Inhibition of Hepatocarcinogenesis by the Deletion of the p50
Subunit of NF-jB in Mice Administered the Peroxisome
Proliferator Wy-14,643
Howard P. Glauert,*
,†
,1Aysegul Eyigor,‡
,¶Job C. Tharappel,*
,† Simon Cooper,‡ Eun Y. Lee,§ and Brett T. Spear*
,†
,‡
*Graduate Center for Nutritional Sciences, †Graduate Center for Toxicology, ‡Department of Microbiology, Immunology, and Molecular Genetics, and §Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, Kentucky 40506; and¶Uludag University, Faculty of Veterinary
Medicine, Department of Food Hygiene and Technology, 16059, Gorukle kampusu, Bursa, Turkey Received November 21, 2005; accepted January 20, 2006
Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic
peroxisome proliferation and tumors in rodents. We previously
showed that peroxisome proliferators increase NF-kB DNA
binding activity in rats, mice, and hepatoma cell lines, and that
mice deficient in the p50 subunit of NF-kB had much lower cell
proliferation in response to the peroxisome proliferator
ciprofi-brate. In this study we examined the promotion of
hepatocarcino-genesis by WY in the p50 knockout (/) mice. The p50 / and
wild type mice were first administered diethylnitrosamine (DEN)
as an initiating agent. Mice were then fed a control diet or a diet
containing 0.05% WY for 38 weeks. Wild-type mice receiving
DEN only developed a low incidence of tumors, and the majority
of wild-type mice receiving both DEN and WY developed tumors.
However, no tumors were seen in any of the p50
/ mice. Cell
proliferation and apoptosis were measured in hepatocytes by
BrdU labeling and the TUNEL assay, respectively. Treatment with
DEN + WY increased both cell proliferation and apoptosis in both
the wild-type and p50
/ mice; DEN treatment alone has no
effect. In the DEN/WY-treated mice, cell proliferation and
apoptosis were slightly lower in the p50
/ mice than in the
wild-type mice. These data demonstrate that NF-kB is involved in
the promotion of hepatic tumors by the peroxisome proliferator
WY; however, the difference in tumor incidence could not be
attributed to alterations in either cell proliferation or apoptosis.
Key Words: NF-kB; peroxisome; carcinogenesis; cell
prolifera-tion; apoptosis.
Peroxisome proliferators are a group of chemically distinct
compounds capable of eliciting a persistent peroxisome
pro-liferation in hepatocytes and promoting liver tumors in rats and
mice (Cattley et al., 1998; Rao and Reddy, 1987). These
chem-icals activate the peroxisome proliferator-activated
receptor-alpha (PPARa), which leads to an increase in the size and
number of peroxisomes as well as activating genes encoding
several enzymes of the peroxisomal b-oxidation pathway
(Eacho and Feller, 1991; Schoonjans et al., 1996). The rate
limiting enzyme of this pathway, fatty acyl CoA oxidase
(FAO), produces hydrogen peroxide (H
2O
2) as a by-product.
The activity of this enzyme is increased 10- to 15-fold by
peroxisome proliferators such as ciprofibrate or Wy-14,643 (Rao
and Reddy, 1987). In contrast, the activity of the H
2O
2-detoxifying enzyme catalase is only increased about two-fold
by peroxisome proliferators (Rao and Reddy, 1987). It has been
proposed that this imbalance in FAO and catalase induction may
result in the accumulation of H
2O
2, which could at least partially
be responsible for the effects of peroxisome proliferators.
Several studies have found that the administration of
peroxi-some proliferators leads to lipid peroxidation and oxidative
DNA damage, but other studies have not (O’Brien et al., 2005).
Peroxisome proliferators have been found to decrease the levels
of several cellular antioxidants and antioxidant enzymes,
in-cluding vitamins C and E, and glutathione peroxidase (O’Brien
et al., 2005).
In addition to these biochemical changes, peroxisome
proliferators increase cell proliferation in the liver soon after
they are administered (Reddy and Lalwani, 1983). Cell
proliferation eventually returns to basal levels for many
peroxisome proliferators, but remains elevated for others (Chen
et al., 1994; Eacho et al., 1991; Marsman et al., 1988; Yeldandi
et al., 1989). In addition to stimulating DNA synthesis,
peroxisome proliferators have been shown to inhibit apoptosis
in normal and preneoplastic hepatocytes (Bayly et al., 1993,
1994; Roberts et al., 1995; Schulte-Hermann et al., 1995). The
withdrawal of peroxisome proliferators leads to rapid reduction
in liver weight, presumably in part by apoptosis
(Schulte-Hermann et al., 1995).
In spite of numerous studies, the link between peroxisome
proliferators and hepatocarcinogenesis on a molecular and
cel-lular level is not fully understood. In addition, the link between
active oxygen production by peroxisome proliferator-induced
en-zymes and carcinogenesis has not been demonstrated. In several
1To whom correspondence should be addressed at University of Kentucky, Graduate Center for Nutritional Sciences, 222 Funkhouser Building, Lexington, KY 40506-0054. Fax: (859) 323-0061. E-mail: hglauert@uky.edu.
Ó The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
studies using rats and mice, we have shown that peroxisome
proliferators increase the DNA binding activity of the
tran-scription factor nuclear factor-jB (NF-jB) (Li et al., 1996;
Nilakantan et al., 1998; Tharappel et al., 2001). NF-jB is
normally found in the cytoplasm as an inactive complex
consisting primarily of two subunits (p50 and p65) which are
bound to an inhibitory subunit, IjB; upon activation, NF-jB is
released from IjB and translocates to the nucleus, where it
increases the transcription of specific genes (Verma et al., 1995).
NF-jB is important in the activation of genes that regulate cell
proliferation and apoptosis in various cell types (Barkett and
Gilmore, 1999; Beg et al., 1995; Demartin et al., 1999; Sha
et al., 1995). Reactive oxygen species, including H
2O
2, are
activators of NF-jB, while the addition of antioxidants such as
vitamin E can block activation of NF-jB (Calfee-Mason et al.,
2004; Gabbita et al., 2000; Li et al., 2000a,b; Meyer et al., 1994;
Nilakantan et al., 1998). This has led us to hypothesize that
peroxisome proliferators activate NF-jB through the induction
of H
2O
2-generating enzymes such as FAO or through the
down-regulation of antioxidants and antioxidant enzymes, such as
vitamin E and glutathione peroxidase. We subsequently found
that NF-jB can be activated by the overexpression of FAO
in Cos cells, and that ciprofibrate-induced NF-jB activation can
be inhibited by vitamin E or N-acetyl cysteine in rat hepatoma
cells, by dietary vitamin E in rats, and by catalase
over-expression in mice (Calfee-Mason et al., 2004; Li et al.,
2000a,b; Nilakantan et al., 1998). Catalase overexpression also
inhibited ciprofibrate-induced cell proliferation in hepatocytes
(Nilakantan et al., 1998).
In this study, we examined the hypothesis that NF-jB
activation is necessary for the promotion of hepatic tumors
by peroxisome proliferators. We used a mouse model that is
deficient in the p50 subunit of NF-jB (Sha et al., 1995). We
recently observed that the induction of cell proliferation by the
peroxisome proliferator ciprofibrate was inhibited in p50
/
mice (Tharappel et al., 2003). Wild-type and p50
/ mice
were injected with diethylnitrosamine (DEN) and then were
administered the peroxisome proliferator Wy-14,643 (WY) for
38 weeks. The incidence of tumors, the rates of cell
pro-liferation and apoptosis, and the activity of the peroxisome
proliferator-induced enzyme fatty acyl CoA oxidase were then
quantified.
MATERIALS AND METHODS
Chemicals. Wy-14,643 (WY) was purchased from Chem-Syn Synthesis (Lenexa, Kansas). Anti-bromodeoxyuridine (BrdU) antibody was purchased from Becton Dickinson (San Jose, CA); all other antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). The Vectastain kit for immunos-taining was obtained from Vector Laboratories (Burlingame, CA). The antigen retrieval solution Citra was purchased from BioGenex (San Ramon, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Experimental design. Mice homozygous for p50 / deletion and B6129SF2/J age-matched wild type controls were obtained from our breeding
colony; founders had been obtained from The Jackson Laboratory (Bar Harbor, ME). After weaning, mice were fed an unrefined diet (Harlan Teklad 2018 Global 18% protein rodent diet) and water ad libitum. When the mice of both strains were eight weeks old, they received either an ip injection of DEN (90 mg/kg) or an equivalent amount of saline. Two weeks later, mice were fed either a diet containing 0.05% WY or a control diet for 38 weeks. Six days before euthanasia, mice were provided drinking water containing bromodeoxyuridine (BrdU, 0.5 mg/ml) (Ledda-Columbano et al., 1998). Mice were euthanized by overexposure to carbon dioxide and livers removed. A portion of the liver was fixed in formalin for histology and the remainder was frozen in liquid nitrogen and then stored at80°C.
BrdU immunohistochemical staining. The paraffin-embedded liver tis-sues were sectioned, stained with an anti-BrdU antibody, and counter-stained with hematoxylin. The staining was carried out using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA), according to the protocol provided by the manufacturer. Cells that had incorporated BrdU were easily identified as those with brown nuclei. At least 3000 hepatocellular nuclei per slide (1000 in each of three lobes) were counted in random fields and the labeling index was expressed as a percentage of the number of labeled hepatocyte nuclei out of total number of hepatocyte nuclei counted.
Apoptosis assay (TUNEL). The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) apoptosis assay kit was purchased from Intergen (Purchase, NY). The assay was performed on paraffin sections following the manufacturer’s protocol. At least 3000 nuclei were randomly counted per slide and the apoptotic index was expressed as the percentage of number of labeled apoptotic bodies of the total number of nuclei counted.
Fatty Acyl CoA Oxidase (FAO) assay. FAO activity in liver tissue homogenates was determined using lauroyl CoA as the substrate as described by Poosch and Yamazaki (1986).
Statistical analyses. Tumor incidence data were analyzed by v2analyses. Body weight, liver weight, FAO, cell proliferation, and apoptosis data were analyzed by two-way analysis of variance (ANOVA). If a significant f value was observed, results were further analyzed using Bonferroni’s test. The results are reported as means ± standard error of mean (SEM).
RESULTS
In this study, we tested whether NF-jB is necessary for the
promotion of DEN-initiated hepatic tumors by peroxisome
proliferators. To accomplish this, we fed an unrefined diet
containing 0.05% WY (or a control unrefined diet) to mice that
were deficient in the p50 subunit of NF-jB (p50
/; Sha et al.,
1995) or to wild-type control mice. After 38 weeks of feeding,
mice were euthanized; mice were given bromodeoxyuridine
in their drinking water six days before euthanasia
(Ledda-Columbano et al., 1998). Several mice died before the end of the
study: one in the wild-type DEN group, two in the wild-type
DEN/WY group, two in the p50
/ DEN group, and two in the
p50
/ DEN/WY group. We examined whether wild-type or
p50
/ mice were responding to WY treatment by the increase
in liver weights and the activity of hepatic fatty acyl CoA
oxidase (FAO). The wild-type control mice or wild-type mice
receiving only DEN weighed significantly more than wild-type
mice fed WY or all p50
/ mice (Table 1). Mice administered
WY, both wild-type and p50
/, had higher liver weights and
liver to body weight ratios than did mice not receiving WY
(Table 1). The activity of FAO was higher in mice administered
WY compared to those fed control diets (Fig. 1).
The incidence of hepatic tumors is shown in Table 1. Only
wild-type mice developed tumors; 25% of the wild-type mice
administered DEN developed tumors, while 62.5% of
wild-type mice receiving both DEN and WY developed tumors. The
incidence of tumors in wild-type mice receiving both DEN and
WY was significantly greater than in p50
/ mice receiving
both DEN and WY. All the tumors in this study were small and
did not show certain histologic features that are known to be
associated with hepatocellular carcinoma. Therefore, all the
tumors in this study were classified as hepatocellular adenomas
(Maronpot et al., 1986).
Peroxisome proliferators in rodents lead to hepatomegaly, in
part, by increasing cell proliferation (Chen et al., 1994; Eacho
et al., 1991; Marsman et al., 1988; Reddy and Lalwani, 1983;
Yeldandi et al., 1989). To examine the role of p50 in DNA
synthesis, we quantified BrdU uptake, which serves as a measure
of proliferation, by staining liver sections with anti-BrdU
antibodies (Fig. 2). In both wild-type and p50
/ mice, the
labeling index was higher in mice treated with both DEN and
WY than in control mice or mice treated with DEN only. There
was no significant difference in the labeling index between the
wild-type and p50
/ mice in the ANOVA, and no significant
interaction was observed. However, the labeling index in the p50
/ mice administered both DEN and WY was about 40%
lower than in wild-type mice adminstered both DEN and WY.
Previous studies have shown that peroxisome proliferator
treatment leads to a decrease in apoptosis in hepatocytes (Bayly
et al., 1993, 1994; Roberts et al., 1995; Schulte-Hermann et al.,
1995). Apoptosis was evaluated in this study with the TUNEL
assay (Fig. 3). In both wild-type and p50
/ mice, the
apoptotic index was higher in mice treated with both DEN and
WY than in control mice or mice treated with DEN only. The
apoptotic index was not significantly affected by the deletion of
the p50 subunit in the ANOVA, and no significant interaction
was observed. However, the apoptotic index in the p50
/
mice administered both DEN and WY was about 60% lower
than in wild-type mice adminstered both DEN and WY.
DISCUSSION
The data presented here indicate that NF-jB plays an
important role in the induction of hepatic tumors by
peroxi-some proliferators. DEN induced a low incidence of tumors in
FAO Activity (nmol/mg/min)
0 2 4 6 8 10 12 WT WT: DEN WT: DEN + WY p50 p50: DEN p50: DEN + WY
*
*
FIG. 1. Effect of Wy-14,643 (WY) on fatty acyl CoA oxidase (FAO) activity in p50/ and wild-type (WT) mice. The activity of FAO was quantified in liver homogenates. Data are means ± standard errors. Values with different asterisks are significantly different from their respective controls, by Bonferroni’s test ( p < 0.05).
TABLE 1
Influence of p50 Deletion and Wy-14,643 (WY) on Liver
and Body Weights and on Tumor Incidence
Final body weight (g) Liver weight (g) Liver wt./body wt. (%) Tumor incidence (%) Wild-type Control 42.5 ± 2.3a 2.00 ± 0.11a 4.71 ± 0.10a 0/3 (0)a,b DEN 45.9 ± 2.8a 2.15 ± 0.18a 4.65 ± 0.15a 2/8 (25)a,b DENþ WY 28.8 ± 0.5b 5.11 ± 0.22b 17.6 ± 0.6b 5/8 (62.5)b p50/ Control 29.4 ± 1.3b 1.75 ± 0.10a 5.94 ± 0.17a 0/4 (0)a DEN 32.3 ± 0.5b 1.97 ± 0.14a 6.11 ± 0.55a 0/8 (0)a DENþ WY 23.9 ± 1.3b 5.44 ± 0.43b 22.6 ± 0.6c 0/4 (0)a Note. Values are means ± standard errors. Values with different superscripts are significantly different from one another ( p < 0.05).
Labeling Index (%)
*
*
0 2 4 6 8 10 12 WT WT: DEN WT: DEN + WY p50 p50: DEN p50: DEN + WY FIG. 2. Effect of Wy-14,643 on hepatocyte proliferation in p50/ and wild-type (WT) mice. Mice were administered diethylnitrosamine (DEN) or saline and then fed a control diet or a diet containing 0.05% Wy-14,643 (WY) for 38 weeks. Six days before euthanasia, mice were administered drinking water containing bromodeoxyuridine (BrdU). Histological sections for the liver were immunohistochemically stained for BrdU, and labeling indexes were determined in hepatocytes to determine the rate of DNA synthesis. Data are means ± standard errors. Values with asterisks are significantly different from their respective controls, using Bonferonni’s test ( p < 0.05).the wild-type mice and further treatment with WY further
increased the tumor incidence, but no tumors were seen in any
of the p50
/ mice. Treatment with WY increased both cell
proliferation and apoptosis in both the wild-type and p50
/
mice; therefore the difference in tumor incidence could not be
attributed to alterations in either of these endpoints.
In the wild-type mice, treatment with DEN induced a low
incidence of tumors. The long-term administration of WY
following DEN treatment resulted in a high tumor incidence in
the wild-type mice. This finding is in agreement with other
studies showing that peroxisome proliferators have promoting
activity in the liver (Borges et al., 1993; Cattley and Popp,
1989; Glauert et al., 1986; Reddy and Rao, 1978). No tumors
were observed in any of the p50
/ mice. Therefore the
presence of intact NF-jB appears to contribute to the
pro-moting activities of peroxisome proliferators. This is the first
in vivo study to show that deletion of an NF-jB subunit results
in the inhibition of tumorigenesis.
WY increased the rate of hepatocyte cell proliferation, which
has been shown previously in several studies. However, no
sig-nificant change was observed between wild-type and p50
/
mice, although cell proliferation was 40% lower in the
p50
/ WY/DEN group than in the wild-type WY/DEN
group. These findings contrast with earlier studies, which imply
that NF-jB is important in the induction of cell proliferation by
peroxisome proliferators. Nilakantan et al. (1998) found that
catalase overexpression decreased both the activation of
NF-jB and the induction of cell proliferation by ciprofibrate in
mice. In studies comparing species that are responsive (rats) or
non-responsive (hamsters) to peroxisome proliferator-induced
carcinogenesis, NF-jB was found to be activated by
peroxi-some proliferators in rats but not in hamsters, which correlated
with the induction of cell proliferation in these two species
(Durnford et al., 1998; Tharappel et al., 2001). In mice treated
with the peroxisome proliferator ciprofibrate for 10 days, cell
proliferation was increased in wild-type mice but inhibited in
p50
/ mice (Tharappel et al., 2003). In the present study,
cell proliferation was only slightly inhibited in the p50
/
mice administered both DEN and WY. However, the mice in
the present study were treated with peroxisome proliferators
for a much longer period of time than the animals in the above
studies; it is likely that hepatocytes from p50
/ in the
present study have been able to overcome the previous
inhibition, by a yet unknown mechanism.
In previous studies, peroxisome proliferators were shown to
inhibit apoptosis in hepatocytes (Lu et al., 2004;
Schulte-Hermann et al., 1995; Tharappel et al., 2003). NF-jB has been
found to have anti-apoptotic activity in several cell types,
including hepatic cell lines, by several agents, including TNF-a
and TGF-b (Barkett and Gilmore, 1999). The deletion of the
p50 subunit was found to result in increased apoptosis in
hepatocytes (Lu et al., 2004; Tharappel et al., 2003). In the
present study, however, apoptosis was not significantly affected
in p50
/ mice compared to wild-type mice (although
apoptosis was lower in p50
/ mice receiving both DEN
and WY compared to wild-type mice receiving the same
treatment), and apoptosis was increased by WY administration.
The mechanism for the differences with previous studies is not
clear. As with cell proliferation, it is likely that long-term
treatment or the age of the animals may be affecting the results.
Also, a different peroxisome proliferator was used in the
present study (WY) than in our previous studies.
Several studies have used genetically modified mice to
examine the role of NF-jB subunits on cell proliferation and
apoptosis in the liver and other tissues. A clear role for NF-jB
in inhibiting apoptosis by TNF-a or other apoptosis inducers in
several cell types, including hepatocytes, has been
demon-strated in studies in which NF-jB activity has been inhibited by
the deletion of one of its subunits, the inhibition of its
translocation, or the expression of a dominant negative form
of IjB (Beg and Baltimore, 1996; Schoemaker et al., 2002;
Vanantwerp et al., 1996; Wang et al., 1996; Xu et al., 1998).
However, DNA synthesis and liver regeneration following
partial hepatectomy or carbon tetrachloride treatment were
not affected by the absence of the p50 subunit. In this latter
study, increased levels of the p65 subunit may have
compen-sated for the lack of p50 (Deangelis et al., 2001). Similarly, the
hepatic-specific expression of a truncated IjBa super-repressor
did not affect DNA synthesis, apoptosis, or liver regeneration
following partial hepatectomy, but led to increased apoptosis
after treatment with TNF-a (Chaisson et al., 2002). Also, the
hepatic inflammatory response after ischemia/reperfusion was
not altered in p50
/ mice (Kato et al., 2002). In the RALA
255–10G rat hepatocyte cell line, expression of an IjB
super-repressor inhibited cell proliferation but not apoptosis by TNF-a
Apoptotic Index (%) 0.0 0.2 0.4 0.6
*
*
WT WT: DEN WT: DEN + WY p50 p50: DEN p50: DEN + WY FIG. 3. Effect of Wy-14,643 on hepatocyte apoptosis in p50/ and wild-type (WT) mice. Mice were administered diethylnitrosamine (DEN) or saline, and then fed a control diet or a diet containing 0.05% Wy-14,643 (WY) for 38 weeks. Histological sections for the liver were used for TUNEL staining, and apoptotic indexes were determined in hepatocytes to determine the rate of apoptosis. Data are means ± standard errors. Values with different asterisks are significantly different from their respective controls, using Bonferroni’s test ( p < 0.05).(Xu et al., 1998). In addition, B cells lacking p50, RelB, or
c-Rel (but not p52 or p65) have decreased proliferation in
response to LPS (Horwitz et al., 1999; Kontgen et al., 1995; Sha
et al., 1995; Snapper et al., 1996a,b). Overall, whether specific
NF-jB subunits are essential for cell proliferation depends on
the tissue and the stimulus for DNA synthesis.
This study shows that the p50 subunit of the NF-jB family is
necessary for the promotion of hepatocarcinogenesis by
peroxisome proliferators. To our knowledge, this represents
the first study to show that deletion of an NF-jB subunit results
in the inhibition of tumorigenesis in vivo. The molecular
mechanisms responsible for these changes, however, are not
clear at this time. We found that Wy-14,643 increased both cell
proliferation and apoptosis, but the changes observed did not
correlate with the effects on tumor induction. This suggests that
NF-jB target genes that regulate proliferation and apoptosis
are not responsible for the inhibition of tumorigenesis. Future
studies will be needed to determine which NF-jB-regulated
genes are responsible for alterations in carcinogenesis that are
induced or promoted by peroxisome proliferators.
ACKNOWLEDGMENTS
This study was supported by National Cancer Institute grant CA74147, National Institute of Environmental Health Sciences grant ES11526, and the Kentucky Agricultural Experiment Station.
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