Vesicular flavonoid in combating diethylnitrosamine induced hepatocarcinoma in rat model

Reactive oxygen species (O2(*-), OH(-), H2O2) are known to play an important role in tumor initiation in hepatocarcinoma. Hepatocarcinoma was developed in the Swiss Albino rats by administration three doses of diethylnitrosamine (DEN) (200 mg/kg b. wt.) (i.p.) at 15 days interval. Quercetin (QC), herbal polyphenolic compound, is a potent anticancer drug. Clinical trials are difficult for its hydrophobic nature. To overcome this problem, our study was aimed to formulate soluble liver specific, galactosylated liposomal QC and to investigate its efficacy against hepatocarcinoma in rat model. Galactosylated liposomal QC was formulated and the suspension was introduced intravenously to rats (8.98 microM/kg) once in a week for 16 weeks. Hepatocarcinoma in rat model and its pathological improvement were evaluated histopathologically, histochemically and electron microscopically. Severe oxidative damage was noticed in the whole liver and its microsomal fraction of DEN treated rats. Huge numbers of hyperplastic nodules (HNs) with pre-neoplastic lesions appeared in rat liver by DEN administration. Galactosylated liposomal QC injections prevented DEN mediated development of hepatocarcinoma and oxidative damage in rat liver. Quercetin in liver specific galactosylated liposomal drug delivery system may be recommended as a potent therapeutic formulation against DEN-induced hepatocarcinoma.     

Delay of diethylnitrosamine-induced hepatoma in rats by carrot feeding

Carcinogenesis and death caused by diethylnitrosamine(DENA)-induced hepatomas in Sprague Dawley rats can be significantly delayed by feeding only carrots for several days a week. As this result is not obtained by supplementing food with even higher concentrations of synthetic beta-carotene, there must be several other factors which--together with the carotenoid--contribute to the positive effect. Several other possibilities such as effects of fasting, direct interactions with the carcinogen, resorption, vitamin A storage in the liver, etc., are proposed and discussed.     

Anticarcinogenic activity of quinacrine in the rat mammary gland

Mammary carcinogenesis studies were conducted to determine thechemopreventive activity of quinacrine, an antimalarial drugwhich suppresses the production of arachidonic acid from phospholipidthrough inhibition of phospholipase A₂. Beginning 1 week aftera single i.v. dose of N-methyl-N-nitrosourea (MNU), female Sprague—Dawleyrats were fed a semi-purified diet supplemented with 0 or 75mg quinacrine/kg diet. Quinacrine reduced cancer incidence andcarcinoma multiplicity in rats administered 20 mg MNU/kg bodywt, but had no inhibitory activity in rats treated with 50 mgMNU/kg. These data suggest that pathways of arachldonic acidmeta-bolism in addition to cyclooxygenase present useful targetsfor mammary cancer chemoprevention. However, the chemo-preventiveactivity of quinacrine may be limited by toxicity.     

Preventive effect of caffeine and curcumin on hepato-carcinogenesis in diethylnitrosamine-induced rats

Chemopreventive effects of caffeine and curcumin were evaluated in the diethylnitrosamine (DEN)-induced hepatocarcinogenic rat model. Animals injected with DEN for 10 weeks (G2-10w) and 14 weeks (G2-14w) were hepato-carcinogenic rats. Animals injected with DEN and treated with curcumin and caffeine for 10 weeks (G3-10w, G4-10w) and 14 weeks (G3-14w, G4-14w) were compared with those in G2. Macroscopic and microscopic features suggested that treatment with caffeine, but not curcumin, for 10 and 14 weeks was effective in inhibiting DEN-induced hepatocarcinogenesis. Immunohistochemical and western blot analysis with proliferating cell nuclear antigen and glutathione S-transferase-P antibodies also showed that expression levels of these hepato-carcinogenic markers were more efficiently reduced by treatment with caffeine than curcumin. Our data demonstrate that caffeine could be a more potent compound than curcumin for prevention of hepatocarcinogenesis in DEN-induced rats.     

南方壁虎制剂对DEN诱导大鼠肝癌癌前病变的探讨

目的:探讨南方壁虎制剂对二乙基亚硝胺(DEN)诱导大鼠肝癌癌前病变的作用.方法:利用Solt-Farber的DEN诱导肝癌癌前病变短期动物模型,在制模过程中给予不同剂量的南方壁虎制剂进行干预,通过组织化学染色观察各组动物肝组织内r-谷氨酰转肽酶阳性肝细胞增生灶(r-GT灶)的数量和面积,以及免疫组化SP法对肝组织中的Ki-67、EFGR和ErbB4蛋白表达进行检测分析.结果:不同剂量南方壁虎实验组γ-GT阳性灶的数量、面积均小于DEN对照组,其中低剂量组显示差异有统计学意义,P<0.05.各组肝组织均表达Ki-67、EGFR和ErbB4.Ki-67在低剂量组中的阳性和强阳性的表达率(20.0%)与阳性对照组(73.3%)差异有统计学意义,P<0.05;EGFR在高剂量和低剂量的阳性和强阳性的表达率分别为22.2%和10.0%,与阳性对照组(40.0%)差异有统计学意义,P<0.05;ErbB4不同剂量组的表达均高于阳性对照组,差异无统计学意义,P>0.05.结论:南方壁虎制荆对DEN诱发大鼠肝癌癌前病变有一定的抑制作用;Ki-67、EGFR可能参与南方壁虎抑制肝癌癌前病变的调节作用.     

Anticarcinogenic and hepatotoxic interactions between retinyl acetate and butylated hydroxytoluene in rats

The natural retinoid, retinyl acetate (RA), and the phenolic antioxidant, butylated hydroxytoluene (BHT), are both effective inhibitors of mammary carcinogenesis in rats. The present study was designed to determine if an increased inhibition of mammary carcinogenesis is obtained when RA and BHT are administered in combination. At age 50 days (time 0), virgin, female Sprague-Dawley rats received a single intragastric instillation of 16 mg of 7,12-dimethylbenz(a)anthracene dissolved in 1 ml sesame oil. Groups of 30 carcinogen-treated rats received Wayne Lab Chow supplemented with (per kg diet) 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT by the following schedule: -2 to +1 week; +1 week until the end of the experiment; -2 weeks to end; or none. Combined administration of RA plus BHT by the -2 weeks to end schedule was more effective in mammary cancer chemoprevention than was RA alone or BHT alone; the interaction of RA and BHT was additive. Similarly, administration of RA plus BHT by the -2 weeks to end protocol was more active in chemoprevention than was RA plus BHT administered either from weeks -2 to +1 or +1 week to end. Chronic exposure to RA plus BHT induced a high incidence of hepatic fibrosis and bile duct hyperplasia; these changes were not observed in controls and were seen in low incidence in animals exposed to RA only or BHT only. These data indicate that enhanced anticarcinogenic activity can be obtained through the use of "combination chemoprevention" regimens; however, chemopreventive compounds may interact not only to inhibit carcinogenesis but also to induce toxicity.     

Anticarcinogenic activity of natural sweeteners,cucurbitane glycosides,from Momordica grosvenori

To search for cancer chemopreventive agents from natural resources, many phytochemicals and food additives have been screened. Consequently, two natural sweeteners, mogroside V and 11-oxo-mogroside V isolated from the fruits of Momordica grosvenori, exhibited strong inhibitory effect on the primary screening test indicated by the induction of Epstein-Barr virus early antigen (EBV-EA) by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). These sweet glycosides, having cucurbitane triterpenoid aglycon, exhibited the significant inhibitory effects on the two-stage carcinogenesis test of mouse skin tumors induced by peroxynitrite (ONOO-) as an initiator and TPA as a promoter. Further, 11-oxo-mogroside V also exhibited the remarkable inhibitory effect on two-stage carcinogenesis test of mouse skin tumor induced by 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.     

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.     

Carcinogenicity examination of quercetin and rutin in ACI rats

Carcinogenicity of quercetin and rutin were examined in inbred ACI strain rats. Rats were given a diet containing 1% or 5% quercetin or 5% rutin for 540 days, or 10% quercetin and 10% rutin for 850 days. Rats in control groups were fed a normal basal diet. Most tumors found in experimental groups were also found in the corresponding control groups. Furthermore, there was no significant difference between the incidence of tumors in the experimental or control groups (P > 0.05). Thus, quercetin and rutin tested were not shown to be carcinogenic to ACI rats.     

Immunohistochemical detection of c-myc and c-erbA products in diethylnitrosamine-induced preneoplastic and neoplastic liver lesions in rats

An immunohistochemical study of c-myc and c-erbA products (p-myc and p-erbA) in preneoplastic and neoplastic rat liver lesions showed that the longer the time of hepatocarcinogenic treatment, the higher the proportion of lesions, whatever their type, showing p-erbA positive cells (p-erbA+). The proportion of p-myc positive foci (the majority of which are also p-erbA+), which was low at the focus stage, increased at the nodule and tumor stages. The incidence of p-myc positivity (alone or combined with erbA positivity) in the nodules decreased from the nodule to the tumor stage. For tumors, three types of altered phenotypes were found, namely: p-myc+, p-erbA+ or p-myc+/p-erbA+. Nevertheless, there were regions in these tumors that were negative. At a given stage, all types of lesions exhibited about the same incidence of immunopositivity for the two oncogene products (expressed either alone or together), the presence of which did not correlate with proliferative activity. Since the fraction of lesions that undergo full malignant progression is much smaller than the proportion of lesions that express c-myc and/or c-erbA proteins, our data exclude the possibility that increased incidence of p-myc/ and/or p-erbA+ cells might be sufficient for inducing full malignancy. A significant proportion of foci and nodules, and of regions of these lesions and of tumors, were unlabeled, whatever the stage at which they are found. This indicates that, if implicated, the positive phenotype(s) would not be required for the maintenance of those liver alterations.     

Lack of carcinogenicity of quercetin in F344/DuCrj rats

Quercetin was administered at dietary levels of 0(control), 1.25 and 5.0% to groups of 50 male and 50 female rats for 104 weeks, and then all animals were maintained without quercetin supplement for a further 8 weeks. At 5.0% quercetin, both sexes showed growth retardation throughout the study. There were no treatment-ascribed effects regarding clinical signs, mortality, urinalyses or hematology. Although serum glucose in 5.0% quercetin-treated males was significantly decreased and some relative organ weights in 5.0% groups showed statistically significant increases, these latter changes seemed to be related to the growth retardation. An increased incidence of non-neoplastic hyperplastic polyps in the cecum was noted in the 5.0% males. The incidences of cystic changes and fibroadenomas of the mammary gland, and foci (areas) of hepatocellular alteration in the 5.0% females, and liver bile duct proliferations in the 5.0% males were significantly decreased. No proliferative lesions of the urinary bladder related to treatment with quercetin were found in any rats. The incidences of several other nonneoplastic and neoplastic lesions which demonstrated statistically significant changes appeared to be related to the growth retardation or to be within the normal range, and therefore none was considered to be significant biologically. Thus, the investigation did not demonstrate any clear carcinogenic effect of quercetin on F344 rats at dietary levels of up to 5.0%.     

Effect of phenobarbital on the activity of RNA polymerase II during diethylnitrosamine-induced hepatocarcinogenesis in the rat

The effects of dietary phenobarbital (PB) on RNA polymerase II (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2. 7. 7. 6) in the liver of male Sprague-Dawley rats previously administered diethylnitrosamine (DENA) were studied. When a diet containing 0.05% phenobarbital was administered, the activity of DNA-dependent RNA polymerase II in the liver nuclei of rats was temporarily increased 1 and 2 weeks after the onset of feeding and expressed to be relatively higher afterwards, compared with the activity in rats fed the control diets.     

槲皮素提高肝癌HepG2细胞热化疗疗效的实验研究

  目的 研究热休克对人肝癌HepG2细胞耐药性的影响，合用槲皮素（Qu）能否提高肝癌细胞热化疗的疗效。方法 42 ℃恒温水浴法热休克传代人肝癌细胞系HepG2细胞90 min。MTT检测半数抑制浓度（IC50），求得耐药倍数、增敏倍数。荧光染色检测凋亡率。流式细胞术检测HSP70和P-gp表达率。结果 Qu能诱导HepG2细胞凋亡。HepG2细胞热休克诱导后4 h对ADM耐药性升高2.78倍（P＜ 0.05），HSP70阳性细胞数升高，4 h达到11.47 ％，升高近1倍（P ＜0.01），P-gp阳性细胞数12 h达到96.31 ％，升高近2倍（P ＜0.01）。热休克前应用Qu能有效抑制热休克诱导HSP70和P-gp的过量表达，抑制细胞对ADM耐药性的产生，起增敏作用（P ＜0.05），呈浓度依赖性。结论 槲皮素可阻断热休克诱导HepG2细胞HSP70和P-gp的过表达，抑制耐药性的产生，起到热化疗的增敏作用，可成为肿瘤耐药逆转剂。     

Invasiveness, proliferative activity and ultrastructural phenotypes of hepatocytes from diethylnitrosamine-induced neoplastic nodules and hepacarcinomas in vitro

The invasive behavior of hepatocytes from diethylnitrosamine (DENA) induced neoplastic nodules and hepatocarcinomas was studied in a confronting culture system in vitro. These observations confirm our previous report, demonstrating that hepatocytes from hepatocarcinomas and from neoplastic nodules invaded into embryonic chick precultured heart fragments (PHF), a property associated with malignancy (Mareel, 1979). We now further demonstrate that: (1) Invasiveness was expressed by the hepatocytes in 10 out of 12 samples from hepatocarcinomas, and in 13 out of 36 confronted nodular samples. The hepatocytes from the other two-thirds of nodule samples died off in the confrontation as did all normal hepatocytes. (2) Invasive hepatocytes from tumors and from nodules showed the same arrangement of invasive liver cells in relation to the heart tissue, and the same ultrastructural phenotypes. The latter did not differ from those in the non-invading subpopulations, with the exception perhaps of a higher proportion of cells with an indented nucleus. (3) None of the scored ultrastructural alterations was present in all the invasive cells, thus excluding any specific requisite in this respect. (4) When 3H-TdR was made continuously available to the cultures, starting at the time of confrontation between heart and liver tissues, unlabelled as well as labelled invasive hepatocytes were found inside the PHF in about equal proportions. It is concluded that nodular hepatocytes deviate from normality by at least 2 different properties that may or may not be related to each other and are revealed under in vitro conditions, namely the ability to survive and to proceed through S-phase and mitosis under such conditions and to actively invade precultured chick heart fragments. The latter property indicates that at least some of the nodules contain hepatocytes that have performed one step in malignant progression.     

Cytotoxicity phase of diethylnitrosamine-induced hepatic neoplasia in medaka

Adult Oryzias latipes were exposed to 50 mg of diethylnitrosamine per liter of water for 5 wk and then transferred to clean water for an additional 15 wk. Response of the liver during the first 6 wk were analyzed by enzyme histochemistry and by high-resolution light and transmission electron microscopy. After 1 wk, cytotoxicity was apparent at the light microscopic level by piecemeal necrosis and phagocytosis apoptosis by adjacent hepatocytes and resident macrophages. Spongiosis hepatis and inflammation, found as early as wk 3, were not widespread until wk 6. Glycogen depletion and multifocal increases in gamma-glutamyl transpeptidase were found as early as 3 wk. At 5 wk, macrophage infiltration and aggregation and hepatocyte lysosome proliferation were revealed by an increase in cells staining for acid phosphatase. In addition, a subpopulation of macrophages stained positively for glucose-6-phosphate dehydrogenase during wk 6. Other histochemical biomarkers (Mg2(+)-ATPase, DT-diaphorase, uridine diphosphoglucuronyl dehydrogenase) were not altered. Mitotic figures were rare for the entire 6-wk period. At the ultrastructural level, necrotic alterations of some hepatocytes were seen within 24 h. Within 48 h, an apparent reduction of hepatocyte glycogen and cell volume characterized the majority of hepatocytes; this was accompanied by an increase in interhepatocytic space and the length and complexity of the hepatocyte microvillous projections found in the space of Disse. Lipid vacuolar inclusions inhabited space previously occupied by glycogen. Margins of hepatocyte nuclei were irregular, and mitochondria were condensed and their shape altered so that crescentric and elongated profiles were abundant. Lysosomes and residual bodies were increased after 1 wk. The cytoplasmic processes delineating spongiotic lesions were identified as originating from Ito cells. After 4 wk, apparent proliferation of smooth endoplasmic reticulum and retention of transport lipid within its cisternae were seen. The toxic depletion of hepatocytes and the attendant altered cellular environment are discussed in relation to cell-to-cell interactions and the possible contribution of stromal and extracellular matrix changes to liver regeneration and neoplasia.     

Cytochrome P-450 isozyme pattern is related to individual susceptibility to diethylnitrosamine-induced liver cancer in rats.

Differences in susceptibility to chemical carcinogenesis between rodent strains and species have been linked to variations in genetically-determined mixed function oxidase activities. In order to verify whether such variations also determine the susceptibility of individual animals of the same strain to a chemical carcinogen, outbred male Wistar rats were administered diethylnitrosamine (DEN) (1, 2, or 3 mg/kg) five times a week for 20 weeks. The relationship was examined between the outcome (i.e., presence or absence of liver tumors, and latency period) and the hepatic activities of mixed function oxidases and conjugating enzymes, as well as of O6-methylguanine-DNA-methyltransferase, measured before the carcinogen treatment. In addition, the metabolic profiles of two model drugs, antipyrine and disopyramide, in the urine were analyzed and correlated with the carcinogen susceptibility. The length of the latency period of hepatocellular tumors in individual rats was negatively related to the activities of hepatic dimethylnitrosamine N-demethylase, aryl hydrocarbon hydroxylase and epoxide hydrolase and positively related to the amount of microsomal protein. Consistent relationships between the other 10 measured parameters and the susceptibility to DEN-induced carcinogenesis were not detected. Long-term treatment with DEN slightly decreased the proportion of metabolism of antipyrine into norantipyrine, and increased the share of 4-hydroxyantipyrine; a decrease in the metabolism of disopyramide to N-deisopropyldisopyramide was also detected. It is concluded that the pattern of cytochrome P-450 isoenzymes is related to differences in individual susceptibility to nitrosamine-induced carcinogenesis. The relationship was most marked at low dose levels, which are the levels at which nitrosamine exposures of humans are known to occur.     

槲皮素对肺癌干细胞活性的影响

目的 探索槲皮素对A549肺癌干细胞活性及对Wnt/β-连环蛋白(β-catenin)和音猬因子(sonic hedgehog,SHH)信号通路的影响。方法 使用不同浓度槲皮素(0、5、10、25、50、100和200μmol/L)处理肺癌A549细胞24、48和72 h,MTT法检测细胞存活率。采用无血清培养法分离富集肺癌干细胞,观察槲皮素对A549悬浮细胞球体积、数量以及肺癌干细胞分子标志物表达的影响;流式细胞术检测细胞凋亡;蛋白质印迹法检测增殖凋亡相关指标以及Wnt/β-catenin和SHH信号通路关键分子的表达变化。结果 槲皮素能够显著抑制A549细胞活力,且呈现时间和浓度依赖性;与空白对照组相比,槲皮素均显著抑制A549成球能力和肺癌干细胞分子标志物[CD133、CD44和醛脱氢酶1A1(ALDH1A1)]表达;同时,肺癌干细胞增殖相关蛋白增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)和B细胞白血病/淋巴瘤-2(Bcl2)水平显著下调,凋亡相关指标Bcl2相关蛋白X(Bax)和半胱氨酰天冬氨酸特异性蛋白酶-3(Cleaved Caspase 3)表达显著...     

Expression of p53 mutant protein(s) in diethylnitrosamine-induced foci of enzyme-altered hepatocytes in male Fischer-344 rats

Several types of human and animal tumors have been shown to carry mutations in the p53 gene. While the translation product of the wild type gene has tumor suppressor properties, mutant alleles of the gene produce proteins that can cooperate with other oncogene products in transforming cells. In this paper, evidence is presented indicating that a p53 gene mutation(s) occurs in foci of enzyme-altered hepatocytes induced by diethylnitrosamine in male Fisher-344 rats. The evidence was obtained by means of immunohistochemical and immunoblotting techniques, using antibodies directed against mutant forms of the p53 protein.