Rat Liver Epithelial Cells Research Articles

The cell adhesion and cytotoxicity effects of the derivate of vitamin E compared for two cell lines using a piezoelectric biosensor

The quartz crystal microbalance (QCM) was used to construct a piezoelectric biosensor utilizing rat liver epithelial cells (WB F344) and lung melanoma cells (B16F10) as biorecognition elements. The cells adhered on the gold surface modified by either hydrophobic polystyrene or fibronectin. The simultaneous monitoring of changes in the resonant frequency Delta f and the resistance Delta R corresponded with the mass and viscoelastic properties of the adhered cells. In addition, the bound cells were stained using fluorescent probes to evaluate their viability on the non-transparent gold surface. Furthermore, this piezoelectric biosensor was used for monitoring of drug-induced apoptosis initiated by micromolar concentrations of alpha-tocopherol amidomalate (alpha-TAM), a potential anticancer drug. The characteristic features of apoptosis as shrinkage and disintegration of cell-cell contacts could be monitored by QCM. The decrease of frequency and increase of resistance characterized gradual death of cells that was confirmed by fluorescence microscopy. The results indicate that the piezoelectric biosensor can be used for the real-time study of the cell adhesion and the screening of biologically active drugs affecting cellular behaviour. (C) 2012 Elsevier B.V. All rights reserved.

Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants – Evidence from in vitro study

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6–24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

Abstract 31: Activated phosphatidylinositol-3 kinase: An oncogene that increases gap junctional, intercellular communication

Abstract Gap junctions are channels that connect the cytoplasm of adjacent cells. Gap junctional, intercellular communication (GJIC) is blocked in cells transformed by oncogenes such as the middle Tumor antigen of polyoma virus (mT), an oncoprotein which associates with and is tyrosine-phosphorylated by cSrc family members. Specific phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) and the SH2 domain of the phosphatidylinositol 3-kinase (mT-tyr315), resulting in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI-3 kinase/Akt, respectively. To examine the effect of these mT-initiated pathways upon gap junctional communication and neoplasia, GJIC was assessed in mT-mutant-expressing, rat liver epithelial T51B cells which normally have extensive GJIC, using a novel technique of in situ electroporation we developed. The results show that although low levels of wt-mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI-3 kinase pathway is not required for GJIC suppression. It is remarkable that T51B cells expressing a mutant deficient in binding of Shc, hence activation of the Ras pathway, are still morphologically transformed and do grow into tumors in syngeneic rats, albeit with an altered morphology. These results indicate that GJIC suppression requires Ras but not PI-3 kinase/Akt signalling, and is independent of full neoplastic conversion in rat liver epithelial cells. Since PI3k activation by mT increases GJIC, we examined whether PI3k might have a positive role upon GJIC. The results showed that PI3k pharmacological inhibition eliminates GJIC, concomitant with apoptosis induction. We next expressed a form of PI3k rendered constitutively active through the addition of a myristylation signal (myr-PI3k). The results demonstrated that myr-PI3k causes an increase in GJIC. Therefore PI3k, although in an activated form it can act as an oncogene, it plays a positive role upon gap junctional, intercellular communication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 31. doi:1538-7445.AM2012-31

Abstract 1810: Mechanistic studies of the dual Akt and JNK inhibitor Chaetoglobosin K: Effect on downstream pathways and AP-1 proteins in tumorigenic cells

Abstract We have previously shown that the anti-tumorigenic indolylcytochalasin Chaetoglobosin K (ChK) concomitantly down-regulates Akt and JNK activation in ras-transformed cells. This dual inhibition was found not be regulated by Rac1, a Rho protein that is involved in both signaling pathways, or MKK4, an upstream kinase of JNK. The purpose of this study was to determine: 1) the effect of ChK on the activation of downstream mediators of the PI3K/Akt (including mdm2), and JNK (including the activator protein-1 (AP-1) proteins and stat3) signaling pathways; 2) whether the upstream mitogen-activated protein kinase kinase 7 (MKK7) is involved in ChK's effect on JNK. The AP-1 transcription factor complex, composed of proteins of the JUN, FOS, and ATF families, has become a promising therapeutic target due to the proposed role of AP-1 activation in tumorigenesis. In these studies, ras-transformed rat liver epithelial cells were grown to confluence and treated with either ChK (5μM or 10μM) or vehicle (DMSO) for 24 hours. Cell proteins were extracted and separated using SDS-PAGE. Western blot analysis was performed using phosphorylation site-specific antibodies to monitor changes in MKK7, c-JUN, ATF-2, mdm2, and stat3 protein activation levels. The results of these studies indicate that ChK treatment decreased c-JUN and ATF-2 phosphorylation, but had no effect on stat3 phosphorylation. Additionally, ChK treatment decreased mdm2 phosphorylation. ChK did not alter MKK7 activation levels and, taking into consideration its lack of effect on MKK4, suggest ChK does not inhibit JNK via its archetypical MAPK signaling pathway. These studies indicate ChK may serve as a small molecule inhibitor of the AP-1 pathway proteins, as well as the Akt/mdm2 pathway. This data further supports ChK's potential as a unique targeted tumor therapy. This project was funded by NIH grant 1R15CA135415 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1810. doi:1538-7445.AM2012-1810

Abstract 3816: Comparative cellular effects of 4-phenyl-3-butenoic acid and suberoylanilide acid in tumorigenic and non-tumorigenic cells

Abstract 4-phenyl-3-butenoic acid (PBA) is a small molecule anti-inflammatory agent which was previously shown to inhibit growth, increase gap junction intercellular communication, increase phosphorylation of p38 mitogen activated protein kinase (p38 MAPK), and decrease phosphorylation of c-Jun N-terminal Kinase (JNK) in tumorigenic cells at concentrations that do not similarly affect non-tumorigenic cells. Suberoylanilide acid (SAHA), which is also known as vorinostat, is a relatively new anticancer agent structurally similar to PBA. The purpose of this study was to compare the effects of these two agents on JNK and p38 activation in vitro, as well as on cell growth. p38 MAPK and JNK are stress-activated members of the MAPK family that play important roles in the control of cell proliferation in a wide variety of cell types. Altered expression or activation of these MAPKs has been observed in numerous tumorigenic cells and human cancers. Our studies utilized human lung carcinoma cells (H2009), transformed rat liver epithelial cells (WB-ras) as well as non-tumorigenic cells from the same line (WB-neo). Cells were grown to 60-70% confluence and were subjected to treatment with PBA (0.1 mg/ml), SAHA (500 nM) or vehicle for 48 hours. Whole cell lysates were extracted and individual proteins were separated by SDS-PAGE, followed by western blotting using phosphorylation site-specific antibodies to p38 and JNK proteins. For cell counting, cells were trypsinized and suspended followed by counting utilizing a Biorad TC-10TM cell counter. PBA and SAHA were shown to have similar effects on JNK and p38 MAPK phosphorylation in tumorigenic WB-ras and H2009 cell lines. These effects included an increase in p38 MAPK phosphorylation and a decrease in JNK phosphorylation. Both compounds were shown to significantly decrease cell growth under these conditions. In untransformed WB-neo cells significant differences in p38 MAPK phosphorylation were not seen with either treatment. However it was shown that a decrease in phosphorylation of JNK occurred following SAHA treatment, but not PBA treatment in the WB-neo cells. Overall these studies demonstrate that PBA has similar effects on JNK and p38 MAPK compared to SAHA in tumorigenic cells, while being less potent in the non-tumorigenic WB-neo cells. This earmarks PBA for further studies as a targeted anti-tumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3816. doi:1538-7445.AM2012-3816

Defective signaling in a spontaneous transformation model of rat prostate epithelial cells

We describe an in vitro model of spontaneous transformation using normal rat prostate epithelial cells (PEC) that culminates at high passage (p>85) in anchorage independent growth when plated on soft agar. High passage PEC also formed large tumors in immunodeficient mice. We identified a small subpopulation of cells derived from the high passage anchorage independent cells that migrate through soft agar and expand on the underlying plastic substratum. In addition, several antibodies generated against rat liver epithelial cells of ductal origin that react strongly with rat prostate cells were examined. OC.2 was expressed by a low percentage of low and high passage PEC but was expressed by 100% of freshly isolated soft agar invasive (SAI) cells. Conversely, OC.4 and OC.5 were expressed by a high percentage of low and high passage PEC but were absent in SAI cells. Tumorigenicity experiments demonstrated that the SAI subpopulation was considerably more tumorigenic than high passage cells, evidenced by decreased latency period and increased tumor size. Immunoblot analysis identified alterations in signaling proteins belonging to EGFR and JAK/STAT pathways between low pass, high pass and SAI cells. We are investigating the role of these pathways on the phenotype of the 3 PEC models by examining anchorage independent growth, invasion, and tumorigenicity. (Supported by COBRE grant 1P20RR17695)

Antioxidant activity of Fragilariopsis pseudonana and protective effect against hydrogen peroxide-induced inhibition of gap junctional intercellular communication

This study was aimed to evaluate antioxidant activities of a methanol extract from the polar microalgae Fragilariopsis pseudonana (FP) and to investigate the protective effect for gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The results indicated that the methanol extract of FP (FPM) scavenged ABTS free radicals and inhibited copper-induced low density lipoprotein oxidation in a dose-dependent manner. Exposing HepG2 cells to FeSO4 increased intracellular reactive oxygen species formation and lipid peroxides, which was significantly reduced by treatment with FPM. Furthermore, FPM increased the activities of superoxide dismutase and glutathione peroxidase in cells. FPM recovered the hydrogen peroxide (H2O2)-induced inhibition of GJIC and attenuated the phosphorylation of connexin 43 (Cx43) and extracellular signal-regulated kinase1/2 (ERK1/2). Taken together, these results demonstrated that FPM has antioxidant activity and protects against H2O2-induced inhibition of GJIC by blocking phosphorylation of Cx43 via activation of ERK1/2.

Tannic Acid Down-Regulates the Angiotensin Type 1 Receptor Through a MAPK-Dependent Mechanism

In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 μg/ml (7.34-58.78 μm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression.

The influence of oracin on reduction and toxicity of doxorubicin in hepatocytes and mammary epithelial cells MCF-10A

The ways, how to increase effectiveness of doxorubicin (DOX) in cancer cells and decrease its toxicity in normal cells, have been intensively studied. In breast cancer cells MCF-7, isoquinoline derivative oracin (ORC) inhibited DOX reduction and increased DOX antiproliferative effect. The aim of this study was to test the influence of ORC on the reduction of DOX and its toxicity in hepatocytes and non-tumourous breast cells.The kinetics of DOX reduction was measured in cytosols from rat liver, human liver and human mammary epithelial cells MCF-10A. Activity and expression of carbonyl reductase 1 (CBR1) were assayed using menadione as a substrate and western blot analysis. End-point tests of viability served for study of cytotoxicity of DOX, ORC and DOX+ORC combinations in rat hepatocytes and MCF-10A cells.The inhibitory effect of ORC on DOX reductases was almost none in MCF-10A cells and mild in liver. CBR1 expression and activity was lower in non-tumourous MCF-10A cells than in cancer MCF-7 cells. Cytotoxicity tests showed that DOX+ORC combinations had significantly lower toxicity than DOX alone in MCF-10A cells as well as in hepatocytes.ORC significantly decreases DOX toxicity in MCF-10A and in hepatocytes. Therefore, concomitant use of ORC and DOX may protect normal cells against DOX toxicity.

A novel antihepatitis drug, bicyclol, prevents liver carcinogenesis in diethylnitrosamine-initiated and phenobarbital-promoted mice tumor model.

Bicyclol, an antihepatitis drug developed by Chinese scientists, has been shown to prevent the malignant transformation induced by 3-methylcholanthrene and 12-O-tetradecanoylphorbol-13-acetate in WB-F344 rat liver epithelial cells. This study provides further evidence on its role as a chemopreventive agent in experimental mice with diethylnitrosamine- (DEN-) initiated and phenobarbital- (PB-) promoted liver carcinoma. Liver tissue and serum were collected. In the two-stage model of hepatocarcinogenesis in mice, oral administration of bicyclol (100, 200 mg/kg) before DEN injection showed significant reduction in the incidence of hepatocellular foci, nodules, or carcinoma. Histopathological examination revealed that there was no hepatocellular carcinoma (HCC) and hepatoma formation in the mice pretreated with bicyclol (200 mg/kg) at week 20, while the mice treated with DEN/PB developed 33.3% HCC and 55.6% hepatoma. Furthermore, the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and α-fetal protein (AFP) in serum significantly increased in the DEN/PB model group in comparison with the control group. Pretreatment with bicyclol showed a marked reduction in the above condition. Bicyclol also decreased the expression of AFP and proliferating cell nuclear antigen level in the liver tissue and attenuated the decrease in body weight. In this study, we also found that 10 weeks after stopping the administration of PB and drugs, the control and bicyclol-treated (200 mg/kg) animals showed no HCC and hepatoma formation at the time of termination whereas DEN/PB-induced mice developed 100% hepatoma and 50% HCC. These results further indicate that bicyclol has the chemopreventive potential for liver carcinogenesis induced by carcinogens.

13-cis-Retinoic acid specific down-regulation of angiotensin type 1 receptor in rat liver epithelial and aortic smooth muscle cells

Transcriptional repression through cis- and trans-acting factors enabling an alternate approach to control angiotensin type 1 receptor (AT1 or AGTR1 as listed in the MGI database) expression has not been studied. In previous investigations, treatment with retinoic acid was found to be associated with enhanced insulin sensitivity. In our previous study, expression of AT1 was found to be inversely correlated with intracellular glucose concentrations. Therefore, we hypothesized that 13-cis-retinoic acid (13cRA), an antioxidant, enhances insulin-sensitive glucose-mediated down-regulation of the AT1. In this study, we used continuously passaged rat liver epithelial cells. Our study shows that cells exposed to 13cRA specifically down-regulated the AT1 protein in a dose- and time-dependent manner, independently of any change in receptor affinity. Down-regulation of the AT1 expression leads to reduced AngII-mediated intracellular calcium release, a hallmark of receptor-mediated intracellular signaling. Similarly with receptor down-regulation, we observed a significant reduction in AT1 mRNA; however, the AT1 down-regulation was independent of insulin-sensitive glucose uptake and retinoic acid receptor activation (RAR/RXR). Treatment with 13cRA resulted in phosphorylation of p42/p44 MAP kinases in these cells. Subsequent studies using MEK inhibitor PD98059 prevented 13cRA-mediated AT1 down-regulation and restored AngII-mediated intracellular calcium response. Furthermore, 13cRA-mediated inhibitory effects on AT1 were validated in primary rat aortic smooth muscle cells. In summary, our results demonstrate for the first time that 13cRA has a glucose- and RAR/RXR-independent mechanism for transcriptional inhibition of AT1, suggesting its therapeutic potential in systems in which AT1 expression is deregulated in insulin-sensitive and -insensitive tissues.

P38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras‐transformed cells by 4‐Phenyl‐3‐butenoic acid

Human lung neoplasms frequently express mutations that down-regulate expression of various tumor suppressor molecules, including mitogen-activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti-tumor therapies. We now report that 4-phenyl-3-butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non-cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell-cell communication. H2009 human lung carcinoma cells and ras-transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras-transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell-cell communication and connexin 43 phosphorylation in ras-transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up-regulation of p38 MAPK activity, altered connexin 43 expression, and down-regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down-regulated p38 MAPK activity and/or activated JNK and altered cell-cell communication.

Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.

Examing the calcium response of white matter glia cells with calcium imaging

Free fatty acids (FFAs) are dietary nutrients which act as ligands for FFAs receptors. G-protein-coupled receptor 120 (GPR120) and GPR40 are activated by long and medium chain FFAs. In the present study, we investigated the role of the GPR120 and GPR40 in cell motile activity stimulated by ethionine in rat liver epithelial WB-F344 cells. Cells were treated with ethionine at a concentration of 10 μM every 24 h for 2 days. The expression levels of the Gpr120 and Gpr40 genes in WB-F344 cells treated ethionine were significantly higher than those in untreated cells. In cell motility assay, the cell motile activity of WB-F344 cells was markedly elevated by ethionine, compared with untreated cells. To evaluate the effects of GPR120 on the cell motile activity by ethionine, we established GPR120 knockdown cells from WB-F344 cells. The cell motile activity stimulated by ethionine was significantly suppressed by GPR120 knockdown. In addition, a potent GPR40 antagonist GW1100 enhanced the cell motile activity by ethionine. These results suggest that opposite effects of GPR120 and GPR40 may be involved in the cell motile activity stimulated by ethionine in WB-F344 cells.

Inhibition of osteoblastic differentiation by warfarin and 18-α-glycyrrhetinic acid

Anticoagulation therapy with vitamin K antagonists such as warfarin is widely used to prevent and treat stroke in patients with chronic atrial fibrillation or mechanical heart valves. Because vitamin K is an essential factor for ggg-carboxylation of osteocalcin, vitamin K antagonists might cause bone loss. Although the association between warfarin use and bone metabolism is still controversial, several studies show that bone mineral density is decreased and fracture risk is increased with warfarin therapy. Meanwhile, attenuation of gap junctional communication (GJC) by warfarin is reported in rat liver epithelial cells. However, the effect of warfarin on osteoblasts, in which GJC is important for osteoblastic differentiation, remains unknown. Here we investigated whether warfarin has an inhibitory effect on osteoblastic differentiation using an osteoblastic cell line (C2C12). Warfarin and 18-α-glycyrrhetinic acid (AGA), which is known as a nontoxic reversible GJC inhibitor, had the same effect on osteoblastic differentiation. Warfarin and AGA inhibited the bone morphogenetic protein (BMP)2-induced mRNA levels of alkaline phosphatase (ALP), collagen I α1, osteocalcin (OC) and osterix, which are specific markers for osteoblastic differentiation, in a dose-dependent manner. Moreover, the activities of OC- and ALP-luciferase reporters, which are induced by BMP2, and the transcriptional activity of Runx2 on OC and ALP promoters were inhibited by warfarin and AGA. The amount and activity of ALP induced by BMP2 were also decreased by warfarin and AGA. These results suggest that warfarin and AGA, a GJC inhibitor, have an inhibitory effect on osteoblastic differentiation.

Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like Cells

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Modulation of gap-junctional intercellular communication by a series of cyanobacterial samples from nature and laboratory cultures

Cyanobacterial extracts have been recently shown to alter two in vitro biomarkers of tumor promotion, namely to cause inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (Blaha et al., 2010a). In the present study, we investigated GJIC-inhibitory potencies of 10 laboratory strains representing common water bloom-forming cyanobacteria (Anabaena, Aphanizomenon, Cylindrospermopsis, Microcystis and Planktothrix) and six natural water bloom samples (dominated by Aphanizomenon sp. or Microcystis). The most pronounced inhibitions of GJIC in a model rat liver epithelial cell line WB-F344 were caused by methanolic extracts of Anabaena flos-aquae UTEX 1444, Aphanizomenon flos-aquae SAG 31.87, Aphanizomenon gracile RCX 06, Microcystis aeruginosa PCC 7806, Cylindrospermopsis raciborskii SAG 1.97, Planktothrix agardhii CCALA 159 and SAG 32.79, whereas weaker effects were induced by Aphanizomenon klebahnii CCALA 009 and no inhibition was induced by extracts of Aph. flos-aquae PCC 7905 and Aph. gracile SAG 31.79. Exudates of the laboratory cultured strains concentrated by solid phase extraction also induced species-specific inhibitory effects, but they did not necessarily correlate with the inhibitory potencies of extracts from the corresponding species. Interestingly, the GJIC-inhibitory effects may not be restricted to cyanobacteria, since exudates of two green alga species also affected GJIC, although their extracts caused no effects. The extracts from different natural water blooms inhibited GJIC with different potencies without apparent relation to bloom-species composition. Since the observed effects on GJIC did not correlate with the content of cyanotoxins microcystins and cylindrospermopsin in the tested samples, they were most likely induced by unknown compound(s). Our results indicate that putative tumor promoting compound(s) could be associated with different species of bloom-forming cyanobacteria, but their production is probably species- and strain-specific.

Abstract 3123: Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication

Abstract Gap junctions are channels that connect the cytoplasm of adjacent cells. Gap junctional, intercellular communication (GJIC) is blocked in cells transformed by oncogenes such as the middle Tumor antigen of polyoma virus (mT), an oncoprotein which associates with and is tyrosine-phosphorylated by cSrc family members. Specific phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) and the SH2 domain of the phosphatidylinositol 3-kinase (mT-tyr315), resulting in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI-3 kinase/Akt, respectively. To examine the effect of these mT-initiated pathways upon gap junctional communication and neoplasia, GJIC was assessed in mT-mutant-expressing, rat liver epithelial T51B cells which normally have extensive GJIC, using a novel technique of in situ electroporation we developed. The results show that although low levels of wt-mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI-3 kinase pathway is not required for GJIC suppression. It is remarkable that T51B cells expressing a mutant deficient in binding of Shc, hence activation of the Ras pathway, are still morphologically transformed and do grow into tumors in syngeneic rats, albeit with an altered morphology. These results indicate that GJIC suppression requires Ras/Erk but not PI-3 kinase/Akt signalling, and is independent of neoplastic conversion in rat liver epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2011-3123

Abstract 2649: Attenuation of JNK phosphorylation by 4-phenyl-3-butenoic acid in tumorigenic cells

Abstract The c-Jun N-terminal kinase (JNK) signaling pathway controls many cellular processes such as cell growth, differentiation, and apoptosis. Aberrant JNK activity has been implicated in various cancer types including but not limited to hepatocellular carcinoma, glioblastoma, leukemia, and lung cancer, presumably through activation of the transcription factor activator protein-1 (AP-1). When activated by JNKs, AP-1 is a positive regulator of cell proliferation and many tumorigenic cell types are found to have increased JNK activity. Phosphorylated mitogen-activated protein kinase kinase 4 (pMKK4) and phosphorylated mitogen-activated protein kinase kinase 7 (pMKK7) are known to activate JNKs via phosphorylation. The purpose of this study was to determine the effects of 4-phenyl-3-butenoic acid (PBA) on JNK pathway proteins. PBA is a small molecule anti-inflammatory agent which was previously shown to inhibit growth, increase gap junction intercellular communication, and increase phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) in tumorigenic cells at concentrations that do not similarly affect non-tumorigenic cells. Our studies utilized tumorigenic ras-transformed rat liver epithelial cells (WB-ras) and human lung carcinoma (H2009) cells. Cells were grown to confluence and were subjected to treatment with PBA (0.1 mg/ml) or vehicle for 48 hours. Whole cell lysates were extracted and individual proteins were separated by SDS-PAGE, followed by western blotting using phosphorylation site-specific antibodies to JNK pathway proteins. Quantitative analysis of western blots was performed utilizing densitometry software. PBA attenuated phosphorylation of JNK approximately two-fold in both WB-ras and H2009 lung carcinoma cells. PBA also attenuated phosphorylation of the JNK downstream effector, activating transcription factor 2 (ATF-2). Phosphorylation of MKK4 was decreased following treatment with PBA, while phosphorylation of MKK7 was unaffected. This data suggests that PBA may inhibit growth of tumorigenic cells in part through decreased activation of the JNK pathway via a MKK4 mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2649. doi:10.1158/1538-7445.AM2011-2649

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1–5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell–cell contact was apparent in CH (0.5–5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.