Loofa sponge as a scaffold for the culture of human hepatocyte cell line

Loofa sponge was investigated as a three-dimensional scaffold for stationary and perfusion culture of human hepatoblastoma cell line C3A/HepG2. In stationary culture, C3A/HepG2 cells in loofa cubes showed higher alpha-fetoprotein and albumin secretion rates than those in polyurethane foam (PU). To use loofa cylinders in a packed-bed reactor, immobilization of C3A/HepG2 cells by recirculating medium at 26 mL/min (superficial velocity = 51.7 cm/min) resulted in a cell loading density of 5.15 x 10(7) cells/cm(3)-loofa. This cell loading density is higher than values reported in the literature for packed-bed reactor intended for bioartificial liver. During 9 days of perfusion culture in the reactor, immobilized C3A/HepG2 showed steady synthesis of albumin with an average synthesis rate at 42.2 microg/10(6) cells/day. These experimental results and observations by SEM suggested that loofa sponge is a suitable scaffold for high-density culture of human hepatocyte cell line and the immobilized cells could express high levels of liver-specific functions.     

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.     

The cytotoxicity of naturally occurring styryl lactones

We extracted and isolated three natural styryl lactones from Goniothalamus griffithii Hook f. Thoms and investigated their cytotoxicity on a panel of three hepatocyte cell lines, HepG2, drug resistant HepG2 (HepG2-R) and primary cultured normal mice hepatocyte in order to find candidates of potential anti-cancer drugs which have low toxicity on normal cells and high effect on tumors or drug resistant tumors. All the three styryl lactones showed evident cytotoxic activities on both HepG2 and HepG2-R cell lines; however, gonithalamin and goniodiol shows less toxicity on normal mice hepatocyte as the IC(50) values of them on normal mice hepatocyte were about three times of that on HepG2. Morphological observation and cell cycle analysis were employed to elucidate the mechanisms of cytotoxicity of the tested compounds. Many apoptotic cells were observed in gonithalamin- and altholactone-treated cells, whereas, cells with chromosomes gathered at the equator were easily found in goniodiol-treated cultures. The analysis of cell cycle showed that G(2)/M arrest contributed to goniothalimin- and gonidiol-caused cell death and apoptosis was the cause of gonithalamin- and altholactone-induced cell death. Our results suggest that the three styryl lactones may be prospectively developed into anti-tumor drugs, especially on treating drug-resistance tumor after structure modification.     

Differential sensitivity of human hepatoma cell line and primary rat hepatocyte culture to benzo(a)pyrene-induced unscheduled DNA synthesis and adduct formation.

We studied the genotoxic potential of a carcinogen in the human hepatoma cell line, HepG2 and in primary rat hepatocyte culture. HepG2 is a well differentiated human hepatoblastoma cell line with biotransforming capacity. Rat hepatocytes were obtained by the standard two-step in situ perfusion technique. Following benzo(a)pyrene treatment, both HepG2 and primary rat hepatocyte culture showed unscheduled DNA synthesis with different sensitivity. In ³²P-postlabelling analysis, the chromatogram revealed quantitative and qualitative differences between HepG2 and primary rat hepatocyte cultures when treated with 10 μM benzo(a)pyrene for 18 hr. The results have demonstrated that the HepG2 cell line may be used in addition to primary rat hepatocytes in risk assessment for detection of environmental carcinogens.     

Panaxydol inhibits the proliferation and induces the differentiation of human hepatocarcinoma cell line HepG2

Panaxydol, a polyacetylene compound isolated from Panax ginseng, exerts anti-proliferative effects against malignant cells. No previous study, however, has been reported on its effects on hepatocellular carcinoma cells. Here, we investigated the effects of panaxydol on the proliferation and differentiation of human hepatocarcinoma cell line HepG2. We studied by electronic microscopy of morphological and ultrastructural changes induced by panaxydol. We also examined the cytotoxicities of panaxydol against HepG2 cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and the effect of panaxydol on cell cycle distributions by flow cytometry. We investigated the production of liver proteins in panaxydol-treated cells including alpha-fetoprotein and albumin and measured the specific activity of alkaline phosphatase and gamma-glutamyl transferase. We further investigated the effects of panaxydol on the expression of Id-1, Id-2, p21 and pRb by RT-PCR or immunoblotting analysis. We found that panaxydol inhibited the proliferation of HepG2 cells and caused morphological and ultrastructural changes in HepG2 cells resembling more mature forms of hepatocytes. Moreover, panaxydol induced a cell cycle arrest at the G₁ to S transition in HepG2 cells. It also significantly decreased the secretion of alpha-fetoprotein and the activity of gamma-glutamyl transferase. By contrast, panaxydol remarkably increased the secretion of albumin and the alkaline phosphatase activity. Furthermore, panaxydol increased the mRNA content of p21 while reducing that of Id-1 and Id-2. Panaxydol also increased the protein levels of p21, pRb and the hypophosphorylated pRb in a dose-dependent manner. These findings suggest that panaxydol is of value for further exploration as a potential anti-cancer agent.     

Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells

Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.     

Effects of dimethyl sulphoxide on the synthesis of plasma proteins in the human hepatoma HepG2. Induction of an acute-phase-like reaction.

Effects of dimethyl sulphoxide (Me2SO) on the synthesis of plasma proteins by the human hepatoma cell line HepG2 were examined. Me2SO treatment resulted in decreased synthesis of albumin and alpha-fetoprotein, and in increased synthesis of haptoglobin. Plasma-protein profiles induced by Me2SO treatment were very similar to those seen in acute-phase reactions.     

Regulation of angiotensinogen gene expression in a human hepatoma cell line

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its synthesis is increased in the liver during acute inflammation. We have used radiolabeled human angiotensinogen cDNA to study the effect of hepatocyte stimulating factor (HSF), a protein synthesized in differentiating monocytes which increases the synthesis of various hepatic proteins during inflammation, on angiotensinogen mRNA levels in human hepatoma cells (HepG2). Our results indicate that angiotensinogen mRNA is present in human hepatoma (HepG2) cells and its levels are decreased when treated with hepatocyte stimulating factor. Although dexamethasone elevated angiotensinogen mRNA levels, HSF reduced this increase. These results suggest that a factor other than HSF may be involved in elevating the angiotensinogen mRNA levels in the liver during inflammation.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.

Two members of the C/EBP family of basic region-leucine zipper proteins enriched in the liver, C/EBP (C/EBP alpha) and CRP2 (C/EBP beta), were previously shown to transactivate the albumin promoter in a cell type-dependent manner. These proteins function efficiently in HepG2 hepatoma cells, but inefficiently in HeLa (epithelial) and L (fibroblastic) cells. Here we have investigated the mechanism for cell-specific control of CRP2 activity. We show that CRP2 contains a negative regulatory region composed of two elements, RD1 and RD2. Deletions of RD2 relieve the inhibition of CRP2 activity in L cells, but do not affect CRP2 function in HepG2 cells. These deletions also increase the DNA binding activity of CRP2 approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for CRP2 inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the CRP2 activation domain, which we show to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but inhibits the transactivation potential of GAL4-CRP2 hybrid proteins by 50-fold. These findings suggest that CRP2 assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain and that to function efficiently in HepG2 cells the protein must undergo an activation step. We propose that relief of inhibition conferred by the regulatory domains also accounts for CRP2 activation in response to extracellular signals.     

Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Our previous studies showed that both the whole HBV genome and its HBx protein enhanced TRAIL-induced hepatocyte apoptosis. We report here that preS2-containing viral proteins are also potent regulators of TRAIL-induced apoptosis. HBV-transfected hepatoma cell line, HepG2.2.15, pretreated with antisense oligonucleotide against preS2 gene, showed a lower sensitivity towards TRAIL-induced apoptosis. However, this effect might not be related with the expression level of TRAIL receptors. These results establish that besides HBx, preS2 viral proteins are also involved in enhancing TRAIL-induced hepatocyte apoptosis. The novel role of preS2 would be useful to further unravel the mechanisms of imbalanced apoptosis during HBV infection and provides a potential therapeutic target for HBV-related diseases.     

The amino-terminal domains of the ezrin,radixin, and moesin (ERM) proteins bind advanced glycation end products,an interaction that may play a role in the development of diabetic complications

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography. NH2-terminal and internal sequencing identified the proteins as the NH2-terminal domains of ezrin, radixin, and moesin (ERM proteins). Using BIAcore biosensor analysis, human N-ezrin-(1-324) bound to immobilized AGE-BSA with a KD of 5.3 +/- 2.1 x 10 -7 m, whereas full-length ezrin-(1-586) and C-ezrin-(323-586) did not bind. Other glycated proteins such as AGE-RNase, N in -carboxymethyllysine (CML)-BSA, and glycated human serum albumin isolated from hyperglycemic diabetic sera competed with the immobilized AGE-BSA for binding to N-ezrin, but non-glycated BSA and RNase did not. Thus N-ezrin binds to AGEs in a glycation- and concentration-dependent manner. Phosphorylated ezrin plays a crucial role in cell shape changes, cell attachment, and cell adhesion. The effect of AGE-BSA on ezrin function was studied in a tubulogenesis model in which LLC-PK1 cell tubule formation is dependent on phosphorylated ezrin. Addition of AGE-BSA completely inhibited the ability of the cells to produce tubules. Furthermore, in vitro tyrosine phosphorylation of N-ezrin and ezrin was also inhibited by AGE-BSA. These proteins represent a novel family of intracellular binding molecules for glycated proteins and provide a potential new target for therapeutic intervention in the prevention or treatment of diabetic complications.     

Down-regulation of CMTM8 induces epithelial-to-mesenchymal transition-like changes via c-MET/extracellular signal-regulated kinase (ERK) signaling

The acquisition of an invasive phenotype is a critical turning point for malignant tumor cells. CMTM8, a potential tumor suppressor, is frequently down-regulated in solid tumors, and its overexpression induces tumor cell apoptosis. Here, we identify a new role for CMTM8 in regulating tumor cell migration. Reducing CMTM8 expression in HepG2 hepatocellular carcinoma cells results in the acquisition of epithelial-to-mesenchymal transition (EMT) features, including a morphological change from organized epithelial sheets to scattered fibroblast-like shapes, reduction of the epithelial marker E-cadherin, and an increased invasive and migratory ability. These phenotypic changes are mediated in large part by the ERK-MAPK pathway, as the MEK inhibitor U0126 and shRNA-mediated knockdown of ERK2 significantly reversed these phenotypes. Hepatocyte growth factor binding to the c-MET receptor is known to induce EMT in HepG2 cells. We found that CMTM8 knockdown in HepG2 cells induced c-MET signaling and ERK activation. Inhibition of c-MET signaling with the small molecule inhibitor SU11274 or c-MET RNAi blocked the EMT-like changes following CMTM8 knockdown. CMTM8 overexpression in HepG2 cells inhibited hepatocyte growth factor-induced EMT-like morphological changes and cell motility. Down-regulation of CMTM8 also promoted an EMT-like change in MCF-10A cells, indicating a broader role for CMTM8 in regulating cellular transformation.     

Differential cellular gene expression induced by hepatitis B and C viruses

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.     

Byakangelicin protects against carbon tetrachloride–induced liver injury and fibrosis in mice

Liver fibrosis is a disease caused by long‐term damage that is related to a number of factors. The current research on the treatment of liver fibrosis mainly focuses on the activation of hepatic stellate cell, in addition to protecting liver cells. byakangelicin has certain anti‐inflammatory ability, but its effect on liver fibrosis is unclear. This study aims to explore whether byakangelicin plays a role in the development of liver fibrosis and to explore its mechanism. We determined that byakangelicin has a certain ability to resist fibrosis and reduce liver cell damage in a model of carbon tetrachloride–induced liver fibrosis in mice. Thereafter, we performed further verification in vitro. The signalling pathways of two important pro‐fibrotic cytokines, transforming growth factor‐β and platelet‐derived growth factor, were studied. Results showed that byakangelicin can inhibit related pathways. According to the hepatoprotective effect of byakangelicin observed in animal experiments, we studied the effect of byakangelicin on 4‐HNE–induced hepatocyte (HepG2) apoptosis and explored its related pathways. The results showed that byakangelicin could attenuate 4‐HNE–induced hepatocyte apoptosis via inhibiting ASK‐1/JNK signalling. In conclusion, byakangelicin could improve carbon tetrachloride–induced liver fibrosis and liver injury by inhibiting hepatic stellate cell proliferation and activation and suppressing hepatocyte apoptosis.     

Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: Morphology and immunocytochemical studies of hepatic-secreted proteins

Summary The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI,-B,-D, and-E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2±1.9; 4.9±1.5; 3.2±0.4; and 10.7±1.7 μg/10⁶ cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.