Metabolic competition studies of 2'-fluoro-5-iodo-1-beta-d-arabinofuranosylcytosine in vero cells and herpes simplex type 1-infected vero cells

2'-Fluoro-5-iodo-1-beta-D-arabinofuranosylcytosine (FIAC) is a potent antiviral agent with minimal cytotoxicity. In Vero cells, incorporation of labeled dCyd and dThd into the acid-insoluble DNA fraction was, respectively, competitively and noncompetitively inhibited by FIAC. In herpes simplex type 1 (HSV-1) infected Vero cells, these inhibition patterns became noncompetitive. The inhibition constants of FIAC on dThd and dCyd incorporation into the acid-insoluble fraction during a 15-min period were greater than 30 microM which were much higher than the antiviral concentration of FIAC (ED90 = 0.003-0.013 microM) for continuous exposure. Incorporation of dUrd into acid-insoluble DNA was inhibited by 10 microM FIAC in HSV-1-infected Vero cells, but not in uninfected cells. The radioactivity of [2-14C]FIAC was incorporated into the acid-insoluble DNA fraction, and this incorporation in uninfected cells was strongly inhibited by 10 microM dCyd but not by dThd. By contrast, the incorporation in HSV-1-infected Vero cells was strongly inhibited by 10 microM dThd but not by dCyd. These data indicate that FIAC behaves metabolically like dThd, dUrd, or 5-iodo-dUrd in HSV-1-infected cells but like dCyd in noninfected cells. Thus, combined use of dCyd and FIAC may reduce cytotoxicity of FIAC or incorporation of FIAC into host cell DNA without affecting its antiviral activity. This finding is of significance since, for practical reasons, incorporation of FIAC into host cell DNA needs to be reduced as much as possible.     

Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines

The effects of the flavonoids from Scutellaria baicalensis Georgi (baicalein, baicalin and wogonin) in cultured human hepatoma cells (Hep G2, Hep 3B and SK-Hep1) were compared by MTT assay and flow cytometry. All three flavonoids dose-dependently decreased the cell viabilities accompanying the collapse of mitochondrial membrane potential and the depletion of glutathione content. However, the influence of baicalein, baicalin or wogonin on cell cycle progression was different. All three flavonoids resulted in prominent increase of G2/M population in Hep G2 cells, whereas an accumulation of sub G1 (hypoploid) peak in Hep 3B cells was observed. In SK-Hep1 cells, baicalein and baicalin resulted in dramatic boost in hypoploid peak, but wogonin made mainly in G1 phase accumulation. These data, together with the previous findings in other hepatoma cell lines, suggest that baicalein, baicalin and wogonin might be effective candidates for inducing apoptosis or inhibiting proliferation in various human hepatoma cell lines.     

Inhibitory effect of rhodamine B on the proliferation of human lip fibroblasts in culture

The effect of the cosmetic dye rhodamine B on the proliferation of human lip fibroblasts (KD cells) was investigated in a culture system. Rhodamine B at 25 micrograms/ml and above significantly decreased the number of the cells after a 72 h culture. A time course study revealed that 50 micrograms/ml of rhodamine B-induced decrease in the cell number occurred after 48 h and longer, suggesting that the dye inhibited cell proliferation without a decrease in cell attachment. The detachment of [3H]thymidine-labeled cells from the monolayer was unaffected by rhodamine B at 100 micrograms/ml and below. The incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of the cell layer was significantly inhibited by 50 micrograms/ml rhodamine B treatment. Histologically, the damage of KD cells was not marked, however, a degenerative change of nuclei and an irregular shape of the cells as well as a decrease in the cell number were caused by 50 micrograms/ml rhodamine B. Rhodamine 6G caused a severe damage of the cells, and rhodamine B significantly decreased the cell number; rhodamine 123 had no significant effect; rhodamine 116 significantly increased the cell number. Furthermore, rhodamine B decreased the number of both vascular endothelial cells from bovine aorta and vascular smooth muscle cells from murine aorta after a 72 h culture. It is concluded that rhodamine B inhibits the proliferation of human lip fibroblasts. This rhodamine B effect may be a warning sign for the dye toxicity.     

The effect of desferrioxamine on cell proliferation in human tumour cell lines

Iron is known to have a stimulatory effect on cell proliferation whereas the iron-complexing agent desferrioxamine (DFO) has an inhibitory influence on the growth of cultured cells. The effect of iron salts and DFO on cell proliferation and DNA synthesis was studied on two established human tumour cell lines, a colon carcinoma (Caco-2) and a hepatoma-derived cell line (Hep.G2). Cell proliferation was estimated by the neutral red method, DNA synthesis by measuring the [³H]thymidine incorporation into the cells. For the analysis of the cell cycle the cells were marked with bromodeoxyuridine for S-phase cells and the monoclonal antibody Ki-67 for all proliferating cells. Ferric chloride stimulated cell proliferation in the Caco-2 line whereas there was no effect in the Hep.G2 line. DFO inhibited cell proliferation and DNA synthesis in both cell lines. Cell cycle analysis revealed a prolongation of the cell cycle by DFO, thereby reducing the number of cells entering the proliferating phases of the cell cycle in both cell lines. The data support the essential role of iron in cell proliferation and tumour growth.     

Effects of the inhibitors of IMP dehydrogenase, tiazofurin and mycophenolic acid, on glycoprotein metabolism

The effects of the inhibitors of IMP dehydrogenase, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and mycophenolic acid, on the synthesis of cellular glycoproteins were evaluated in Sarcoma 180 cells. Both tiazofurin and mycophenolic acid decreased the rate of incorporation of [2-3H]mannose and [2-3H]fucose into acid-precipitable glycoproteins within 4 hr of exposure; this inhibitory activity was concentration dependent and occurred in the absence of a significant effect on the incorporation of labeled glucosamine and leucine into acid-insoluble material. Interference with the utilization of [3H]mannose for the formation of glycoproteins was paralleled by an inhibition of [3H] mannose incorporation into their lipid-linked oligosaccharide precursors following treatment with cytotoxic concentrations of tiazofurin (100 microM) or mycophenolic acid (10 microM); these actions occurred within 3 hr of exposure to these agents, with maximal reductions being observed at 12 hr. Under these conditions, intracellular GTP levels were reduced by 80%, whereas ATP pools remained unaffected ant UTP levels were markedly increased. Guanosine (100 microM) prevented the cytotoxic actions of tiazofurin and mycophenolic acid and reversed the drug-induced decrease in GTP pools and in the incorporation of mannose and other metabolic precursors into acid-insoluble material. Inhibition of fucose and mannose incorporation into lipid-linked oligosaccharides and glycoproteins were preceded by decreases in the labeling of their respective guanosine nucleotide sugars and were followed sequentially by alterations in the plasma membrane as detected by both the binding and the rate of cell agglutination caused by the plant lectin, concanavalin A. The findings that tiazofurin and mycophenolic acid produce alterations in the utilization of [3H]mannose for the formation of glycoproteins and in membrane architecture are indicative of metabolic lesions induced by agents that selectively depress guanine nucleotide synthesis through inhibition of IMP dehydrogenase.     

Comparison of metabolic activities in two human hepatoma cell lines, HEP 3B and HEP G2. Application to lindane

In a preliminary investigation, the evolution with time of the levels of ATP and glutathione species [reduced glutathione (GSH) and glutathione disulphide (GSSG)], glutathione reductase activity and RNA synthesis rate were studied in two human hepatoma cell lines, Hep 3B and Hep G2. Both cell lines exhibited higher cellular activities, during the first 48 hr. In a second investigation, the cells were exposed to lindane. Different cytotoxicological parameters such as viability, lactate dehydrogenase release, ATP, GSH and GSSG contents, RNA synthesis rate and glutathione reductase activity, were measured. Results showed that Hep 3B cells were more susceptible to lindane than Hep G2 cells, which perhaps have superior defence capacity and metabolism.     

Rhodamine B inhibits collagen synthesis by human lip fibroblasts in culture.

To investigate the effect of the cosmetic dye, rhodamine B, on the metabolism of collagen in fibroblasts, confluent KD cells, an established cell line of fibroblasts from human lip, were cultured for 6 h in a serum-free medium in the presence of the dye at 100 micrograms/ml and below. It was found that rhodamine B significantly decreased the content of procollagen type I C-terminal peptide antigen in both the cell layer and the medium with an only slight decrease in the cell number. Rhodamine B significantly decreased the incorporation of [3H]proline into either the collagen-digestible protein or the non-collagen protein in the cell layer. The incorporation of both [3H]thymidine and [14C]leucine into the acid-insoluble fraction of the cell layer was significantly decreased by rhodamine B; the activity of lactate dehydrogenase that leaked into the medium was not changed by the dye. From these results, it was suggested that rhodamine B has the capacity of decreasing the collagen content of the fibroblast cell layer of the human lip, which may result from a non-specific inhibition of protein synthesis without non-specific cell damage. Rhodamine B may impair the formation of extracellular matrix which is important for the maintenance of the lip tissue.     

p21基因对肝癌细胞的生物学影响

目的探讨逆转录病毒介导的p21基因对肝癌Hep3B细胞的杀伤作用。方法通过脂质体将有p21基因的逆转录病毒载体MMLV-p21转染肝癌Hep3B细胞系,并用G418筛选。试验分为转p21基因的Hep3B-P21组及相同遗传背景及培养过程的两对照组肝癌Hep3B系细胞组、转Hep3B-MMLV组3组。采用RT-PCR检测实验组p21基因的表达;应用流式细胞计数分别分析3组细胞周期;MTT法检测细胞增殖并比较细胞抑制率。结果野生p21基因阻止Hep3B系细胞进入S期,停滞在G1期,Hep3B-P21组细胞增殖比两对照组明显受到抑制（P〈0.05）。结论逆转录病毒介导p21基因可有效抑制肝癌Hep3B系细胞的增殖并诱导肝癌细胞的凋亡。     

Anticancer activity evaluation of the solanum glycoalkaloid solamargine. Triggering apoptosis in human hepatoma cells

Solamargine, an herbal and molluscicidal medicine derived from Solanum incanum, is a steroidal alkaloid glycoside. To characterize the anticancer mechanism of solamargine on human hepatoma cells (Hep3B), changes of cell morphology, DNA content, and gene expression of cells after solamargine treatment were studied. The appearance in solamargine-treated cells of chromatin condensation, DNA fragmentation, and a sub-G(1) peak in a DNA histogram suggests that solamargine induces cell death by apoptosis. The maximum number of dead Hep3B cells was detected within 2 hr of incubation with constant concentrations of solamargine, and no further cell death was observed after an extended incubation with solamargine, indicating that the action of solamargine was irreversible. To determine the susceptibility of cell phases to solamargine-mediated apoptosis, Hep3B cells were synchronized at defined cell cycles by cyclosporin A, colchicine, and genistein, followed by solamargine treatment. The IC(50) values of solamargine for control, G(0)/G(1)-, M-, and G(2)/M-synchronized Hep3B cells were 5.0, > 10, 3.7, and 3.1 microg/mL, implying that cells in the G(2)/M phases are relatively susceptible to solamargine-mediated apoptosis. In addition, a parallel up-regulation of tumor necrosis factor receptor (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I or -II specific antibody. Therefore, these results reveal that the actions of TNFR-I and -II on Hep3B cells may be independent, and both are involved in the mechanism of solamargine-mediated apoptosis.     

Influence of trapidil and derivatives on cholesterol synthesis and esterification in cultured cells

The effect of trapidil (Rocornal) and its derivatives AR 12456 and AR 12463 on endogenous cholesterol synthesis and on cholesterol esterification rate was studied in human skin fibroblasts (HSF), in human hepatoma cell line Hep G2 and in primary culture of peritoneal macrophages from mouse (PMM). The cholesterol esterification rate was not influenced by the drugs in the tested cell lines. The incorporation of [14C]acetate into cholesterol in HSF was inhibited by AR 12463 and AR 12456, but not by trapidil. The inhibitory potency of AR 12456 in HSF was enhanced after preincubation of the drug with Hep G2 and removal of the medium to HSF, suggesting that the formed metabolite(s) are more potent inhibitors than the parent substance. The metabolite(s) formed seem(s) to influence the first steps in the endogenous formation of cholesterol, because the incorporation of [14C]mevalonate into cholesterol was not significantly inhibited. These findings suggest that the demonstrated inhibition of the endogenous cholesterol synthesis by AR 12456, especially after transformation into a probably more active substance(s), together with the recently described enhanced expression of LDL receptors in Hep G2 cells may partially explain the hypocholesterolaemic activity of AR 12456.     

Feasibility of a simple double-layered coculture system incorporating metabolic processes of the intestine and liver tissue: application to the analysis of benzo[a]pyrene toxicity.

A simple double-layered coculture system using Caco-2 cell and Hep G2 cell, which mimic metabolic processes occurring in humans such as absorption through the intestine and cytochrome P450 1A1/2 involving biotransformation in both the intestine and liver cells, was used to investigate the toxicity of model chemical, benzo[a]pyrene (B[a]P). It was found that both Caco-2 and Hep G2 cells can metabolize B[a]P to toxic metabolites including B[a]P-7,8-hydrodiol (7,8-diol), an immediate precursor to the highly-reactive ultimate toxicant of B[a]P, B[a]P-7,8-hydrodiol-9,10-epoxide (BPDE), possibly mediated by cytochrome P450 1A1/2 activity. However, in a double-layered coculture system, no significant reduction of Hep G2 cell viability was found, although an approximately 50% reduction in viability was observed in pure Hep G2 cells. HPLC analysis showed that Caco-2 cells transfer B[a]P and its toxic metabolites back to the apical side, thus decreasing the concentrations of toxic metabolites including B[a]P-7,8-hydrodiol (7,8-diol) in cocultured Hep G2 cells. These results appear to be correlated with in vivo data on the effects of orally administered B[a]P, that is, low (10%) bioavailability in the rats and almost no acute lethal toxicity in rats or mice. As such, the simple double-layered coculture system can provide more accurate information regarding the toxic actions of the hazardous chemicals in humans than a pure culture system, as it also gives the final toxicity as a result of many complicated phenomena such as selective permeation in the intestine and biotransformation in the intestine and liver.     

Modulation of lipoprotein production in Hep G2 cells by fenofibrate and clofibrate.

Fenofibrate and other fibrate derivatives are commonly used to treat hyperlipidemia. It is not yet clear how they exert their modulatory effects on plasma lipoproteins. To investigate whether these drugs act on the liver to primarily inhibit very low density lipoprotein production, we utilized the highly differentiated human hepatoma cell line, Hep G2. At concentrations greater than 15 micrograms/mL, fenofibrate caused a 30% decrease in secreted apolipoprotein B (apo B) after 4 days of treatment. Pulse-chase studies demonstrated that this was not due to inhibition of apo B synthesis. Triglyceride synthesis by fenofibrate-treated Hep G2 cells was decreased by 30%, and the amount secreted into the medium was reduced by 50%. At a low concentration of drug (5 micrograms/mL), triglyceride secretion was reduced markedly while apo B secretion remained unchanged. Thus, apo B secretion is less sensitive to fenofibrate than the synthesis and secretion of triglyceride, and may be secondary to changes in the latter. Fenofibrate has also been shown to raise plasma high density lipoprotein concentrations. We found that low concentrations of fenofibrate caused a 20-101% increase in secreted apolipoprotein AI (apo AI), and pulse-chase immunoprecipitation studies showed that this was due to an increase in apo AI synthesis. Fenofibrate was compared to clofibrate to investigate whether their relative effects on lipoprotein production in Hep G2 cells were comparable to their relative effects on plasma lipoproteins. Both fibrates decreased the secretion of apo B to the same extent, but only fenofibrate increased apo AI secretion. Fenofibrate was more effective than clofibrate in inhibiting the secretion of lipids by these cells. Thus, the known effects of fenofibrate on plasma lipoproteins can be attributed to its direct modulation of lipoprotein synthesis in the liver cell. Hep G2 cells may thus be useful in testing the relative efficacy of fibric acid derivatives in vitro.     

Cytotoxic effect induced by retinoic acid loaded into galactosyl-sphingosine containing liposomes on human hepatoma cell lines

Two retinoids, ATRA and 13cisRA, were incorporated into liposomes of different composition and charge and added to two hepatoma cell lines with different degree of transformation to measure cytotoxicity by MTT assay. Retinoid-free cationic liposomes were more toxic than the other kinds (anionic and made only of PC) but were also the best delivery system for retinoic acid to induce specific cytotoxic effects on these tumor hepatoma cell lines. Galactosyl-sphingosine containing cationic liposomes increased the cytotoxic effect induced by ATRA on Hep3B cells when compared to glucosyl-sphingosine cationic liposomes, but did not improve the effect induced by free retinoid or ATRA loaded into liposomes without glycolipids. This suggests that in this cell line, ATRA is being incorporated by a mechanism mediated by the asialoglycoprotein receptor, but at the same time, non-specific sugar-independent capture is also taking place as well as free diffusion of ATRA directly through the membrane. Galactose-specific effect was not observed in HepG2 cells treated with ATRA or both cell lines treated with 13cisRA. In fact, treatment of HepG2 cells with retinoids entrapped into liposomes likely induces proliferation instead of cytotoxicity, a result that interferes with the measurement of cell death by MTT. Compared to the specific effect of ATRA entrapped into cationic liposomes, vesicles made only by PC, did not mediate a specific mechanism, since differences between ATRA in galactosyl- and glucosyl-shpingosine PC-liposomes were not statistically significant. The specific mechanism was not present in the myoblastic cell line C2C12, where ATRA incorporated into galactosyl- and glucosyl-sphingosine containing cationic and PC-liposomes, was able to induce cytotoxicity at the same extent. Micelles containing ATRA and galactosyl-sphingosine had a significantly more toxic effect than the retinoid administered together with glucosyl-sphingosine, in Hep3B cells. Also, micelles containing ATRA were more toxic than glycolipid-containing liposomes with ATRA, for both kinds of sphingosines. The same effect was not observed in C2C12 cells, where glycolipid-containing liposomes worked better than micelles, and a sugar-specific mechanism was not seen. This suggests that, even though galactose-containing cationic liposomes could be a promising approach, a galactose-specific emulsion system could be the best strategy to specifically deliver retinoic acid to liver tumor cells, since it shows tissue specificity (perhaps induced by ASGPR-mediated internalization) and a stronger cytotoxic effect than the retinoid incorporated into liposomes.     

Albumin modulates docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma cell lines

Fish oil-containing diets rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) provide protection against tumorigenesis. The mechanisms of the cytotoxic effects of DHA include the production of reactive oxygen species (ROS). Albumin has antioxidant property and binds fatty acids, it may protect the cells against the DHA-induced cytotoxicity. In this study, we compared the susceptibility of three human hepatocellular carcinoma (HCC) cell lines (HepG2, Hep3B, Huh7) to the cytotoxic effects of DHA, and examined the changes in the susceptibility following albumin overexpression using transfection vectors or albumin downregulation using small interfering RNA (siRNA). HepG2 cells were the most susceptible to DHA-induced cytotoxicity and increased oxidative activities by DHA compared to Hep3B and Huh7 cells. The cytotoxic effects of DHA were concentration-dependently abrogated by typical antioxidants, a radical scavenger, an iron chelator and incubation with exogenous albumin. Overexpression of albumin in HepG2 cells markedly attenuated DHA-induced oxidative activities and cytotoxicity. Furthermore, knockdown of albumin in both Hep3B and Huh7 cells significantly enhanced the effects of DHA. The results of our in vitro experiments indicate that the cytotoxic effects of DHA on HCC cell lines are modulated by albumin.     

Cytotoxic effects of cantharidin on the growth of normal and carcinoma cells

Cantharidin is isolated from Mylabris phalerata Pallas and is a potent inhibitor of hepatocellular carcinoma cells (Hep 3B cells). In the present study, the IC(50) values of cantharidin on Hep 3B cells and normal Chang liver cells were found to be 2.2 and 30.2 microM for 36 h, respectively. Furthermore, cantharidin-treated Hep 3B cells induced cell death within 1 h (IC(50)=52.8 microM), suggesting that cantharidin is an acute cytotoxic agent. We found that although cantharidin could induce cell death, it could not directly inhibit the activity of nucleic acid biosynthesis by the cellular incorporation of 3H-thymidine, 3H-uridine or 3H-leucine. Cantharidin-treated Hep 3B cells showed no evidence of major alterations in the cell cycle distribution within 1 h. However, examination of cells after treatment for 36 h showed that cantharidin regulated the cell cycle at the G(2)/M phase. Moreover, the treated Hep 3B cells had a rounded and shrunken appearance. The microvilli of treated Hep 3B cells were reduced in number and replaced by numerous blebs. Other ultrastructural changes following cantharidin treatment included the presence of lipid droplets, swelling of the mitochondria and accumulation of glycogen particles. The findings of damaged mitochondria in the cantharidin treated Hep 3B cells in this study suggest that cantharidin can induce acute and lethal toxic effects on Hep 3B cells by inhibiting the mitochondria energy system. In conclusion, this study had demonstrated that cantharidin could inhibit progression of all phases of the Hep 3B cell cycle.     

Metabolism of a novel antitumor agent, crisnatol, by a human hepatoma cell line, Hep G2, and hepatic microsomes. Characterization of metabolites.

Metabolism of the anticancer agent crisnatol was investigated using a human hepatoma cell line, Hep G2, and human liver microsomes. Crisnatol was metabolized extensively by both systems. The TLC/autoradiographic analysis showed that the crisnatol metabolite profile was similar for both systems and the major metabolites were shown to have structural characteristics similar to those formed by the rat. The Hep G2 cells formed three isomeric dihydrodiols; one of these has been identified by GC/MS and 1H-NMR as the crisnatol 1,2-dihydrodiol. Human liver microsomes also formed two isomeric dihydrodiols with 1,2-dihydrodiol as the major isomer and, in addition, produced 1-hydroxycrisnatol. Crisnatol concentrations of 1.3 micrograms/mL completely inhibited the replication of Hep G2 cells as measured by thymidine incorporation and cell growth kinetics and, at this concentration, cell viability decreased by only 35% as determined by vital staining of cells using neutral red dye.     

化合物E-2-（3’，5’-二甲氧基苯亚甲基）-环己酮的体外抗肿瘤作用州

目的：研究化合物E-2-（3＇,5＇-二甲氧基苯亚甲基）-环己酮（IV8）对人类宫颈癌（HeLa）、肝癌（HepG2）、前列腺癌（Dul45）和胃癌（SGC7901）细胞系的抗肿瘤活性。方法：采用MTT法评价IV8对上述4株肿瘤细胞株增殖的影响,并在光学显微镜下观察lV8对HepG2细胞形态学上的变化,以及采用AO／EB双荧光染色法评价IV8对HepG2细胞的凋亡作用。结果：IV8抑制HeLa,HepG2,Dul45和SGC7901细胞的IC。值分别为2．10,11．32,36．50和89．53gmol／L;光学显微镜下观察IV8处理的细胞出现明显增殖抑制和形态学改变;AO／EB双荧光染色IV8组可见凋亡细胞。结论：药理实验结果表明IV8具有较好的体外抗肿瘤的活性,值得进一步研究。