Chemopreventive N-(4-hydroxyphenyl)retinamide (fenretinide) targets deregulated NF-{kappa}B and Mat1A genes in the early stages of rat liver carcinogenesis

Maria M. Simile1, Gabriella Pagnan2, Fabio Pastorino2, Chiara Brignole2, Maria R. De Miglio1, Maria R. Muroni1, Giuseppina Asara1, Maddalena Frau1, Maria A. Seddaiu1, Diego F. Calvisi1, Francesco Feo1,*, Mirco Ponzoni2 and Rosa M. Pascale1

1 Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Italy and 2 Differentiation Therapy Unit, Laboratory of Oncology, G. Gaslini Children's Hospital, Genoa, Italy

* To whom correspondence should be addressed Email: feo{at}uniss.it


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor {kappa}B (NF-{kappa}B) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the ‘resistant hepatocyte’ model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7–147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-{kappa}B expression and binding activity, and under-expression of the inhibitor {kappa}B-{alpha} (I{kappa}B-{alpha}) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-L-methionine, enhances I{kappa}B-{alpha} expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-{kappa}B, nitric oxide and S-adenosyl-L-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

Abbreviations: AAF, 2-acetylaminofluorene; AI, apoptotic index; DENA, diethylnitrosamine; FAH, foci of altered hepatocytes; GST-P, glutathione S-transferase (placental); HCC, hepatocellular carcinoma; HPR, N-(4-hydroxyphenyl)retinamide; I{kappa}B-{alpha}, inhibitor {kappa}B-{alpha}; LI, labeling index; Mat, methionine adenosyl transferase; NF-{kappa}B, nuclear factor {kappa}B; SAM, S-adenosyl-L-methionine; SL, stabilized liposomes


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is the fifth most frequent solid tumor worldwide, whose etiology includes viral hepatitis, Aflatoxin B1, alcohol abuse and some metabolic diseases (1). The morbidity for HCC corresponds to mortality and new preventive and therapeutic approaches are necessary for this tumor. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide; HPR), inhibits the incidence of carcinogen-induced tumors in different tissues and suppresses in vitro growth and induces apoptosis of a variety of human tumor cell lines (25). HPR effects on hepatocarcinogenesis, however, are poorly documented. Dietary HPR partially prevents the development of foci of altered hepatocytes (FAH), induced by diethylnitrosamine (DENA) or a methyl-deficient diet in rats (6). The molecular mechanisms involved and the long-term effect on HCC development have not been evaluated. The evolution of preneoplastic lesions to HCC is associated with cell-cycle deregulation (79). Nuclear factor {kappa}B (NF-{kappa}B) transactivates numerous genes involved in the regulation of cell proliferation and cell death (1013). It is up-regulated in liver tumors of c-myc/Tgf-{alpha} transgenic mice (14) and in human HCC (15). The observation that NF-{kappa}B is up-regulated during hepatitis induced by hepatitis B (16) and hepatitis C (17) viruses, and HBx protein interacts with I{kappa}B-{alpha} and favors NF-{kappa}B translocation into the nucleus (16,18), suggests that NF-{kappa}B activation could be an early event of human carcinogenesis. However, the studies on NF-{kappa}B activation during the early stages of hepatocarcinogenesis are scanty, and did not clearly prove the presence of this activation in pre-cancerous liver lesions. NF-{kappa}B activation was found in rat liver during oxidative stress induced by a short treatment with thioacetamide, in the absence of the development of preneoplastic lesions (19). Nuclear accumulation of NF-{kappa}B was observed 25 days after initiation in the hepatocytes of rats subjected consecutively to DENA, 2-acetylaminofluorene (AAF) and partial hepatectomy (20). At this stage of the process only 2% of the liver was occupied by FAH, and reparative liver growth was presumably not yet complete due to the mitoinhibitory effect of AAF (21). The administration of caffeic acid phenethyl ester, during AAF treatment and after partial hepatectomy, before the development of FAH, inhibited nuclear accumulation of NF-{kappa}B in liver cells and reduced expression of FAH biomarkers. This could indicate that the chemopreventive agent inhibits the strong promoting stimulus, represented by liver reparative growth, thus preventing the development of FAH. Recently, NF-{kappa}B activation, paralleled by down-regulation of I{kappa}B-{alpha} and over-expression of iNos, has been found in liver homogenates from carcinogen-treated rats 28 weeks after initiation when dysplastic nodules and HCCs were present in the liver (22). To better understand the role of the deregulation of NF-{kappa}B and some related pathways in hepatocarcinogenesis, the present paper is designed to evaluate whether these changes are present in early pre-cancerous lesions of rat liver, and the interference of HPR with these molecular mechanisms blocks the evolution of these lesions and prevents HCC development. We demonstrate the existence in early preneoplastic lesions and neoplastic (dysplastic) nodules of up-regulation of functional NF-{kappa}B and under-regulation of the Mat1A gene, encoding liver-specific methionine adenosyltransferase I/III (MatI/III). The reaction product of this enzyme, S-adenosyl-L-methionine (SAM), may regulate NF-{kappa}B activity, by enhancing the expression of the inhibitor {kappa}B-{alpha} (I{kappa}B-{alpha}) (10). Furthermore, we demonstrate that chemopreventive HPR attenuates these changes, inhibits DNA synthesis and induces apoptosis associated with a decrease in the expression ratio between anti-apoptotic and pro-apoptotic genes of Bcl-2 family. Overall, these findings indicate that early changes in NK-{kappa}B and Mat1A gene expression and function may affect the development of early pre-cancerous lesions, they show a partially new mechanism of HPR chemopreventive effect and suggest that HPR administration might have clinical relevance in HCC treatment.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
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Animals and treatments
Male F344 rats (140–160 g) were fed a standard diet (type 48; Piccioni, Gessate, Milano, Italy) ad libitum, and were housed at a constant temperature (22°C) and humidity (55%), with a 12-h light/dark cycle (06.00–18.00). The rats were randomly divided into three groups (Figure 1). Group 1 (eight rats) consisted of normal control rats and groups 2 (40 rats) and 3 (190 rats) were subjected to the ‘resistant hepatocyte’ (RH) protocol that included initiation by DENA (150 mg/kg i.p.) followed 2 weeks later by a 15-day feeding of a hyperprotein diet (type 52; Piccioni) containing 0.02% AAF, with a partial hepatectomy at the midpoint of this feeding (21). Sterically stabilized liposomes (SL) were synthesized using a 2:1:0.1 composition of hydrogenated soy phosphatidylcholine:cholesterol:1,2-distearoylglycero-3-phosphatidyl-ethanolamine-N-polyethylene glycol-2000 (23), and SL-HPR were prepared as described (4). Preliminary experiments were performed in which carcinogen-treated rats were given, between 4 and 25 weeks after initiation, three i.p. injections of 0.4 ml/100 g body wt of empty SL or SL containing 70–350 nmol of HPR. The animals were killed at the end of HPR administration. The highest HPR effects on mean volume of lesions and apoptosis, in the absence of evident toxicity, were found at the dose of 280 nmol (data not presented). Therefore, we used SL containing this HPR dose for further experiments with group 3 animals. Two subgroups of carcinogen-treated animals (five rats each) received, between 4 and 25 weeks after initiation, i.p. 280 nmol/100 g body wt of HPR in fetal calf serum (free HPR) or serum alone. Twenty-five to thirty percent of animals in groups 2 and 3 died 1–3 weeks after partial hepatectomy without any difference between HPR-treated and untreated rats. When indicated (Figure 1) the surviving rats were killed, under ether anesthesia, by bleeding through the thoracic aorta. The livers and isolated nodules were immediately collected and frozen at –80°C until used for molecular biology and biochemical determinations. Nodules from single animals were pooled when <5 mm in diameter. All animals received humane care, and the study protocols were in compliance with our institution's guidelines for use of laboratory animals.



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Fig. 1. Study design. Male F344 rats, initiated with DENA, were fed a hyperprotein diet containing 0.02% AAF for 2 weeks with a partial hepatectomy at the mid-point of this feeding. Then the rats received a standard basal diet (BD). When indicated, sterically SL or SL containing 280 nmol/100 g body wt of HPR (SL-HPR), were injected i.p. into rats three times a week. Upward arrows represent time points at which rats were killed for analysis.

 
Histology and immunohistochemistry
Small portions of liver and isolated neoplastic (dysplastic) nodules were fixed in buffered formaldehyde, embedded in paraffin, serially sectioned into 5-µm-thick slices and used for hematoxylin/eosin and propidium iodide staining, and glutathione S-transferase (placental; GST-P) immunohistochemistry (24). Morphometric analysis was made to evaluate the number/cm3 of liver and mean volume of GST-P(+) lesions, and volume fraction (VF) (25). Re-modeling lesions were identified in GST-P immunostained sections as areas lacking uniformity of immunostaining and exhibiting irregular margins (26,27). The lesions were classified as re-modeling nodules when at least 20% of their surface was not immunostained. Labeling index (LI) was determined after i.p. injection into the rats of 5 mg/100 g body wt of 2-bromo-3-deoxyuridine (BrdU), 2 h before killing, and nuclear BrdU incorporation was evaluated by the ‘cell proliferation kit’ (Amersham Biotech, Cologno Monzese, Italy). From 2000 to 4000 GST-P(+) hepatocytes per liver were counted, and the data were expressed as the percentage of cells that incorporated BrdU. Apoptotic bodies, stained by hematoxylin/eosin, and nuclear changes representing apoptosis (chromatin condensation, margination and fragmentation), revealed by propidium iodide, were determined by scoring 5000 preneoplastic or neoplastic cells per liver, or 5000 hepatocytes in non-adjacent sections of liver, and data were expressed as percentage of total hepatocytes (apoptotic index, AI). Pre-cancerous lesions were identified as follows (28): FAH and early nodules were mostly eosinophilic/clear cell lesions without evident atypical pattern. Twenty-five weeks after initiation, nodules with neoplastic (dysplastic) features were present in the liver. Nodules showed distortion of plate arrangements, thickened plates, some hepatocytes in nests, sinusoidal dilatation and infrequent nuclear atypia. Well-differentiated HCC, present at 50 weeks, exhibited thickened and blunted haphazardly arranged plates, hepatocytes in nests, sinusoidal dilatation and frequent nuclear atypia. Some HCCs showed a pseudoglandular pattern.

Comparative reverse transcription–polymerase chain reaction
Tissue homogenization, RNA extraction and semi-quantitative RT–PCR were performed as described (7). Briefly, 18 µl aliquots of reaction mixture (Titan One Tube RT–PCR System, Boehringer, Roche Diagnostics S.p.A., Monza, Italy), containing dNTPs (200 µM each), and 5'-cyanine5-conjugated forward primers and the correspondent unconjugated reverse primers (20 pmol each) were added to a series of three tubes per tissue, followed by 2 µl aliquots of three appropriate dilutions of total RNA master solutions (4 µg/ml). The following forward/reverse primers were used: NF-{kappa}B, 5'-ACCTGAGTCTTCTGGACCGCTG/5'-CCAGCCTTCTCCCAAGAGTCGT (bp 472), I{kappa}B-{alpha}, 5'-CACACGTGTGCATATGTACTGC/5'-TCAGCTTCTAGCTGCAGCCTCCA (bp 191), Mat1A, 5'-CTCTGGAGCAACAGTCCCCA/5'-TGGCATAGCCGAACATCAGAC (bp 102), iNos, 5'-CCAGAAGAGTTACAGCATCTGG/5'-CAAAGTGCTTCAGTCGGGTGGTTC (bp 279), c-myc, 5'-CAGCTGCCAAGAGGGCCAAGTTG/5'-GTCAGAAGGAACCGTTCTCCTTACAC (bp 422), Cyclin D1, 5'-CTTACTTCAAGTGCGTGCAGAGG/5'-GCTTGTTCACCAGAAGCAGTTCC (bp 315), Vegf-A, 5'-GAAGTTCATGGACGTCTACCAG/5'-CATCTGCTATGCTGCAGGAAGCT (bp 261), Bcl2, 5'-GCAGCTTCTTTCCCCGGAAGGA/5'-AGGTGCAGCTGACTGGACATCT (bp 374), Bax, 5'-AGGATGCATCCACCAAGAAGCTG/5'-ACCCTGGTCTTGGATCCAGACAA (bp 314), Bcl-xL, 5'-CTGGAGTCAGTTTAGCGATGTCG/5'-AGGTAGGTGGCCATCCAACT (bp 430), Bad, 5'-CGAGTGAGCAGGAAGACGCTAGT/5'-CAGGACTGGATAATGCGCGTCC (bp 421). Calibration of mRNA/cDNA concentration was made for each tissue, with primer pairs specific for RNR-18 reference gene (5'-GGCCCGAAGCGTTTACTTTGAA/5'-GCATCGCCAGTCGGCATCGTTTAT; bp 316).

After correction of RNA amounts used, to equalize signal intensities in the different tissue samples, a second calibration was made, if necessary, followed by enzymatic amplification, in the presence of specific primers. Cycling parameters for RT–PCR analyses were: 30 min at 55°C, followed by 2 min at 95°C, 1 min at 55°C, 1 min at 72°C, for 30 cycles in a GeneAmp PCR system 9700 (Perkin Elmer Applied Biosystems, Applera Italia, Monza, Italy). PCR products were run on denaturating 6% polyacrylamide gel in ALFwin Analyzer (Amersham), and analyzed by Allelelinks software.

Protein extraction
Protein for immunoprecipitation was extracted as published (8). For electrophoretic mobility shift analysis (EMSA), the samples were dounced up to cell lysis as described (29), and centrifuged for 10 min at 5000 g, at 4°C. The pellet was re-suspended in the homogenization buffer and centrifuged at 2700 g for 3 min at 4°C. The pellet was re-suspended in the lysis buffer (10 mM Tris–Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, plus protease inhibitors) (30) and incubated 30 min on ice and then centrifuged at 15 000 g for 30 min. The supernatant was stored at –80°C. Proteins were determined according to Lowry et al. (31) using bovine serum albumin as a standard.

Immunoprecipitation analysis
Immunoprecipitation of protein extracts with 4 µg of agarose-conjugated antibodies/mg of protein (Santa-Cruz Biotechnology, Santa Cruz, CA; Table I), separation of immunoprecipitated samples by SDS–PAGE, and treatment with biotinylated secondary antibody (Vector Laboratories, D.B.A. Segrate, Milano, Italy) were made as described previously (8). Immunocomplexes were revealed and quantified by enhanced chemiluminescence (Amersham). To evaluate NF-{kappa}B/I{kappa}B-{alpha} complex, immunoprecipitates with I{kappa}B-{alpha} primary antibody, were separated by SDS–PAGE and transferred onto PVDF membranes (Amersham), probed with 1.8 µg/ml of p65 NF-{kappa}B antibody, washed and incubated with secondary antibody. Immunocomplexes were visualized by enhanced chemiluminescence.


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Table I. Primary antibodies used in immunoprecipitation experiments

 
EMSA and supershift assay
This was performed as described (30). Briefly, nuclear extracts were incubated 30 min at 23°C with poly(dI–dC) (1 µg, Amersham) in binding buffer (4 mM Tris–Cl, pH 7.5, 2 mM HEPES, pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DDT, 0.2 mM PMSF, 12% glycerol) containing 2 ng of a consensus NF-{kappa}B probe (consensus binding sites of NF-{kappa}B: (5'-AGTTGAGGGGACTTTCCCAGGC, Santa-Cruz Biotechnology)), end-labeled with [{gamma}-32P]adenosine triphosphate by T4 polynucleotide kinase (Roche Diagnostics S.p.A.). For the competition assay, proteins were pre-incubated with a 100-fold excess of double-stranded unlabeled NF-{kappa}B binding oligodeoxy nucleotide, before addition of the labeled probe. For supershift analysis, treatment of samples with 2 µg of p65 NF-{kappa}B antibody (sc 372 Santa Cruz) preceded the addition of the radiolabeled probe. Samples were separated on non-denaturating 4.5% polyacrylamide gel. Dried gels were analyzed by ImageAnalyzer (Packard BioScience, Milano, Italy).

Enzyme assays
iNos activity was determined with the NOSdetect Kit (Stratagene, LaJolla, CA), by measuring the [3H]citrulline produced from [3H]arginine, according to the manufacturer's instruction, in tissue extracts of normal liver and nodules (60 000 g supernatants of homogenates in 25 mM Tris–HCl/1 mM EDTA/1 mM EGTA, pH 7.4). Liver-specific MatI/III activity of tissue extracts (175 000 g supernatants of homogenates in 80 mM Tes buffer, pH 7.4) was determined in a reaction mixture containing 80 mM Tes, pH 7.4, 50 mM KCl, 40 mM MgCl2, 5 mM ATP, 5 mM methionine, and 0.5 microCi of L-[methyl-3H]methionine, by measuring selective binding of labeled SAM to phosphocellulose filters (32). The release of sorbitol dehydrogenase activity into the serum was evaluated by following NADH oxidation at 366 nm, at 23°C (33). One unit of enzymatic activity corresponds to 1 µmol of oxidized NADH/min.

Statistical analysis
Data are expressed as means ± SD. Data relative to body and liver weights, food consumption, number and volume of liver lesions, and re-modeling and LI (total lesion averages) were analyzed by ANOVA. The significance of the differences between the means was evaluated by the Student's t-test or, in the case of multiple comparisons, by the Tukey–Kramer (TK) test. Tumor yield was analyzed by {chi}2 test. GraphPad InStat 3 (GraphPad Software, San Diego, CA) was used for statistical evaluation of the results. We selected P < 0.05 as the minimum level of significance.


    Results
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General findings
No variation among rat groups occurred for diet consumption and final body weight (not shown). Twenty-five and fifty weeks after initiation the liver weights were: 3.2 ± 0.6 and 3.5 ± 0.7 g/100 g body wt, respectively, in SL-HPR-treated rats, against 5.6 ± 1.0 and 5.7 ± 1.2 g/100 g in SL-treated rats (means ± SD, n = 9–20; P < 0.001). This suggests partial inhibition of liver regeneration by SL-HPR, after AAF-release in carcinogen-treated rats (7).

Maximum liposome uptake 24 h after injection to rats (25 weeks after initiation) of a single i.p. dose of SL containing 200 nmol of cholesteryl-[1,2-3H-(N)]-labeled phospholipids/100 g body wt, was 23.5 ± 2.3 and 14.3 ± 1.7 nmol/g of surrounding liver and isolated nodules, respectively (means ± SD, n = 3). This corresponds to a HPR liver uptake (assuming that HPR does not modify SL uptake) of 74 nmol/g, 21.9 nmol/g of which is in nodules. Liposomal delivery of HPR to the liver did not result in relevant hepatoxicity: 3 weeks after starting SL and SL-HPR treatments serum sorbitol dehydrogenase activity was 2.29 ± 0.05 and 2.3 ± 0.13 mU/ml, respectively, against 1.23 ± 0.06 mU/ml in untreated controls (means ± SD, n = 3).

Chemoprevention of hepatocarcinogenesis
Three days after arresting AAF, several GST-P(+) foci were present in rat liver (Figure 2). Their number progressively decreased thereafter while lesion volume increased. SL-HPR did not influence lesion number but induced significant volume decreases, at 14 and 147 days. A 1.8-fold decrease in VF occurred at 147 days. Treatment of rats with free HPR for 147 days was without effect. Number/cm3 of liver (N), mean volume (V) and VF values were: 4900 ± 780, 4.2 ± 1.6 x 10–4 and 68 ± 8%, respectively, in HPR-treated, against 5200 ± 677, 4.5 ± 0.8 x 10–4 and 76 ± 12% in serum-treated controls (means ± SD, n = 4). Based on these findings SL-HPR was used for further experiments.



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Fig. 2. Effect of HPR on number/cm3 of liver (N), mean volume (V, cm3 x 104), VF and LI of GST-P(+) preneoplastic lesions in the liver of rats subjected to the RH protocol. At the end of AAF feeding (fourth week) the rats received, three times a week, 0.4 ml/100 g of SL without ({bgh315in1}) or with ({bgh315in2}) 280 nmol of HPR and were killed 3, 7, 14 and 147 days after starting the treatment. Inset represents LI values in surrounding liver. Data are means ± SD (n = 5, at 3, 7 days and 9, at 14, 147 days ± HPR). The TK test showed significant differences between SL-HPR and SL subgroups for V at 14 days, and for V and VF at 147 days (P < 0.001), for LI at 7–147 days (at least P < 0.05).

 
Three to seven days after AAF treatment, GST-P(+) lesions and, at a lower extent, the surrounding liver of carcinogen-treated rats showed great increases in LI. This was followed by a progressive decrease in both tissues, although LI remained ~10-fold higher in GST-P(+) lesions than in surrounding liver. A comparative analysis of carcinogen-treated rats ± SL, excluded a liposome effect (not shown). Treatment with SL-HPR for 7–147 days strongly inhibited LI of both GST-P(+) and surrounding liver.

Apoptotic bodies were easily recognizable, 25 weeks after initiation, in hematoxylin/eosin-stained liver sections of rats subjected to RH protocol (Figure 3A and B). Numerous apoptotic bodies were visible in neoplastic nodules, in rats treated with SL-HPR, in which small clusters of apoptotic bodies could be occasionally seen (Figure 3B), whereas single and less frequent apoptotic bodies were present in rats receiving SL alone (Figure 3A). Nuclear changes characteristic of apoptosis, shown by propidium iodide staining, also indicated the presence in neoplastic nodules of more abundant apoptotic cells in SL-HPR-treated (Figure 3E and F) than in SL-treated (Figure 3D) rats or in nodules of untreated rats (group 1; Figure 3C). Figure 3A and B also shows a lack of any histological change suggestive of HPR toxicity. Micro-vacuolization of some nodular hepatocytes, observed in rats treated with SL ± HPR, was presumably consequent to massive endocytosis of liposomes. In the parenchyma surrounding nodules of rats receiving SL ± HPR some micro-vacuolization of hepatocytes could be seen, whereas necrotic spots and inflammatory infiltration were absent (not shown).



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Fig. 3. Histological pattern of GST-P(+) liver lesions 25 weeks after initiation/selection according to RH protocol. Details of neoplastic nodules stained with hematoxylin/eosin (A and B) or propidium iodide (CF), in rats killed 147 days after starting treatment with SL containing (B, E and F) or not containing (A and D) HPR. (C) Carcinogen-treated rats without SL ± HPR. Magnification: (A and B) x300; (C–F) x400.

 
Quantitative analysis of apoptosis, as measured by counting apoptotic bodies (Figure 4) showed significant increase in AI of GST-P(+) lesions with respect to surrounding liver (Figure 4, inset). Short SL-HPR treatment (3–7 days) did not significantly modify the AI of GST-P(+) lesions, whereas 81 and 127% increases occurred in these lesions 14 and 147 days after starting SL-HPR administration, respectively. Small and not significant effect of SL-HPR on AI was observed in surrounding parenchyma. The AI of isolated nodules, in sections stained with propidium iodide, essentially corresponded to that found in GST-P(+) lesions of hematoxylin/eosin-stained sections: AI was 2.8 ± 1.2 and 6.4 ± 1.8 in rats subjected for 147 days to SL and SL-HPR administration, respectively (mean ± SD, n = 9, P < 0.0001).



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Fig. 4. Effect of SL-HPR on AI of GST-P(+) preneoplastic lesions and surrounding liver (inset) of F344 rats subjected to RH protocol. At the end of AAF feeding (fourth week) the rats received, three times a week, 0.4 ml/100 g of SL without ({bgh315in3}) or with ({bgh315in4}) 280 nmol of HPR and were killed 3, 7, 14 and 147 days after starting the treatment. Data are means ± SD (n = 5, at 3, 7 days and 9, at 14, 147 days ± HPR). The TK test showed significant differences for GST-P(+) lesions between SL-HPR and SL subgroups at 14 and 147 days (P < 0.001) and no differences for surrounding liver at all times tested.

 
Re-modeling of preneoplastic liver lesions has been attributed to re-differentiation (26,27), a mechanism involved in retinoids antitumor effect (34). Days 3, 7, 14 and 147 after arresting AAF feeding, the percentages of re-modeling lesions in SL-treated rats were 16.97 ± 5.9, 33.2 ± 8.06, 31.0 ± 6.7 and 17.1 ± 3.8, respectively. These figures were not significantly modified by SL-HPR administration (not shown), thus excluding a role of re-differentiation in the HPR action, in our experimental system.

In order to evaluate if the inhibition of the development of early preneoplastic liver by HPR is a long-lasting effect, we examined tumor development 25 weeks after arresting SL-HPR administration. At this time, there occurred a significant decrease in HCC incidence, without changes in FAH and nodule incidence (Table II). Moreover, SL-HPR induced a decrease in nodule and HCC multiplicity, and an increase in FAH multiplicity suggesting a block of FAH progression.


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Table II. Incidence and multiplicity of liver lesions in rats subjected to SL-HPR and their controls

 
HPR prevents the deregulation of NF-{kappa}B activity
NF-{kappa}B mRNA and protein levels were 180 and 73% higher (Figure 5A) and 128 and 87% higher (Figure 5B) in preneoplastic liver (liver of carcinogen-treated rats, 5 weeks after initiation) and nodules (at 25 weeks), respectively, than in normal liver. SL-HPR administration caused 38–40% decrease in mRNA and protein contents, at 5 and 25 weeks. I{kappa}B-{alpha} mRNA and protein levels underwent 14 and 25% decreases, respectively, in preneoplastic liver, and 28 and 37% decreases in nodules. SL-HPR induced ~133 and 100% increases in I{kappa}B-{alpha} mRNA and protein levels, at 5 weeks, and 120 and 130% increases, at 25 weeks. Accordingly, the NF-{kappa}B/I{kappa}B-{alpha} complex slightly decreased in carcinogen-treated rats receiving SL, and underwent evident increases in those treated with SL-HPR, at both 5 and 25 weeks (Figure 5C). NF-{kappa}B binding experiments (Figure 5D) revealed a complex, super-shifted by p65 antibody, whose level was higher in preneoplastic liver and nodules than in normal liver, and underwent a consistent decrease in SL-HPR-treated rat, especially at 25 weeks.



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Fig. 5. Effect of SL-HPR on the expression of NF-{kappa}B, I{kappa}B-{alpha} and NF-{kappa}B/I{kappa}B-{alpha} complex, and NF-{kappa}B activity in normal liver (C), liver of rats subjected to the RH protocol, 5 weeks after initiation, and isolated nodules, 25 weeks after initiation. At the end of AAF feeding the rats received three times a week 0.4 ml/100 g of SL (S) without (liposomes) or with 280 nmol of HPR (H; liposomes + HPR) and were killed 7 (5 weeks) and 147 days (25 weeks) after starting the treatment. Control rats were killed at these time points and the results were considered together due the absence of significant differences. (A) RT–PCR products were separated by electrophoresis into denaturating polyacrylamide gel, run in an ALFwin Fragment Analyzer and analyzed by Allelelinks software. (Left) Representative reproduction of RT–PCR products. (Right) Quantitative analysis showing mean product values ± SD of 8, 11 and 9 rats, for C, 5 and 25 weeks ± HPR, respectively, normalized to RNR-18 values (relative amounts). The results of control rats at 5 and 25 weeks (four rats each) are considered together. The TK test showed: NF-{kappa}B, SL versus C and SL versus SL-HPR, P < 0.001 at 5 and 25 weeks; I{kappa}B-{alpha}, C versus SL, P < 0.01 at 5 and 25 weeks; SL versus SL-HPR, P < 0.001 at 5 and 25 weeks. (B) Detection by immunoprecipitation of NF-{kappa}B and I{kappa}B-{alpha}. Sample proteins and blocking peptides were co-immunoprecipitated with antibodies against p65 or I{kappa}B-{alpha} and separated by SDS–PAGE. (Left) Representative immunoprecipitation analysis, with control proteins (blocking peptides) in the last lane. (Right) Chemiluminescence analysis showing mean values ± SD of 4, 5 and 9 rats, for C, 5 and 25 weeks ± HPR, respectively, normalized to control proteins (arbitrary units). The TK test showed: NF-{kappa}B, SL versus C, and SL versus SL-HPR, P < 0.001 at 5 and 25 weeks; I{kappa}B-{alpha}, C versus SL, P < 0.01, at 5 and 25 weeks; SL versus SL-HPR, P < 0.001 at 5 and 25 weeks. (C) Reproduction of NF-{kappa}B/I{kappa}B-{alpha} complexes. Proteins were immunoprecipitated by antibody against I{kappa}B-{alpha}. Separated by SDS–PAGE, and probed with p65 antibody. Enhanced chemiluminescence was used to visualize the immune complex. (D) NF-{kappa}B binding activity. Nuclear extracts were isolated from the different tissues. EMSA was performed using the upstream double stranded oligodeoxynucleotides for the consensus binding sites of NF-{kappa}B as a probe. (a) NF-{kappa}B binding; (b) competition assay; (c) super-shift analysis.

 
Expression of NF-{kappa}B related genes
NF-{kappa}B transactivates various growth-related genes, including iNos, c-myc and Cyclin D1 (1012,34). iNos and MatI/III activities may modulate NF-{kappa}B activity (10,35). This suggests the existence of a cross-talk between iNos, Mat1A and NF-{kappa}B genes and their products whose deregulation may affect the growth capacity of preneoplastic liver lesions. As shown in Figure 6A, iNos mRNA level, very low in normal liver, underwent an ~100% increase in preneoplastic liver and nodules, and decreased to normal liver values in SL-HPR-treated rats. Mat1A expression showed an ~50% decrease in preneoplastic liver and nodules. SL-HPR treatment induced partial recovery in preneoplastic liver, and a 200% rise in nodules. Figure 6A also shows great increases in the expression of c-myc, cyclin D1 and Vegf-A genes, in preneoplastic liver and nodules of SL-treated rats, with respect to normal liver. SL-HPR administration resulted in a marked decrease in the expression of both c-myc and cyclin D1, at 5 and 25 weeks, and in the expression of Vegf-A, at 25 weeks. Immunoprecipitation experiments (Figure 6B) confirmed the existence of iNos, c-Myc, Cyclin D1 and Vegf-A up-regulation in nodules of SL-treated rats, 25 weeks after initiation. SL-HPR caused a significant decrease in the expression of all genes tested. In the absence of specific antibodies, no immunoprecipitation analysis of Mat1A gene product was made.



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Fig. 6. Effect of SL-HPR on the expression of growth-related genes in normal liver (C), liver of rats subjected to the RH protocol, 5 weeks after initiation, and isolated nodules, 25 weeks after initiation. At the end of AAF feeding (fourth week) the rats received three times a week 0.4 ml/100 g of SL without (liposomes) or with 280 nmol of HPR (liposomes + HPR) and were killed 7 (5 weeks) and 147 days (25 weeks) after starting the treatment. Control rats were killed at these time points and the results were considered together due the absence of significant differences. (A) RT–PCR products were separated and analyzed as in Figure 5A. (Left) Representative reproduction of RT–PCR products. (Right) Quantitative analysis showing mean product values ± SD of five, seven and five rats for C, 5 and 25 weeks ± HPR, respectively, normalized to RNR-18 values (relative amounts). The TK test showed: SL versus C at least P < 0.05 for all genes tested at 5 and 25 weeks. SL versus SL-HPR: P < 0.001 for iNos, Mat1A, c-myc and Cyclin D1, not significant for Vegf-A, at 5 weeks; at least P < 0.05 for all genes tested, at 25 weeks. (B) Detection by immunoprecipitation of iNos, c-Myc, Cyclin D1 and Vegf-A. Sample proteins and blocking peptides were co-immunoprecipitated with antibodies against iNos, Myc, Cyclin D1 and Vegf-A and separated by SDS–PAGE. (Left) Representative immunoprecipitation analysis with control proteins (blocking peptides) in the last lane. (Right) Chemiluminescence analysis showing mean values ± SD of three and five rats, for C and 25 weeks ± HPR, respectively, normalized to control proteins (arbitrary units). The TK test showed: SL versus C: P < 0.001 for all genes tested. SL versus SL-HPR: P < 0.001 for iNos, and c-Myc, P < 0.05 for Cyclin D1, and Vegf-A.

 
Activities of iNos and MatI/III
Liver SAM level plays a central role in the regulation of reparative and neoplastic liver growth (26,36). HCCs exhibit low activity of the MatI/III (37,38) that can be modulated by nitric oxide (10). Since SL-HPR inhibits iNos expression, we determined the HPR effect on iNos and MatI/III activities to get insights on the interference of HPR with SAM synthesis in preneoplastic liver tissue. As shown in Table III, iNos activity was 1.9-fold higher in nodules than in normal liver, and exhibited a 35% decrease in SL-HPR-treated rats, whereas SL alone was ineffective. MatI/III activity was significantly lower in nodules than in normal liver. It increased 16-fold in nodules of SL-HPR-treated rats and did not change in SL-treated rats.


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Table III. Effect of SL-HPR on iNos and MATI/III activities

 
Expression of apoptosis-related genes
Bcl-2 expression was 306 and 130% higher in preneoplastic liver and nodules, respectively, than in normal liver. It was not affected by SL-HPR administration for 7 days, whereas it returned to normal liver values after administration for 147 days (Figure 7A). The expression of the pro-apoptotic Bax gene was higher in preneoplastic liver and nodules than in normal liver, and was not modified by SL-HPR. Increase in Bcl-xL mRNA with respect to control was only found in preneoplastic liver, whereas Bad expression increased both in preneoplastic liver and nodules. SL-HPR treatment induced a decrease in both Bcl-xL and Bad mRNA levels at 5 and 25 weeks. As a consequence of these changes, the Bcl-2/Bax and Bcl-xL/Bax mRNA expression ratios decreased ~2- and 2.2–3-fold, respectively, at 5 and 25 weeks (Table IV). No significant changes of the Bcl-2/Bax mRNA ratio occurred, whereas Bcl-xL/Bad ratio decreased ~1.5-fold. Immunoprecipitation analysis (Figure 7B) confirmed these results at the protein level, for Bcl-2 and Bax, showing an ~3-fold decrease in the Bcl-2/Bax ratio.



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Fig. 7. Effect of SL-HPR on apoptosis-related genes in normal liver (C), liver of rats subjected to the RH protocol, 5 weeks after initiation, and isolated nodules, 25 weeks after initiation. At the end of AAF feeding (fourth week) the rats received three times a week 0.4 ml/100 g of SL without (liposomes) or with 280 nmol of HPR (liposomes + HPR) and were killed 7 (5 weeks) and 147 days (25 weeks) after starting the treatment. Control rats were killed at these time points and the results were considered together due the absence of significant differences. (A) RT–PCR products were separated and analyzed as in Figure 5A. (Left) Representative reproduction of RT–PCR products. (Right) Quantitative analysis showing mean product values ± SD of five, seven and five rats, for C, 5 and 25 weeks ± HPR, respectively, normalized to RNR-18 values (relative amounts). The TK test showed: SL versus C at least P < 0.05 for all genes tested at 5 and 25 weeks. SL versus SL-HPR: P < 0.001 for Bcl-xL and Bad, not significant for Bcl2 and Bax, at 5 weeks. At least P < 0.05 for Bcl-2 and Bad, not significant for Bcl-xL and Bax, at 25 weeks. (B) Detection by immunoprecipitation of Bcl-2 and Bax in homogenates of normal liver (C) and neoplastic nodules 25 weeks after initiation. Sample proteins and blocking peptides were co-immunoprecipitated with antibodies against Bcl-2 and Bax and separated by SDS–PAGE. (Left) Representative immunoprecipitation analysis, with control proteins (blocking peptides) in the last lane. (Right) Chemiluminescence analysis showing mean values ± SD of four and five rats, for C and 25 weeks ± HPR, normalized to control proteins (arbitrary units). The TK test showed: SL versus C, P < 0.05 for Bcl-2 and Bax. SL versus SL-HPR, at least P < 0.01 for both genes.

 

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Table IV. mRNA expression ratio between anti-apoptotic Bcl2 and Bcl-xL and pro-apoptotic Bax and Bad genesa

 
A comparative analysis of gene expression, at mRNA and protein level, in liver and nodules of carcinogen-treated rats with/without SL excluded an effect of liposomes on the expression of all genes tested (not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our observations demonstrate that SL-HPR treatment, during early stages of hepatocarcinogenesis, causes growth inhibition and apoptosis of initiated cells associated with a great decrease in the volume of liver lesions, without apparent changes in lesion number. A reduced coalescence of lesions, consequent to decrease in volume, however, could mask the eventual decrease in this parameter. The strong chemopreventive effect of SL-HPR treatment persisted 25 weeks after arresting its administration. At this time a decrease in HCC incidence and in nodule and HCC multiplicity was associated with an increase in FAH multiplicity, indicating a block of the evolution of preneoplastic lesions to more malignant conditions.

According to our results, an increase in NF-{kappa}B gene expression, associated with a decrease in the NF-{kappa}B/I{kappa}B-{alpha} complex, NF-{kappa}B activation and decrease in Mat1A expression occur in pre-cancerous liver lesions (Figure 8). These alterations may be at least in part attributed to the presence of rapidly growing GST-P(+) lesions occupying ~50% of liver, 5 weeks after initiation. At this time surrounding liver was still proliferating, due to the delay in liver regeneration induced by AAF (21), and may also transiently contribute to changes of NF-{kappa}B and Mat1A expression (39,40). However, these changes persisted in isolated nodules, at 25 weeks, when they were absent in non-proliferating surrounding liver (data not presented), suggesting that these alterations are constitutive of pre-cancerous cells. The genes controlling G1–S transition are deregulated in initiated liver cells (7,8,41). NF-{kappa}B activation is involved in the entry into G1 phase of cells primed to proliferate (11). Thus, NF-{kappa}B activation maintains initiated cells in early G1 phase and ensures G1 progression to S phase, by transcriptional activation of c-myc and cyclin D1 (9,1113). In this way NF-{kappa}B up-regulation may contribute to the fast growth of pre-cancerous cells, which represents a prerequisite for their evolution to carcinoma, as well as to the high HCC yield (7,8,41).



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Fig. 8. Schematic representation depicting some possible molecular correlations between iNos, Mat1A and NF-{kappa}B genes, in pre-cancerous liver, with sites of HPR action. NF-{kappa}B is over-expressed in pre-cancerous liver lesions. This results in enhanced transactivation of iNos, c-myc and Cyclin D1 genes. Generation of nitric oxide by iNos activates HIF-1 that mediates Vegf over-expression, and I{kappa}B kinase (IKK) with consequent I{kappa}B-{alpha} phosphorylation and ubiquitination. MatI/III activity is low in preneoplastic lesions, due to the under-expression of Mat1A gene, and may be further reduced by the oxidation of its catalytic site, mediated by nitric oxide. This should lead to a decrease in SAM production, independently of the activity of MatII, due to the inhibition of this enzyme by the reaction product. Decrease in SAM level may contribute to the down-regulation of I{kappa}B-{alpha} gene. Up-regulation of Mat1A and down-regulation of NF-{kappa}B genes, by HPR, may modify the interactions between iNos, Mat1A and NF-{kappa}B gene products. Positive and negative interactions are indicated by black arrows and blunt arrows, respectively. Upward and downward white arrows indicate increase and decrease in expression or activity, respectively, in pre-cancerous liver lesions with respect to normal liver. Dotted lines indicate HPR effect.

 
Mat1A down-regulation in pre-cancerous cells may participate in this scenario by contributing to maintain active NF-{kappa}B (Figure 8). Mat1A expression and MatI/III activity are essential for the maintenance of a high SAM liver level (40,42). In response to liver injury or during reparative growth, Mat1A gene is down-regulated whereas the Mat2A, a gene encoding the extrahepatic isoform MatII, induced by NF-{kappa}B (43), is switched on (40,42). Down-regulation of Mat1A and over-expression of Mat2A genes, associated with a fall in SAM level, were also found in HCC (33,3638,42). According to our results under-expression of Mat1A and reduction in activity of MatI/III occur in pre-cancerous liver lesions. We did not determine MatII activity, but this enzyme is inhibited by the reaction product and its up-regulation does not lead to an increase in SAM liver content (42). Under-expression of Mat1A may be implicated in the pathogenesis of HCC. Mice lacking Mat1A show reduced SAM liver content and spontaneously develop HCC (44). Since SAM enhances the synthesis of the NF-{kappa}B inhibitor, I{kappa}B-{alpha}, probably by targeting I{kappa}B-{alpha} gene (10), low MatI/III activity and SAM content in pre-cancerous liver (26,36) should contribute to the observed relatively low levels of NF-{kappa}B/I{kappa}B-{alpha} complex as well as to NF-{kappa}B activation and over-expression of genes targeted by the nuclear factor, such as c-myc, Cyclin D1, iNos and VegfA. A role of Mat1A in cyclin D1 regulation has been documented during liver regeneration (45). On the other hand, transactivation of iNos gene by NF-{kappa}B (10) and iNos over-activity, in preneoplastic lesions, result in nitric oxide overproduction that may inhibit hepatocyte MatI/III (50) and SAM production (42). Furthermore, nitric oxide activates I{kappa}B kinase with consequent I{kappa}B-{alpha} phosphorylation and ubiquitination (30). Thus, overproduction of nitric oxide can contribute, by modulating SAM level and I{kappa}B kinase activity, to decrease in MatI/III activity and increase in NF-{kappa}B level (Figure 8). Finally, nitric oxide up-regulates the hypoxia inducible factor 1 that mediates Vegf-A over-expression (46). According to recent observations, iNos is located mainly in oval cells (47). Nevertheless, the effect of nitric oxide on hepatocyte enzymes could result from the high diffusibility of this compound, or from eventual differentiation of oval cells into preneoplastic hepatocytes (48), supported by NF-{kappa}B and STAT3 (49). Overall, our observations indicate that up-regulation of NF-{kappa}B, associated with the down-regulation of Mat1A, and consequent changes in the cross-talk between NF-{kappa}B, iNos and MatI/III (Figure 8) may represent a condition favorable to fast growth of pre-cancerous liver lesions and their progression to HCC.

HPR may interfere at various levels with the NF-{kappa}B-related pathways (Figure 8). SL-HPR treatment induced opposite effects on NF-{kappa}B and Mat1A genes, resulting in NF-{kappa}B under-expression and Mat1A up-regulation in preneoplastic liver and dysplastic nodules. This could account for various effects of the retinoid at molecular and cellular level, including down-regulation of cell-cycle key genes targeted by NF-{kappa}B, observed in the present and previous (46,50,51) research, and inhibition of DNA synthesis in proliferating cells. The stimulation of MatI/III activity, in SL-HPR-treated rats, may contribute to NF-{kappa}B inactivation by increasing SAM content and, consequently, the levels of I{kappa}B-{alpha} and NF-{kappa}B/I{kappa}B-{alpha} complex (Figure 8). Decrease in iNos activity, by SL-HPR, should cause Vegf-A down-regulation. However, although most of the effects of SL-HPR at a molecular level can be explained by changes in the expression of NF-{kappa}B and Mat1A genes, it cannot be excluded that SL-HPR directly affects iNos, I{kappa}B-{alpha}, c-myc, cyclin D1 and/or Vegf-A expression.

Suppression of apoptosis by activation of anti-apoptotic Bcl-2 and Bcl-XL genes (52) is one of the mechanisms whereby NF-{kappa}B facilitates cell transformation (13). Induction of apoptosis by HPR includes rise in endogenous ceramide levels and in reactive oxygen species (53,54). These biochemical events stimulate a receptor-independent pathway, under the control of Bcl-2 family genes implicating a contribution of the apoptogenic Bax and Bad genes, leading to cytochrome c release from mitochondria and Caspases 9 and 3 activation (55,56). The interference of HPR with this pathway during hepatocarcinogenesis is shown by a decrease in Bcl-2/Bax, Bcl-xL/Bax and Bcl-xL/Bad mRNA ratios, coincident with a significant stimulation of apoptosis, 5 and 25 weeks after starting SL-HPR administration. However, the implication of other molecular pathways in the apoptogenic effect of HPR cannot be excluded. Recent findings (57) showed that the induction of apoptosis by HPR, in in vitro growing human hepatoma cells, is mediated by the over-expression of GADD153, and is independent of BCL-2 activity.

The molecular mechanisms underlying the control of gene expression by HPR are not clear. Different retinoids induce gene activation through the nuclear retinoic acid and retinoic X receptors, and down-regulation by interaction with transcription factors (34) or stimulation of negative regulators (57). According to our results HPR up-regulates the Mat1A gene and down-regulates NF-{kappa}B and possibly some other growth- and apoptosis-related genes. However, the implications of nuclear receptors and/or other regulators of gene expression, in these HPR effects, are not known and need further research.

In conclusion, our results demonstrate that up-regulation of NF-{kappa}B and under-expression of Mat1A and low activity of MatI/III, described previously in HCCs (14,15,37,38), represent early events of hepatocarcinogenesis. They also show a deregulation of some molecular pathways controlled by NF-{kappa}B and MatI/III activities in early stages of rat liver carcinogenesis. The molecular mechanisms underlying the changes in NF-{kappa}B and Mat1A expression in preneoplastic liver have not been investigated in the present work. Recent results indicate that NF-{kappa}B induction may be mediated by the mitogen-activated protein kinase cascade (59), a molecular pathway that is up-regulated in preneoplastic and neoplastic rat liver lesions (60). Furthermore, our data demonstrate a strong and persistent chemoprevention of hepatocarcinogenesis by SL-HPR and suggest a new mechanism of the antiproliferative effect of HPR, based on NF-{kappa}B inactivation and stimulation of MatI/III activity, which could be extended to other cancer types. Overall, our results strongly support a pathogenic role of the deregulation of NF-{kappa}B and Mat1A genes and some related pathways, in early stages of hepatocarcinogenesis. Taken together with the observation of NF-{kappa}B up-regulation in human viral hepatitis (16,17), they suggest the need of clinical trials to assess the efficacy of SL-HPR to prevent HCC in patients with active hepatitis/cirrhosis.


    Acknowledgments
 
Supported by grants from Associazione Italiana Ricerche sul Cancro, Compagnia S. Paolo, MIUR (PRIN, FIRB) and RAS.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 22, 2004; revised September 20, 2004; accepted October 10, 2004.