Normal Rat Hepatocytes Research Articles

Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

Platelet-Derived Microvesicles Promote Selective Cell Viability in Hepatocellular Carcinoma: Differences in Properties between Normal Platelets and Tumor Educated Platelets

Tumor cells educate platelets by modulating their RNA profiles and phenotypes. These platelets are termed "tumor educated platelets: TEPs", and this concept is widely accepted. We have previously reported that platelets are activated in microvessels within liver tumors, leading to the progression of hepatocellular carcinoma (HCC). When platelets are activated, platelet-derived microvesicles (PDMV) containing platelet-derived nucleic acids and proteins are released. The contents in these are known to vary depending on the various pathological conditions of the host. It is generally believed that when cells take up extracellular vesicles, they are affected by the action of the encapsulated substances. However, it is still unclear what biological properties of PDMV are released by TEPs and how they are involved in carcinogenesis. We hypothesized that the release of PDMV by activated platelets in liver tissue and the uptake of its contents by normal hepatocytes and HCC cells may be involved in the progression of hepatocarcinogenesis, and we analyzed their role in carcinogenesis. The rat HCC model was prepared according to the Solt-Farber protocol. Briefly, male F344 rats were injected intraperitoneally with diethylnitrosamine at 4 weeks of age, then fed a powdered diet containing 2-acetylaminofluorene to induce liver damage, followed by partial hepatectomy. The rats were then kept under normal conditions for more than one month. Abdominal ultrasonography was performed on rats, and those with obvious tumors with a diameter of 2 mm or more were used for the experiments. Peripheral blood was collected from the tail veins of normal rats or HCC model rats, and platelets were isolated. The platelets were labeled with the fluorescent substance CMRA, activated in vitro with thrombin, and the PDMV in the supernatant was collected by polyethylene glycol precipitation and used as CMRA-labeled PDMV in the experiments. When PDMV was added to primary cultured rat hepatocytes and cultured for 24 hours, fluorescence was observed in the hepatocytes. The fluorescence was also observed in hepatocytes in the portal region of liver tissue collected 24 hours after injection of CMRA-labeled PDMV from the mesenteric vein of rats. These indicate that PDMV released into the liver tissue is taken up by hepatocytes. Peripheral blood platelets from normal rats or HCC model rats were isolated and PDMV was purified from each (N-PDMV and HCC-PDMV). These PDMVs were added to cultures of normal rat hepatocytes and hepatocellular carcinoma cell lines to analyze their effects on cell survival. N-PDMV did not affect the viability of normal hepatocytes, but mildly enhanced the survival of HCC cells. On the other hand, HCC-PDMV inhibited the viability of normal hepatocytes, but promoted viability of HCC cells more strongly than N-PDMV. These results indicate that the contents of PDMV differ between normal and HCC rats, and in particular that PDMV from rats with HCC is taken up by normal and HCC cells and is associated with selective survival of HCC cells.

Aspirin Eugenol Ester Attenuates Paraquat-Induced Hepatotoxicity by Inhibiting Oxidative Stress.

Aspirin eugenol ester (AEE) is a new potential drug with anti-inflammatory and antioxidant stress pharmacological activity. Paraquat (PQ) is an effective and commercially important herbicide that is widely used worldwide. However, paraquat is highly toxic and can cause various complications and acute organ damage, such as liver, kidney and lung damage. The purpose of this study was to investigate whether AEE has a protective effect on hepatotoxicity induced by PQ in vivo and in vitro. Cell viability, apoptosis rate, mitochondrial function and intracellular oxidative stress were detected to evaluate the protective effect of AEE on PQ-induced BRL-3A (normal rat hepatocytes) cytotoxicity in vitro. In vivo, AEE pretreatment could attenuate oxidative stress and histopathological changes in rat liver induced by PQ. The results showed that AEE could reduce the hepatotoxicity induced by PQ in vivo and in vitro. AEE reduced PQ-induced hepatotoxicity by inhibitingoxidative stress and maintaining mitochondrial function. This study proved that AEE is an effective antioxidant and can reduce the hepatotoxicity of PQ.

Kinetic modeling of glucose central metabolism in hepatocytes and hepatoma cells

BackgroundKinetic modeling and control analysis of a metabolic pathway may identify the steps with the highest control in tumor cells, and low control in normal cells, which can be proposed as the best therapeutic targets. MethodsEnzyme kinetic characterization, pathway kinetic modeling and control analysis of the glucose central metabolism were carried out in rat (hepatoma AS-30D) and human (cervix HeLa) cancer cells and normal rat hepatocytes. ResultsThe glycogen metabolism enzymes in AS-30D, HeLa cells and hepatocytes showed similar kinetic properties, except for higher AS-30D glycogen phosphorylase (GP) sensitivity to AMP. Pathway modeling indicated that fluxes of glycogen degradation and PPP were mainly controlled by GP and NADPH consumption, respectively, in both hepatocytes and cancer cells. Likewise, hexose-6-phosphate isomerase (HPI) and phosphoglucomutase (PGM) exerted significant control on glycolysis and glycogen synthesis fluxes in cancer cells but not in hepatocytes. Modeling also indicated that glycolytic and glycogen synthesis fluxes could be strongly decreased when HPI and PGM were simultaneously inhibited in AS-30D cells but not in hepatocytes. Experimental assessment of these predictions showed that both the glycolytic and glycogen synthesis fluxes of AS-30D cells, but not of hepatocytes, were inhibited by oxamate, by inducing increased Fru1,6BP levels, a competitive inhibitor of HPI and PGM. ConclusionHPI and PGM seem suitable targets for decreasing glycolytic and glycogen synthesis fluxes in AS-30D cells but not in hepatocytes. General significanceThe present study identified new therapeutic targets within glucose central metabolism in the analyzed cancer cells, with no effects on non-cancer cells.

Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

Insulin is a critical signaling molecule in reducing blood glucose levels, and pyruvate dehydrogenase (PDH) is an essential enzyme in regulating glucose metabolism. However, the insulin effect on PDH function has not been well established. We observed that insulin attenuated the phosphorylation (p) of Ser264 (p-Ser264) in the PDH E1α subunit (PDHA1) in normal rat hepatocyte. In contrast, insulin induced an increase of p-Ser264 PDHA1 levels in hepatocellular carcinoma HepG2 and Huh7 cells. Insulin activated RhoA and Rho-dependent coiled coil kinase, an effector protein of active RhoA, which regulated p-Ser264 PDHA1 levels, along with both p-Ser9 and p-Tyr216 forms of glycogen synthase kinase-3β (GSK-3β) in HepG2 cells. Only p-Tyr216 GSK-3β, the active form was involved in an increase of p-Ser264 PDHA1. Akt was also engaged in p-Ser9 of GSK-3β, but neither in p-Tyr216 of GSK-3β nor p-Ser264 of PDHA1 upon insulin. Reconstituted dephospho-mimic forms PDHA1 S264A and GSK-3β Y216F impaired, but wild-types PDHA1 and GSK-3β and phospho-mimic forms PDHA1 S264D and GSK-3β Y216E increased cell proliferation upon insulin through expression of c-Myc and cyclin D1. Therefore, we propose that insulin-mediated p-PDHA1 is involved in the regulation of HepG2 cell proliferation through RhoA signaling pathway.-Islam, R., Kim, J.-G., Park, Y., Cho, J.-Y., Cap, K.-C., Kho, A.-R., Chung, W.-S., Suh, S.-W., Park, J.-B. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

Anti-proliferative effect of ridaifen-B on hepatoma cells.

Ridaifens (RIDs), a novel series of tamoxifen derivatives, exhibit a potent growth-inhibitory effect against numerous tumor cells regardless of the expression of estrogen receptors, and are thus promising candidates as novel anti-tumor drugs. RID-B is a first generation RIDs, and inhibits the proliferation of several tumor cell lines. However, the potentially growth inhibitory effect of RID-B against hepatoma cells, and the detailed mechanism underlying RID-B-mediated tumor cell death remain to be elucidated. The purpose of the current study was to evaluate the anti-proliferative effect of RID-B against hepatoma cells. The anti-proliferative effect of RID-B against human hepatoma Huh-7 cells was investigated by cell proliferation assay using WST-1 reagent, and caspase-3 activity was evaluated by using specific fluorescent substrate. In addition, DNA fragmentation in Huh-7 cells induced by RID-B was estimated by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay, and binding of RID-B to double-stranded DNA was confirmed by mass spectrometry. RID-B (0.5, 1 and 2 µM) inhibited the growth of Huh-7 cells, seemingly dose-dependently, but did not inhibit the growth of normal primary rat hepatocytes in the same concentration range. Furthermore, the caspase-3 activity of Huh-7 cells was increased by RID-B (0.5 and 5 µM), and the anti-proliferative effect of RID-B (1 µM) on Huh-7 cells was partially suppressed by the addition of the caspase inhibitor, Z-VAD-FMK. Additionally, RID-B (10 µM) directly bound to double-stranded DNA, and the addition of DNA suppressed RID-B-mediated cell growth inhibition and DNA fragmentation in Huh-7 cells. From these data, it may be concluded that RID-B inhibited cell growth and induced apoptosis via activating caspase-3 and binding to DNA directly, leading to DNA fragmentation in hepatoma cells.

Iron(III)-Tannic Molecular Nanoparticles Enhance Autophagy effect and T1 MRI Contrast in Liver Cell Lines

Herein, a new molecular nanoparticle based on iron(III)-tannic complexes (Fe–TA NPs) is presented. The Fe–TA NPs were simply obtained by mixing the precursors in a buffered solution at room temperature, and they exhibited good physicochemical properties with capability of inducing autophagy in both hepatocellular carcinoma cells (HepG2.2.15) and normal rat hepatocytes (AML12). The Fe–TA NPs were found to induce HepG2.2.15 cell death via autophagic cell death but have no effect on cell viability in AML12 cells. This is possibly due to the much higher uptake of the Fe–TA NPs by the HepG2.2.15 cells via the receptor-mediated endocytosis pathway. As a consequence, enhancement of the T1 MRI contrast was clearly observed in the HepG2.2.15 cells. The results demonstrate that the Fe–TA NPs could provide a new strategy combining diagnostic and therapeutic functions for hepatocellular carcinoma. Additionally, because of their autophagy-inducing properties, they can be applied as autophagy enhancers for prevention and treatment of other diseases.

On reprogramming of tumor cells metabolism: detection of glycogen in the cell lines of hepatocellular origin with various degrees of dedifferentiation.

The reprogramming of cancer cells includes shifts in glucose and glycogen metabolism. The aim of our work was to check the ability of forming glycogen grains in hepatocellular tumor cell lines of various dedifferentiation levels. We studied the monolayer culture established in vitro after explanting cells from rat ascites Zajdela hepatoma strain C (ZH-C) as a "parental" line and its five daughter clonal sublines: the holoclonal sublines 3H, 5F, 6H and the meroclonal ones 1E, 9C, which possess, respectively, the properties of cancer stem-like cells (CSLCs) and cancer progenitor-like cells(CPLCs). Besides, we studied four permanent cell lines of a rat hepatoma HTC, two murine hepatomas BWTG3 and MH-22a,and human hepatoblastoma HepG2. We used normal rat hepatocytes as positive control cells that form glycogen. We estimated relative cell dedifferentiation levels of the studied lines via analysis of cell morphology, morphometry and motility character on stained cell preparations and lifetime video files. Glycogen in the cells was detected using a Schiff type Au-SO2 reagent. All studied hepatocellular tumor lines were not of equal dedifferentiation level as manifested by different nucleus-to-cytoplasm ratio, by epithelium-like or fibroblast-like morphology, by tight or loosen intercellular contacts, by cell migration of collective or individual types. Glycogen fluorescence of uneven intensity was observed in all normal rat hepatocytes, but only in some cell groups or in single cells of hepatocellular tumor lines. The large or small fluorescent grains were found not only in relatively less dedifferentiated parental ZH-C line, BWTG3 and HepG2 lines, but also in moderately dedifferentiated 1E and HTC lines and even in severely dedifferentiated 3H, 5F and 6H sublines, as well as in the islets of the rat ascites hepatoma induced in vivo by the injection of 3H cells (the tumor-initiating cells). On the other hand, MH-22 and 9C lines, being relatively less and moderately dedifferentiated, showed no glycogen fluorescence. Thus,in 10 tumor cell lines of hepatocellular origin, an ability to reserve glycogen manifested no obvious dependency on their dedifferentiation level. Glycogen grains were detected in some cells even of the severely dedifferentiated lines: in single CSLCs of holoclonal ZH sublines grown in vitro and in a majority of tumor-initiating cells derived from ascites hepatoma in vivo. We suggest that dynamic changes in glycogen formation in CSLCs and tumor-initiating cells might be of importance for their dedifferentiation, self-renewal in vitro, survival and metastasis in vivo. The role of glycogen in maintaining viability and metastasis of tumor cells is to be further studied.

Microarray analysis reveals Tmub1 as a cell cycle-associated protein in rat hepatocytes.

Transmembrane and ubiquitin-like domain containing protein 1 (Tmub1), formerly known as hepatocyte odd protein shuttling (HOPS) has been recognized as a ubiquitously expressed shuttling protein that moves between the nucleus and cytoplasm in hepatocytes. Tmub1 is involved in liver regeneration and functions as a bridging protein in tumor cell proliferation. To investigate the transcriptional profile and potential biological processes affected by Tmub1 expression in normal rat hepatocytes, microarray and bioinformatics experiments were used to identify 127 mRNAs differentially expressed between Tmub1-overexpression, Tmub1-knockdown and normal BRL-3A cells (fold-change ≥2.5). The expression levels of 17 key node genes associated with the cell cycle were confirmed by reverse transcription-quantitative polymerase chain reaction analysis. Flow cytometry, 5-Ethynyl-20-deoxyuridine, Cell Counting Kit-8 and western blotting experiments revealed the effects on the cell cycle and the inhibition of proliferation in BRL-3A cells overexpressing Tmub1. Further co-immunoprecipitation assays demonstrated that Tmub1 interacts with cyclin A2 during the cell cycle and that the overexpression of Tmub1 may postpone cyclin A2 and cyclin B1 degradation in the M phase. The results of the present study indicated that Tmub1 functions as a cell proliferation inhibitor and cell cycle-associated protein.

Combination of Paracetamol and the Glutathione Depleting Agent Buthionine Sulfoximine Show Differential Effect on Liver Cancer Cells and Normal Hepatocytes

Background: Paracetamol exerts toxic effects on liver cells through its metabolism into N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified by conjugation with cellular glutathione (GSH). Once GSH is depleted, NAPQI stimulates a range of oxidative reactions that result in cell necrosis. The aim of the present investigation is to find a new strategy that would selectively protect normal hepatic tissues and sensitize liver cancer cells to the toxic effects of paracetamol or its metabolite. This may lead to the development of a targeted therapy for liver cancer. Methods: The anti-proliferative effects of paracetamol and buthionine sulfoximine BSO (a glutathione depleting agent) alone and in combination on the liver cancer cells HepG2 and normal rat hepatocytes were investigated by sulphorhodamine-B assay. Effects on cell cycle regulation and induction of apoptosis were tested by flow cytometry. The level of prostaglandin expression was measured by ELISA. Results: The present study showed that both agents alone or in combination have anti-proliferative effects on both cell types. Surprisingly, BSO showed a cytoprotective effects on normal hepatocytes treated with high concentrations (1.75 and 2 mM) of paracetamol. This was confirmed by cell cycle analysis that recorded decreased fraction of sub-G1 cells indicating reduction of apoptosis in normal hepatocytes. Analysis of prostaglandin E2 revealed differential effects of paracetamol on normal and liver cancer cells. A significant increase in PGE2 level over the control was observed in normal hepatocytes whereas a significant decrease was seen in HepG2 cells after treatment with paracetamol. Conclusion: These results indicate that combination of paracetamol/BSO has differential effects on liver cancer cells and normal hepatocytes, which opens the avenue for a new effective and selective combination for management of liver cancer.

Fatty acid esters of phloridzin induce apoptosis of human liver cancer cells through altered gene expression.

Phloridzin (phlorizin or phloretin 2′-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated through the attenuated expression of several key proteins involved in cell cycle regulation, DNA topoisomerases IIα activity and epigenetic mechanisms followed by cell cycle arrest and apoptosis.

AD-O53.2--a novel recombinant fusion protein combining the activities of TRAIL/Apo2L and Smac/Diablo, overcomes resistance of human cancer cells to TRAIL/Apo2L.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein-the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Composite fatty acid ether amides suppress growth of liver cancer cells in vitro and in an in vivo allograft mouse model

The heterogeneity of liver cancer, in particular hepatocellular carcinoma (HCC), portrays the requirement of multiple targets for both its treatment and prevention. Multifaceted agents, minimally or non-toxic for normal hepatocytes, are required to address the molecular diversity of HCC, including the resistance of putative liver cancer stem cells to chemotherapy. We designed and synthesized two fatty acid ethers of isopropylamino propanol, C16:0-AIP-1 and C18:1-AIP-2 (jointly named AIPs), and evaluated their anti-proliferative effects on the human HCC cell line Huh7 and the murine hepatoma cell line BNL 1MEA.7R.1, both in vitro and in an in vivo allograft mouse model. We found that AIP-1 and AIP-2 inhibited proliferation and caused cell death in both Huh7 and BNL 1MEA.7R.1 cells. Importantly, AIP-1 and AIP-2 were found to block the activation of putative liver cancer stem cells as manifested by suppression of clonal 'carcinosphere' development in growth factor-free and anchorage-free medium. The AIPs exhibited a relatively low toxicity against normal human or rat hepatocytes in primary cultures. In addition, we found that the AIPs utilized multifaceted pathways that mediate both autophagy and apoptosis in HCC, including the inhibition of AKTs and CAMK-1. In immune-competent mice, the AIPs significantly reduced BNL 1MEA.7R.1 cell-driven tumor allograft development, with a higher efficiency than sorafenib. A combination of AIP-1 + AIP-2 was most effective in reducing the tumor allograft incidence. AIPs represent a novel class of simple fatty acid derivatives that are effective against liver tumors via diverse pathways. They show a low toxicity towards normal hepatocytes. The addition of AIPs may represent a new avenue towards the management of chronic liver injury and, ultimately, the prevention and treatment of HCC.

Antihepatoma and Liver Protective Potentials of Ganoderma Lucidum (靈芝 Ling Zhi) Fermented in a Medium Containing Black Soybean (黑豆 Hēi Dòu) and Astragalus Membranaceus (生黃耆 Shēng Huáng Qí)

The antihepatoma activity and liver protective function of the fermentation products (5 L fermenator) of Ganoderma lucidum (GL; 靈芝 Ling Zhi) cultivated in a medium containing black soybean (BS; 黑豆 Hēi Dòu) and Astragalus membranaceus (AM; 生黃耆 Shēng Huáng Qí) at different fermentation temperatures were investigated in this study. Hep 3B cells pretreated with lovastatin were used to study the antihepatoma activity, and possible active components were analyzed by reverse-phase high-performance liquid chromatography. Carbon tetrachloride (CCl4)-induced primary rat hepatocyte injury was further used to evaluate the liver protective activity of the fermentation products. While all the GL broth filtrates do not inhibit the growth of Hep 3B cells, the ethanolic extract from GL-2 mycelia (GL-2-mE), cultivated in the medium containing BS (50 g/L) and AM (20 g/L) at 24°C for 11 days showed the best antihepatoma activity (IC50 26.6 μg/mL) than the other ethanolic extracts from GL mycelia, GL fruiting body, BS, and AM did. The antihepatoma activities were correlated with some unknown active components in these samples. Furthermore, GL-2-mE (100 μg/mL) without harmful effect on the growth of normal primary rat hepatocytes significantly maintained cell viability, reduced lactate dehydrogenase leakage, lowered lipid peroxidation, and increased glutathione peroxidase and glutathione S-transferase activities in the CCl4-induced damaged primary rat hepatocytes.

Implications of differential stress response activation following non-frozen hepatocellular storage.

Hepatocytes are critical for numerous cell therapies and in vitro investigations. A limiting factor for their use in these applications is the ability to process and preserve them without loss of viability or functionality. Normal rat hepatocytes (NHEPs) and human hepatoma (C3A) cells were stored at either 4°C or 37°C to examine post-processing stress responses. Resveratrol and salubrinal were used during storage to determine how targeted molecular stress pathway modulation would affect cell survival. This study revealed that storage outcome is dependent upon numerous factors including: cell type, storage media, storage length, storage temperature, and chemical modulator. These data implicate a molecular-based stress response that is not universal but is specific to the set of conditions under which cells are stored. Further, these findings allude to the potential for targeted protection or destruction of particular cell types for numerous applications, from diagnostic cell selection to cell-based therapy. Ultimately, this study demonstrates the need for further in-depth molecular investigations into the cellular stress response to bioprocessing and preservation.

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

BackgroundIt is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.MethodsThe rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.ResultsIn the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.ConclusionThe results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).

Molecular mechanism for the selective impairment of cancer mitochondrial function by a mitochondrially targeted vitamin E analogue

The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca2+ uptake, by collapsing the membrane potential at low doses (1–10μM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES>α-TEA>α-TOS. At high doses (>10μM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O2 uptake, collapsed ∆ψm, inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.

Analysis of Lymphocyte Specific Protein‐1 (LSP‐1) expression in normal primary rat hepatocytes and liver following partial hepatectomy

Hepatocellular carcinoma (HCC), the most frequently diagnosed form of liver cancer, is associated with high mortality rates. A recent finding of our laboratory shows that a portion of the gene for lymphocyte‐specific protein 1 (LSP1), an intracellular F‐actin binding protein expressed in neutrophils, macrophages and endothelial cells, is deleted or amplified in a majority of HCCs evaluated. The deletions of LSP‐1 correlated with large tumor size indicating that LSP‐1 may play an inhibitory role in hepatocyte growth. Based on these findings, we hypothesize that LSP‐1 expression is a critical regulator of cellular growth underlying HCC development. However, the expression of LSP‐1 in hepatocytes and normal liver remains to be elucidated. Utilizing immunoblotting and Reverse Transcriptase PCR (RT‐PCR), we examined the total protein and mRNA expression of LSP‐1 in primary rat hepatocytes in vitro and in rat liver tissue after partial hepatectomy. In the rat, LSP‐1 has three known splicing variants; therefore we will examine the expression of the isoforms of LSP‐1 using RT‐PCR with isoform specific LSP‐1 primers. Results from both immunoblotting and RT‐PCR experiments indicate that primary rat hepatocytes and tissue from partial hepatectomized livers express LSP‐1 and that the expression correlates inversely with the rate of growth. Future studies aim to address the function of LSP‐1 in normal and diseased liver states and whether LSP‐1 plays a critical role in hepatocyte growth. Research supported by grant 5T32HL094295‐02.

Direct Cytotoxicity of <i>Lentinula edodes</i> Mycelia Extract on Human Hepatocellular Carcinoma Cell Line

Lentinula edodes mycelia (L.E.M.) is a dried powder extracted from shiitake mushrooms (Lentinula edodes). We previously demonstrated that it has immunomodulatory effects. In this paper, the direct cytotoxic effects of the polysaccharide-rich fraction of L.E.M. (L.E.M. ethanol precipitate; LEP) on HepG2 human hepatocellular carcinoma (HCC) cells were investigated. LEP directly killed the HepG2 cells efficaciously, but had only minor effects on normal rat hepatocytes and normal mouse dermal cells under the same conditions. Characteristic morphological changes associated with apoptosis such as shrinkage, rounding, and floating as well as chromatin condensation were confirmed; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was positive as determined by fluorescence microscopy analyses. The caspase-3 and -8 death receptor pathway was found largely responsible for the apoptotic death of HepG2 cells treated with LEP. In conclusion, LEP can directly induce apoptosis of HepG2 cells, and thus may have potential chemotherapeutic applications for the treatment of HCC.