Aflatoxin B1-induced Hepatocellular Carcinoma Research Articles

Protective and therapeutic effects of sildenafil and tadalafil on aflatoxin B1-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has been classified as one of the most common forms of liver cancer occurring worldwide, and risk factors include hepatitis B & C virus, alcoholism, and dietary carcinogens like aflatoxin B1 (AFB1), which is produced by fungus Aspergillus flavus and Aspergillus parasiticus. Metabolism of AFB1 resulted into the formation of AFB1-exo-8, 9-epoxide which is largely responsible for HCC development. So far conventional cytotoxic chemotherapy has not provided much benefit in HCC, necessitating the need for newer treatment modalities. Recent reports suggest that phosphodiesterase-5 inhibitors (PDE5i) may have anticancer activity, but till date, the anticancer property of PDE5i (tadalafil & sildenafil) has not been evaluated in HCC. The present study was aimed to define the anticancer property of tadalafil and sildenafil against AFB1-induced HCC rats. Rats were randomly divided into five groups with five rats in each group. Except normal control group, rats of all other groups were fed with 5% alcohol via drinking water for 3weeks. After 3weeks, two successive dose of AFB1 (1mg/kg bw, ip) was administered on subsequent days followed by the administration of PDE5i (tadalafil & sildenafil, 10mg/kg bw) along with drinking water after 6weeks of treatment with AFB1 for 2weeks. An in-depth investigation into its mechanistic aspect revealed that development of HCC induced by aflatoxin B1, decreased the mRNA expression and activity of antioxidant enzyme SOD, GPx, catalase, GR and GST, and GSH content with a concomitant increase in the level of lipid peroxidation. Post-treatment with PDE5 inhibitor (tadalafil & sildenafil) restored the above parameters towards normal, and this result was more effective in case of sildenafil. Thus, results from the above studies suggest that PDE5 inhibitors may act as anticancer agents by preventing the development and progression of HCC by modulating the key parameters of antioxidant pathway.

Anticarcinogenic activity of kalpaamruthaa, a modified Siddha preparation, against aflatoxin–B1-induced hepatocellular carcinoma in rats

To investigate the potential anticancer effect of kalpaamrutha (KA), a modified Siddha preparation, against aflatoxin–B1(AFB1)-induced hepatocellular carcinoma (HCC) in rats, KA (200 mg/kg body weight/day) was administered to AFB1-induced (2 mg/kg body weight i.p.) HCC rats, orally for 28 days. At the end of the experimental period, changes in body weight were recorded; the levels of alpha-fetoprotein and total protein was estimated in the serum; activities of marker enzymes were assayed in serum and total protein; DNA and RNA contents were estimated in liver tissue; activities of glycolytic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes were assayed in liver tissue of control and experimental rats. Further histopathological examination of the liver sections was carried out to support the anticancer effect of KA against AFB1-induced HCC. Administration of KA overall increase in glycolytic enzymes with a subsequent reduction in gluconeogenic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes was observed in HCC-induced rats. These altered enzyme activities were effectively counteracted by supplementation with KA and also prevented the body weight loss by enhancing the host energy metabolism. Histological studies supported the biochemical findings. The results of the present study reveal that the drug KA has potential anticancer effect against AFB1-induced HCC rats.

Aflatoxin B1-induced hepatocellular carcinoma in developing countries: Geographical distribution, mechanism of action and prevention

Hepatocellular carcinoma (HCC) is the most well-known primary liver malignancy worldwide. Its incidence is rising at alarming rates and has become a public concern globally. It is more frequent in developing countries than in industrialized countries with respect to geographical variation, ethnic disparities and socioeconomic status. Dietary exposure to aflatoxins is among the major HCC risk factors. Aflatoxin B1, which is a genotoxic hepatocarcinogen, which presumptively causes cancer by inducing DNA adducts leading to genetic changes in target liver cells. AFB1 is metabolized by cytochrome-P450 enzymes to the reactive intermediate AFB1-8, 9 epoxide (AFBO) which binds to liver cell DNA, resulting in DNA adducts. DNA adducts interact with the guanine bases of liver cell DNA and cause a mutational effect in the P53 tumor suppressor gene at the codon 249 hotspot in exon 7, which may lead to HCC. Approximately 4.5 billion of the world’s population is exposed to aflatoxin-contaminated food, particularly in low-income countries. Prevention involves treating crops that are susceptible to fungal contamination, appropriate handling of foodstuffs and the use of chemopreventive intervention. Moreover, an integrated network collaboration of different sectors, including public health, agricultural departments and mass media, is required to ensure effective food regulation systems so as to minimize the contamination of food by aflatoxins.

Immunomodulatory Activity of Semecarpus anacardium Linn. Nut Milk Extract in Aflatoxin B1-induced Hepatocellular Carcinoma in Rats

The immunmodulatory activity of Semecarpus anacardium nut milk extract (Serankottai nei), an Indian Siddha medicinal preparation, was studied in male Wistar rats bearing aflatoxin B1-induced hepatocellular carcinoma. Immunomodulatory activity was assessed by measuring serum immunoglobulin (Ig) levels in control and experimental animals. Reduced IgG and elevated IgA and IgM in the hepatocellular carcinoma condition were returned to near normal levels in rats treated with 200 mg kg−1S. anacardium nut milk extract. This significant modulation of immune reactivity in drug-treated animals may play a role in the anticarcinogenic potency of S. anacardium nut milk extract against aflatoxin B1-induced hepatocellular carcinoma.

A Novel Binding Factor of 14-3-3β Functions as a Transcriptional Repressor and Promotes Anchorage-independent Growth, Tumorigenicity, and Metastasis

The 14-3-3 proteins form a highly conserved family of dimeric proteins that interact with various signal transduction proteins and regulate cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the beta isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3beta-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3beta-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3beta with FBI1. The strong expression of FBI1 was observed in several tumor cell lines but not in non-tumor cell lines. Forced expression of antisense FBI1 in K2 cells inhibited anchorage-independent growth but had no significant effect on cell proliferation in monolayer culture. Down-regulation of FBI1 also inhibited tumorigenicity and metastasis accompanying a decrease in MMP-9 (matrix metalloproteinase-9) expression. In addition, the duration of ERK1/2 activation was curtailed in antisense FBI1-expressing K2 cells. A luciferase reporter assay revealed that the FBI1-14-3-3beta complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3beta complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3beta were presented on the MKP-1 promoter. These results indicate that FBI1 promotes sustained ERK1/2 activation through repression of MKP-1 transcription, resulting in promotion of tumorigenicity and metastasis.

Cancer stem cells in aflatoxin B1-induced rat hepatocellular carcinoma K2 cells

In this study, we performed the side population (SP) cell analysis and the expression analysis of stemness genes to identify stem-like cells (cancer stem cells) in aflatoxin B1-(AFB1) induced rat hepatoma K2 cells. Using the flow cytometric analysis with rhodamine 123, the SP cells, which are thought to be cancer stem cells, were detected with high frequency and estimated to be approximately 89 % of K2 cell population. Furthermore, K2 cells expressed stemness genes including the sox2, nanog, klf4, the ras family E-ras and the polycomb family bmi1 genes, which are implicated in the maintenance of pluripotency and self-renewal of embryonic stem (ES) cells. On the other hand, in K2 cells the expression levels of mature hepatocyte-specific marker genes such as albumin and tyrosine aminotransferase (TAT) were very low. Thus, these results imply that K2 cells possess the typical properties of cancer stem cells through the acquisition of stemness gene expressions.

Use of a Rainbow Trout Oligonucleotide Microarray to Determine Transcriptional Patterns in Aflatoxin B1-Induced Hepatocellular Carcinoma Compared to Adjacent Liver

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.

Deregulated expression of a novel gene, NC33, in aflatoxin B1-induced rat hepatocellular carcinoma K2 cells

Aflatoxin B1 (AFB1) is known to be one of the most potent hepatocarcinogens and causes hepatocellular carcinomas in various experimental animals. We have previously reported that AFB1-induced rat hepatocellular carcinoma K2 cells over-express 14-3-3 β and c-myc genes which are the key regulators of in vitro and in vivo growth of K2 cells. Nonetheless, it has been speculated that in addition to these genes, some other genes also implicate in AFB1 hepatocarcinogenesis, because AFB1 induces genomic alterations. Here, we report the cloning of cDNA encoding rat nuclear protein NC33 (nuclear coiled-coil protein 33 kDa) as a new candidate responsible for AFB1 hepatocarcinogenesis. The expression level of NC33 mRNA in K2 cells was extremely higher than that in normal rat liver. The colony forming ability of K2 cells in semi-solid medium as a hallmark of tumor cells in vitro was diminished by the down-regulation of NC33 gene expression. Thus, it is most likely that the deregulated expression of NC33 gene is involved in AFB1 hepatocarcinogenesis.

Potency of Semecarpus anacardium Linn. Nut milk extract against aflatoxin B1-induced hepatocarcinogenesis: reflection on microsomal biotransformation enzymes

The effect of Semecarpus anacardium Linn. nut milk extract on host detoxification system in aflatoxin B1induced hepatocellular carcinoma, which is a vital mechanism in cancer treatment, was studied in male albino rats. Oral administration of nut extract (200 mg kg−1body weight per day for 14 days) is found to be highly effective in inducing phase I and phase II biotransformation enzymes. The obtained results have shown an overall decrease of liver microsomal cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, and aniline hydroxylase with a subsequent decrease of phase II enzymes, glutathione-S-transferase and UDP-glucuronyl transferase in cancer-bearing animals. The Semecarpus anacardium nut extract affords anticancer activity by enhancing both phase I and phase II enzymes to near normal levels. We propose that, much of the anticarcinogenic potency of Semecarpus anacardium nut extract on aflatoxin B1-induced hepatocarcinogenesis is mediated through the induction of hepatic biotransformation enzymes.

MODULATING ROLE OF SEMECARPUS ANACARDIUM L. NUT MILK EXTRACT ON AFLATOXIN B1BIOTRANSFORMATION

As part of a substantial effort to curtail the adverse health effects posed by aflatoxin B1, studies have been conducted to elucidate the possible mechanism for the anticarcinogenic action of Semecarpus anacardium nut extract against aflatoxin B1-induced hepatocellular carcinoma. Rats are monitored for levels of urinary, serum and liver biomarkers, namely, unmetabolised aflatoxin B1, and its metabolites aflatoxin M1, and aflatoxin Q1, over the course of 2 weeks with nut extract therapy following a single-exposure to aflatoxin B1. Due to the administration of nut extract, the excretion of unmetabolised aflatoxin B1was increased in day 1 urine when compared with rats without drug treatment. In serum and liver which were collected on day 16 and the rest of periodical urine samples showed aflatoxin B1and its metabolites in undetectable levels. The nut extract administration induced cytochrome P450, glutathione, and glutathione- S -transferase levels in liver homogenates of aflatoxin B1-treated rats. These data seem to indicate that anticarcinogenic action by Semecarpus anacardium nut extract is possibly via suppression of aflatoxin B1activation and through interaction with microsomal-activating components. Previous evidence from this laboratory about the potency of Semecarpus anacardium nut extract against aflatoxin B1-induced hepatocellular carcinoma together with the present results suggest that extremely effective therapeutic protection can be achieved by this drug against aflatoxin B1-mediated ill effects.2000 Academic Press@p$hr

アフラトキシンB<SUB>1</SUB>誘導ラット肝癌細胞で見られる14-3-3βの発現亢進とその役割

アフラトキシンB<SUB>1</SUB>誘導ラット肝癌細胞で見られる14-3-3βの発現亢進とその役割

Regulation of Mineral Status by Semecarpus anacardium Linn. Nut Milk Extract in Aflatoxin B1-Induced Hepatocellular Carcinoma.

Semecarpus anacardium Linn, nut milk extract, a herbal formulation, was investigated for its modulating effect on mineral levels in experimental hepatocellular carcinoma. Hepatocellular carcinoma was induced in rats by a single intraperitoneal injection of aflatoxin B1. Sodium, potassium, calcium, and magnesium contents were analyzed in serum and liver homogenate samples. A decreased level of sodium and increased levels of potassium, calcium, and magnesium were observed in carcinoma-bearing rats. Oral administration of Semecarpus anacardium nut milk extract (200mg/kg body weight/day) significantly reversed the mineral contents to near normal levels. These results suggest the improvement of mineral status by the extract, and indirectly indicate the curative efficacy of Semecarpus anacardium nut extract against aflatoxin B1-induced hepatocellular carcinoma. Possible reasons for its restorative potency are discussed.

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.

アフラトキシンB<SUB>1</SUB>誘発ラット肝癌由来Kagura-2細胞のグルココルチコイドによる増殖制御

アフラトキシンB<SUB>1</SUB>誘発ラット肝癌由来Kagura-2細胞のグルココルチコイドによる増殖制御

Iron exclusion from aflatoxin B1-induced hepatocellular carcinoma in rats

Mixed-cell hepatocellular adenocarcinomas, induced by aflatoxin B1 (AFB1) in weanling rats, were allowed to develop for 18 months; age- and sex-matched controls were maintained. After 18 months, treated and control rats were administered iron dextran (ID), intraperitoneally (i.p.) as Nonemic® . No gross or microscopic lesions were observed in control animals, while hepatic tumors were observed grossly in all AFB1 -treated rats. Areas of apparently normal tissue, in histologie sections from control animals and in non-tumor sites of sections from AFB1-treated animals, were deeply stained for iron, both in endothelial cells and in hepatocytes, as visualized by Peris iron stain. Varying degrees of exclusion of stainable iron material were noted in histologie sections of lesions from AFB1-treated rats, as were alterations in endothelial cell staining characteristics. Iron staining clearly demonstrates the presence of varying cell populations within the induced mixed cell hepatocellular adenocarcinoma.

Alpha benzene hexachloride inhibition of aflatoxin B1-induced hepatocellular carcinoma. A preliminary report.

Alpha benzene hexachloride protected against the development of liver carcinoma in male albino Fisher rats ingesting aflatoxin B1.