The expression level of TIP30 in HCC was inversely correlated with serum alpha‐fetoprotein (AFP) levels, HBV infection, and tumor differentiation. Multivariate analysis for survival indicated that serum HBV infection was the most significant predictor of poor prognosis in HCC (P = 0.0023), and TIP30 expression and tumor differentiation were also independent indicators in this respect (P = 0.0364 and P = 0.0397, respectively). Patients with medium or high expression levels of TIP30 (TIP30++/+++) had a better 5‐year overall survival rate than those with low/negative (TIP30+/−) expression (P < 0.001). TIP30+/−/HBV+ patients had the worst 5‐year overall survival rate, whereas TIP30++/+++/HBV− patients had the best. To further explore the correlation between TIP30 and HBV infection in HCC, HBV+ hepatoblastoma cell‐line HepG2 2.2.15 and HCC cell‐line Hep3B were used. Upon silencing of HBV, we observed an upregulation of TIP30 and decreased cell proliferation. In the in vivo studies, we found that the mice inoculated with HepG2 2.2.15 cells with HBV silencing had a prolonged tumor latency and a longer life span, as compared to the control mice inoculated with untreated control cells. In conclusion, the results suggest that downregulation of TIP30 may result from HBV infection, and subsequently promotes the progression of HCC.

Evaluation of Polybrominated Diphenyl Ether Toxicity on HepG2 Cells – Hexabrominated Congener (BDE‐154) Is Less Toxic than Tetrabrominated Congener (BDE‐47)

Apoptotic cell death is one of the main consequences of exposure to brominated flame retardants, including polybrominated diphenyl ethers. However, few of these compounds have had their potential toxicity investigated. BDE-154 is one of the most poorly studied polybrominated diphenyl ether (PBDE) congeners, but its level in the environment and in biological fluids is rising. In addition, its chemical structure differs from the other congeners with well-documented toxicity, so BDE-154 may display a distinct toxicity pattern. This study has evaluated how BDE-154 affects the human hepatoblastoma cell line (HepG2) and has looked into the impact of this congener on human health. In addition, this study has related the effects of BDE-154 with the effects of BDE-47 to clarify the mechanism of PBDE toxicity. The HepG2 cell line was exposed to BDEs for 24 and 48 hr and submitted to assays to examine proliferation, viability, mitochondrial membrane potential, reactive oxygen species accumulation, phosphatidylserine exposure, nuclear fragmentation and evaluation of pro-caspase 3, pro-caspase 9, cytochrome c release, and apoptosis inductor factor release by Western blot analysis. BDE-154 induced mitochondrial damage and led to apoptotic death of HepG2 cells, but these effects were less intense than the effects promoted by BDE-47. Unlike other extensively reported congeners, BDE-154 was only toxic at the higher tested concentrations, whereas BDE-47 cytotoxicity was evident even at lower concentrations. Hence, like the toxicity pattern of other classes of substances such as polychlorinated biphenyls, the toxicity pattern of BDEs also depends on their chemical structure and aromatic substituent.

Treatment of multi-drug-resistant hepatoblastoma cells using unique sequence of high-intensity focused ultrasound and low-dose generic chemotherapeutic.

e22008 Background: High-intensity focused ultrasound (HIFU) is a noninvasive, radiation-free, and precisely targeted form of cancer therapy. In this study, I evaluate a new strategy in which the unique sequence of HIFU and low-dose generic chemotherapeutic is utilized to kill multidrug-resistant (MDR) hepatoblastoma cells. Methods: My strategy, motivated by clinical HIFU trials in adult hepatocellular cancers, led to the in vitro study in which human hepatoblastoma cell line HepG2 (ATCC HB8065), known for resistance to 5-fluorouracil (5-FU), was paradoxically treated with low-dose 5-FU in the following sequence. Day 0: Each data sample consisted of ~10K HepG2 cells grown inside a well of 8-well glass slides (Lab-Tek). Each well was then filled with growth media and incubated at 37ºC for 24h. Day 1: Each slide was sealed, submerged in a 37ºC degassed water bath and secured onto a fixture. The targets for HIFU therapy are the center points of the 4 quadrants of each well's base and the common origin. The constant HIFU parameters for each slide were acoustic pressure 4.5 MPa, repetition rate 1 Hz, focal-zone depth and 30 sec/sonication/target while HIFU duty cycle (DC) ranged 0-50% among slides. After HIFU treatment, the slides were incubated for 24h. Day 2: Cell media for each slide was replaced with fresh media containing [5-FU] 0-400 uM prior to repeating HIFU procedure from day 1. Day 4: After 48h of exposure to 5-FU, cell survivability study using imaging-based live cell count was performed to determine the contributions of HIFU-mediated necrosis and 5-FU-mediated apoptosis/necrosis. Results: Cell survivability of MDR HepG2 cells decreased further using HIFU/5-FU sequence than with either HIFU or 5-FU alone as shown below (Table). Conclusions: The unique sequence of HIFU and low-dose 5-fluorouracil was successful in eliminating MDR hepatoblastoma cells on day 4. Treatment of HepG2 hepatoblastoma cells using HIFU/5-FU sequence. [5-FU], (uM) 0 1 10 100 200 400 95% CI of cell survivability (%) on day 4 = estimate ± margin of error;HIFU DC 0%, N=4 100± 12 81± 37 44± 13 30± 4 34± 5 39± 3 95% CI of cell survivability (%) on day 4 = estimate ± margin of error; HIFU DC 50%, N=4 30±5 7±1 10± 4 10± 2 11± 3 8±3

Lipid disorder and intrahepatic renin-angiotensin system activation synergistically contribute to non-alcoholic fatty liver disease.

This study aimed to investigate the possible synergistic effects of lipid disorder with renin-angiotensin system (RAS) activation in non-alcoholic fatty liver disease (NAFLD). Apolipoprotein E gene-knockout mice, angiotensin II (Ang II) type 1 receptor (AT1) gene-knockout mice and human hepatoblastoma cell line (HepG2) were used for experiments. Lipid accumulation was examined by Filipin staining and intracellular cholesterol quantitative assay. The gene and protein expression of molecules involved in RAS and low-density lipoprotein receptor (LDLr) pathway was examined by real-time PCR, immunofluorescent staining and Western blot. There was significantly increased expression of RAS components and extracellular matrix (ECM) in livers of high-fat-diet-fed apolipoprotein E gene-knockout mice compared with controls. Upregulation of RAS components was positively associated with increased plasma levels of lipid profile. The in vitro study further confirmed that cholesterol loading increased supernatant renin activity and Ang II level of HepG2 cells, accompanied by increased ECM production that was positively associated with increased expression of intracellular RAS components. Interestingly, Ang II treatment increased lipid accumulation in livers of C57BL/6 mice and HepG2 cells. Furthermore, Ang II treatment increased gene and protein expression of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), SREBP-2 and LDLr, which were mediated by enhanced SCAP/SREBP-2 complex translocation from endoplasmic reticulum to Golgi. However, LDLr pathway was accordingly downregulated in livers of AT1 gene-knockout C57BL/6 mice or in HepG2 cells treated by telmisartan. These findings demonstrate that lipid disorder and intrahepatic RAS activation synergistically accelerate NAFLD progression.

Abstract B29: MDM4 is a major p53 regulator in hepatoblastoma

Abstract Purpose: Unresectable or metastatic hepatoblastoma generally confers a poor survival. MDM4 and MDM2 are the major negative regulators of p53, which has a predominantly wild type status in hepatoblastoma. Having previously shown that MDM4 has copy gain/amplification associated with the 1q32.1 amplicon in hepatoblastoma, we hypothesize that MDM4 is the predominant negative regulator of p53 function in hepatoblastoma and that blocking MDM4 will cause tumor cell death due to uninhibited p53 tumor suppressor activity. Methods: An MTT assay was used to measure NSC207895 (MDM4 inhibitor) and Nutlin-3a (MDM2 inhibitor) cytotoxicity on hepatoblastoma cell lines (HepG2, HepT1, and Huh-6) as well as a patient-derived hepatoblastoma cell line (PDCL-1). CCK-8 was used to assess effects of low dose NSC207895 on HB cell line proliferation. Soft agar was used to assess the effect of NSC207895 on hepatoblastoma colony formation. Immunoblotting and quantitative RT-PCR was used to measure expression of p53 downstream targets in hepatoblastoma after treatment with NSC207895. P53 knockdowns were created to assess the effect of NSC207895 in hepatoblastoma in the absence of p53. Cell cycle analysis was performed using BRDU. Results: Huh-6, HepG2, and HepT1 were all tested with Nutlin-3a and did not show significant cell death (IC50 &amp;gt;10μΜ) inhibition with NSC207895 caused significant cell death in Huh-6 (IC50=1.27μΜ), HepG2 (1.62μΜ), and HepT1 (2.05μΜ). A decrease in cell proliferation was noted with concentrations of NSC207895 as low as 0.05μΜ, 0.1μΜ and 0.3μΜ in HepG2, HepT1, and Huh-6 respectively. An increase in IC50 was seen in the p53 knockdowns compared to each respective control (HepG2: 12.6μΜ vs 3.38μΜ; HepT1: &amp;gt;50μΜ vs 1.45μΜ; Huh-6: 20.2μΜ vs 7.52μΜ). Increased PARP cleavage products and decreased MDM4 expression were seen on immunoblotting after treatment with NSC207895. By both immunoblotting and qPCR, the p53 downstream transcriptional targets, p21, BAX and PUMA, were increased after exposure to NSC207895 in all three cell lines. Cell cycle analysis showed an increased percentage of cells in G1/G0-phase and decreased percentage of actively replicating S-phase cells in HepG2 with NSC207895 treatment. Conclusion: Our data supports the hypothesis that in hepatoblastoma, MDM4 may be a dominant inhibitor of p53 and inhibition of MDM4 causes p53-mediated cell death and growth inhibition. Therefore, inhibition of MDM4 may be a viable drug target for treating hepatoblastoma. Citation Format: Yan Shi, Roma Patel, Emporia Hollingsworth, Stephen Sarabia, Jingling Jin, Dolores Lopez-Terrada, Sanjeev Vasudevan. MDM4 is a major p53 regulator in hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B29.

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes.

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A-induced toxicity was investigated using Hepatoblastoma Cell Line HepG-2 cells. Disruption of cholesterol-containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time-dependent manner. Isobaric tags for relative and absolute quantitation-based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology "transport", "cell adhesion" and "vesicle-mediated transport". In conclusion, lipid rafts play a key role in OTA-induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.