Diallyl trisulfide induces apoptosis of human basal cell carcinoma cells via endoplasmic reticulum stress and the mitochondrial pathway

Diallyl trisulfide (DATS), an active component of garlic oil, has attracted much attention because of its anticancer effect on several types of cancers. However, the mechanism of DATS-induced apoptosis of basal cell carcinoma (BCC) is not fully understood. In the present study, we revealed that DATS-mediated dose-dependent induction of apoptosis in BCC cells was associated with intracellular reactive oxygen species accumulation and disrupted mitochondrial membrane potential. Western analysis demonstrated concordant expression of molecules involved in mitochondrial apoptosis, including DATS-associated increases in phospho-p53, proapoptotic Bax, and decreases in antiapoptotic Bcl-2 and Bcl-xl in BCC cells. Moreover, DATS induced the release of cytochrome c, apoptosis-inducing factor, and HtrA2/Omi into the cytoplasm, and activated factors downstream of caspase-dependent and caspase-independent apoptosis, including nuclear translocation of apoptotic-inducing factor and endonuclease G and the caspase cascade. These results were confirmed by pretreatment with the antioxidant N-acetyl-L-cysteine and the caspase inhibitor (z-VAD-fmk), the latter of which did not completely enhance the viability of DATS-treated BBC cells. Exposure to DATS additionally induced endogenous endoplasmic reticulum stress markers and intracellular Ca2(+) mobilization, upregulation of Bip/GRP78 and CHOP/GADD153, and activation of caspase-4. Our findings suggest that DATS exerts chemopreventive potential via ER stress and the mitochondrial pathway in BCC cells.     

Dual effect of ethanol on cell death in primary culture of human and rat hepatocytes

AIMS: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. METHODS: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200-250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0-10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium-DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. RESULTS: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1-2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. CONCLUSIONS: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.     

Diallyl disulfide and diallyl trisulfide suppress oxidized LDL-induced vascular cell adhesion molecule and E-selectin expression through protein kinase A- and B-dependent signaling pathways

Uptake of oxidized LDL (ox-LDL) by vascular endothelial cells is a critical step in the initiation and development of atherosclerosis. Adhesion molecules are upregulated by ox-LDL and numerous inflammatory cytokines and play a pivotal role in atherogenesis. In this study, we examined whether diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), 3 major organosulfur compounds of garlic oil, reduce adhesion molecule expression induced by ox-LDL and, if so, through what mechanism. Human umbilical vein endothelial cells were preincubated with 1 mmol/L DAS, 200 mumol/L DADS, or 100 mumol/L DATS for 16 h and then with 40 mg/L ox-LDL for an additional 24 h. ox-LDL induction of cellular and cell surface expression of E-selectin and vascular cell adhesion molecule (VCAM)-1 was suppressed by garlic allyl sulfides in the order DATS > DADS > DAS. The adhesion of HL-60 cells to endothelial cells was inhibited 27 and 33% and the production of cellular peroxides was inhibited 43 and 50% by DADS and DATS, respectively (P < 0.05). ox-LDL alone dephosphorylated protein kinase B (PKB) and cAMP responsive element binding protein (CREB); such deactivation was reversed by DADS and DATS. Electrophoretic mobility shift assay showed that the activation of CREB binding to DNA was consistent with changes in CREB phosphorylation. The protein kinase A (PKA) inhibitor H89 reversed the suppression of VCAM-1 by DADS and DATS, but the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect. In contrast, wortmannin abolished DADS- and DATS-induced suppression of ox-LDL-induced E-selectin expression. These results suggest that the suppression of ox-LDL-induced E-selectin and VCAM-1 expression by DADS and DATS and, thus, monocyte adhesion to endothelial cells is likely dependent on the PI3K/PKB or PKA/CREB signaling pathway in an adhesion molecule-specific manner. To our knowledge, this is the first report that garlic modulates ox-LDL-mediated leukocyte adhesion to human endothelial cells through the PKB and PKA pathways.     

Consequences of acrylonitrile metabolism in rat hepatocytes: effects on lipid peroxidation and viability of the cells

Acrylonitrile caused thiobarbituric acid-positive reactants time- and concentration-dependently in isolated hepatocytes. This effect was markedly enhanced by gassing of the medium with 95% oxygen-5% CO2 gas mixture. Glycidonitrile, an acrylonitrile metabolite, proved more potent in this respect than the parent acrylonitrile or its end metabolite, cyanide anion. The latter decreased greatly the viability of isolated liver cells but caused thiobarbituric acid-positive reactants only in the presence of diethylmaleate. Acrylonitrile caused also a decrease in the concentration of nonprotein sulfhydryl groups but the oxidation of glutathione (GSH) to GSSG (oxidized glutathione) was not the major mechanism. This might indicate the consumption of GSH in the glutathione S-transferase catalyzed reactions. In contrast to cyanide anion-induced effects acrylonitrile did not affect markedly the viability of hepatocytes.     

Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes

The effects of extracellular magnesium (Mg) concentration on the rate of apoptosis in rat hepatocytes in primary culture were examined. After overnight attachment, incubations were conducted for up to 72 h in serum-free media containing low (0-0.4 mmol/L), physiological (0.8 mmol/L) or high (2 and 5.6 mmol/L) Mg concentrations. At 72 h, we observed numerous rounded hepatocytes on top of a shrunken cell monolayer at extracellular Mg concentrations < 0.8 mmol/L. These morphological features were associated with Mg-dependent differences in the total protein levels. The various Mg concentrations did not affect DNA synthesis; however, at a concentration < 0.8 mmol/L, the susceptibility of cultured rat hepatocytes to oxidative stress was increased as shown by the reduced glutathione concentration (10.6 +/- 2.8 vs. 37.3 +/- 4.1 nmol/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05) and increased lipid peroxidation (0.36 +/- 0.03 vs. 0.21 +/- 0.01 nmol malondialdehyde/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05). Fluorescence microscopy after Hoechst dye staining revealed numerous apoptotic figures in Mg-free monolayers compared with 0.8 and 5.6 mmol/L Mg conditions. These observations were confirmed quantitatively by flow-cytometric analysis after propidium iodide staining. The proportion of subdiploid cells decreased with increasing Mg concentration; for example, it was greater at 72 h in Mg-free cultures (76%) than in cultures containing 0.8 mmol/L or 5.6 mmol/L Mg (28%; P < 0.05). Caspase-3 was highly activated in Mg-free cultures after 48 h of treatment compared with 0.8 and 5.6 mmol/L conditions (P < 0.05). Overall, these results show that extracellular Mg deficiency has a negative effect on the survival of cultured rat hepatocytes by inducing apoptosis; however, supplementation of extracellular Mg did not reduce the spontaneous apoptosis that occurred over time in rat hepatocyte cultures.     

Effects of beta-carotene on cell viability and antioxidant status of hepatocytes from chronically ethanol-fed rats

The purpose of the present study was to evaluate the effects of beta-carotene on the cell viability and antioxidant status of hepatocytes from chronically ethanol-fed rats. Rats in the ethanol group were given an ethanol-containing liquid diet that provided 36 % of total energy as ethanol, while rats in the control group were fed an isoenergetic diet without ethanol. After 4 weeks, hepatocytes were taken out and cultured for 24 h. Hepatocytes from the rats in the control and ethanol groups were cultured in medium without (HC, HE) or with beta-carotene (HC+B, HE+B). The results showed that lactate dehydrogenase leakage was significantly increased in the HE compared with that in the HC group. However, lactate dehydrogenase leakage of the HE+B group was similar to that of the HC group. When compared with the HC group, activities of glutathione peroxidase and catalase in the HE group were significantly decreased by 54 and 31 %, respectively. Catalase activity in the HE+B group was significantly increased by 61 % compared with that in the HE group. However, activities of glutathione reductase and superoxide dismutase showed no difference among the groups. The level of glutathione in the HC+B and HE+B groups was significantly increased to 155 and 143 % compared with those in the HC and HE groups, respectively. The concentration of lipid peroxides showed no difference among the groups. The present results demonstrate that beta-carotene improved the cell viability of hepatocytes, and increased catalase activities and glutathione levels in hepatocytes from chronically ethanol-fed rats.     

Cytotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin on primary cultures of adult rat hepatocytes

Primary cultures of adult rat hepatocytes were used to study the cytotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Rats were treated with a single oral dose of 5, 10 or 25Μg/kg of TCDD. At 2, 6, 10, 20, 30 days post-treatment, hepatocytes were isolated by a collagenase perfusion technique and maintained 48 hr as monolayers in serum-free medium in collagen-coated culture dishes. Uptake of ouabain was depressed in hepatocyte cultures isolated from all three doses of TCDD-treated rats. The effect was detected two days after TCDD-treatment and continued up to 30 days. The magnitude of the depression was directly related to the dose of TCDD. Hepatocyte cultures from TCDD-treated rats also showed depression in hormonally induced uptake ofα-aminoisobutyric acid (AIB) and tyrosine aminotransferase (TAT) activity at 10 and 25Μg/kg but not at 5Μg/kg. These cytotoxic effects of TCDD could be demonstrated only when TCDD was given to rats prior to isolation of hepatocytes; no toxic effects were observed when TCDD was added directly to the hepatocyte cultures. Pretreatment of rats with 1,3,6,8-tetrachlorodibenzo-p-dioxin had no effect on ouabain uptake, AIB transport, or TAT activities.     

Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2

We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells.     

Toxicity assessments of chemical substances using primary culture of rat hepatocytes

A primary hepatocyte culture was used as a model system to assess the toxicity of various chemical substances. Chemical substances tested in this experiment included 14 kinds of organic solvents, arsenic acid and N-nitrosodiumethylamine, most of which are known as hepatotoxic materials. Enzyme (GPT and LDH) leakage and albumin secreted from the hepatocytes to culture medium were measured to evaluate the cell damage after exposure to the chemical substances at various concentrations for 3 d. Cell counts and protein contents before and after exposure to the chemical substances were also measured during the course of these experiments. The extent of LDH leakage from hepatocytes was parallel to that of GPT leakage after exposure to the chemicals. Albumin secreted from the hepatocytes in the culture medium evidently decreased at concentrations of the chemicals which increased the enzyme leakage. Viable cell counts were significantly decreased by chemicals that increased the enzyme leakage, although the cell protein contents were not significantly affected. The minimum concentration of the chemicals at which enzyme leakage from hepatocytes was significantly increased was defined as the lowest toxic concentration (TCL0). Judging from TCL0 values, the degree of toxicity in these chemicals seems to be almost identical to the degree of in vivo hepatotoxicity reported previously. We, furthermore, observed that there is a positive correlation (r = 0.780, p less than 0.01) between PT50 calculated by the LD50 value reported previously and -Log magnitude of TCL0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes.

Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation.     

Hormones and nutrients regulate acetyl-CoA carboxylase promoter I in rat primary hepatocytes

This study investigated the regulation of acetyl-CoA carboxylase (ACC) promoter activity by hormones and nutrients. Genomic clones including promoter I (PI) of the ACC gene were isolated and sequenced. ACC PI fragments (-1,049/+100 or -220/+21 bp) were subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. The ACC PI/luciferase chimeric plasmids were transfected into primary rat hepatocytes using lipofectin. Insulin treatment increased the activity of -1,049/+ 100 and -220/+21 ACC PI by 3.0- and 3.5-fold, respectively, compared to the control. The activity of both constructs was also increased by dexamethasone (Dex) and triiodothyronine (T3), with the greatest effects seen with all three hormones present. With -1,049/+100 or -220/+21 ACC PI, the addition of glucose increased luciferase activity compared to glucose-free control (p<0.05). On the other hand, polyunsaturated fatty acids (PUFA) reduced the activity of the -1,049/+100 ACC PI construct, with eicosapentaenoic acid and docosahexaenoic acid showing the greatest effect (about 70% of the control). However, the addition of PUFA to the culture media did not affect the activity of -220/+21 ACC PI. Therefore, insulin, Dex, T3, glucose, and PUFA regulate ACC gene expression, at least in part, through the PI promoter.     

氟化物对原代培养大鼠肝细胞酶活力及超微结构的影响

目的研究不同剂量的氟化物对原代培养大鼠肝细胞存活率、酶活力及超微结构的影响。方法采用半原位胶原酶消化法分离大鼠肝细胞;MTT法检测细胞存活率;赖氏法检测培养液中谷草转氨酶(AST)和谷丙转氨酶(ALT)活性;透射电镜观察细胞超微结构改变。结果氟化钠染毒24小时后肝细胞存活率下降,且呈现明显的剂量-效应关系;2mmolL和4mmolL染氟组肝细胞培养液中ALT和AST的活性显著升高(P<005);透射电镜下染氟组肝细胞线粒体肿胀,内质网排列紊乱或断裂。结论过量氟化物对原代培养大鼠肝细胞有明显的毒作用,其主要作用方式是引起细胞膜和细胞器质膜损伤。     

Gene expression analysis of the hepatotoxicant methapyrilene in primary rat hepatocytes: an interlaboratory study

Genomics technologies are used in several disciplines, including toxicology. However, these technologies are relatively new, and their applications require further investigations. When investigators apply these technologies to in vitro experiments, two major issues need to be clarified: a) can in vitro toxicity studies, in combination with genomics analyses, be used to predict the toxicity of a compound; and b) are the generated toxicogenomics data reproducible between laboratories? These questions were addressed by an interlaboratory study with laboratories of four pharmaceutical companies. We evaluated gene expression patterns from cultured rat primary hepatocytes after a 24-hr incubation with methapyrilene (MP). Extensive data analysis showed that comparison of genomics data from different sources is complex because both experimental and statistical variability are important confounding factors. However, appropriate statistical tools allowed us to use gene expression profiles to distinguish high-dose-treated cells from vehicle-treated cells. Moreover, we correctly identified MP in an independently generated in vitro database, underlining that in vitro toxicogenomics could be a predictive tool for toxicity. From a mechanistic point of view, despite the observed site-to-site variability, there was good concordance regarding the affected biologic processes. Several subsets of regulated genes were obtained by analyzing the data sets with one method or using different statistical analysis methods. The identified genes are involved in cellular processes that are associated to the exposure of primary hepatocytes to MP. Whether they are specific for MP and are cause or consequence of the toxicity requires further investigations.     

Sulfur amino acid restriction induces the pi class of glutathione S-transferase expression in primary rat hepatocytes

The regulation of genes by amino acids is attracting increasing attention. In the present study, we investigated the restriction of expression of the pi class of glutathione S-transferase (GST Yp) by sulfur amino acids. Hepatocytes isolated from male Sprague-Dawley rats were cultured with L-15-based medium containing low (LSAA; 0.1 mmol/L L-methionine and 0.1 mmol/L L-cysteine) or high (HSAA; 0.5 mmol/L L-methionine and 0.2 mmol/L L-cysteine) amounts of sulfur amino acids for up to 6 d. Cellular protein contents did not differ between LSAA- and HSAA-treated cells over the entire period. In contrast, glutathione concentrations were suppressed by the LSAA medium and on d 6 were only 20% of those of HSAA-treated cells (P < 0.05). As shown by immunoblot analysis, GST Yp protein levels were greater in LSAA-treated cells than in HSAA-treated cells (P < 0.05). The induction of GST Yp by L-methionine and L-cysteine restriction was not affected by insulin and dexamethasone, but the latter suppressed GST Yp expression (P < 0.05). LSAA increased GST Yp mRNA levels and GST activity toward ethacrynic acid (P < 0.05). GST Yp induction occurred only in cells with a limited supply of L-methionine; restriction of L-isoleucine, L-leucine, L-lysine, and L-phenylalanine had no significant effect. In contrast with the induction of GST Yp, the expression of the GST isoforms Ya and Yb was not changed by amino acid restriction. In conclusion, hepatic GST Yp gene expression is upregulated by a limited availability of sulfur amino acids.     

Metabolism of gamma-linolenic acid in primary cultures of rat hepatocytes and in Hep G2 cells

Incorporation and metabolism of gamma-linolenic acid (GLA) in both rat hepatocytes and Hep G2 cells were compared to those of oleic (OA), linoleic (LA), alpha-linolenic (LLA), and dihomo-gamma-linolenic (DGLA) acids. The incorporation of GLA into both types of cells was higher than LLA and DGLA, but lower than OA and LA. It was efficiently converted into DGLA in both types of cells and increased the concentration of DGLA. LLA was converted to a small amount of C20:4 (n-3) only in Hep G2 cells. Incubation with LA, GLA, LLA, and DGLA did not increase the concentration of arachidonic acid (AA) in both types of cells. LA. GLA, LLA, and their metabolites were incorporated into phosphatidylcholine, but only GLA and its metabolite, DGLA, were also incorporated into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The coexistence of GLA and LLA during their catabolism diminished the amounts of respective metabolite in Hep G2 cells. The presence of GLA inhibited completely the formation of C20:4(n-3) from LLA. The results indicate that GLA is more effective in raising the ratio of DGLA/AA. Also, polyunsaturated fatty acids of n-3 and n-6 series have competitively catabolized in both types of hepatocytes.     

Carnitine alters binding of aflatoxin to DNA and proteins in rat hepatocytes and cell-free systems.

The objective of this study was to determine effects of L-carnitine on aflatoxin B(1) (AFB(1))-DNA adduct formation in isolated rat hepatocytes, its dose response, specificity and mode of action. All experiments were conducted in either freshly isolated rat hepatocytes or cell-free systems. There was negative linear correlation between the dosage of carnitine and formation of [(3)H]AFB(1)-DNA adducts in the hepatocytes; however, the partitioning of AFB(1) into cellular compartments was not affected by carnitine. The attenuating effect of carnitine on AFB(1)-DNA adduct formation was also present in a cell-free system, but there was lack of specificity because acetylcarnitine and gamma-aminobutyric acid (GABA) were equally effective. Carnitine appears to interfere with bioactivation of AFB(1) and binding of AFB(1)-epoxide to DNA. On the contrary, carnitine enhanced the binding of AFB(1) and its epoxide to microsomal proteins, plasma proteins and bovine serum albumin. These results indicate that carnitine diverts AFB(1)-epoxide away from DNA by promoting binding to proteins. We conclude that modulation of AFB(1) binding to proteins and DNA by carnitine alters the carcinogenic and hepatotoxic potential of AFB(1) and poses concerns about the human AFB(1)-exposure data based on the AFB(1)-albumin adduct concentrations as a biomarker.