Diethylnitrosamine-induced hepatocarcinogenesis is ... - Oxford Journals

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Nov 24, 2011 - and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development. Introduction. Liver cancer is ...
Carcinogenesis vol.33 no.2 pp.268–274, 2012 doi:10.1093/carcin/bgr275 Advance Access publication November 24, 2011

Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin:retinol acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration Yohei Shirakami, Max E.Gottesman1 and William S.Blaner Department of Medicine, College of Physicians and Surgeons and 1 Department of Microbiology and Institute of Cancer Research, Institute of Cancer Research, Columbia University, New York, NY 10032, USA  To whom correspondence should be addressed. Department of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA. Tel: þ1 212 305 5429; Fax: þ1 212 305 2801; Email: [email protected]

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

Introduction Liver cancer is a major health care problem worldwide and is the fifth most common diagnosed cancer in men and the second most leading cause of cancer death (1). Hepatocellular carcinoma (HCC) accounts for 70–85% of the primary malignant tumors of the liver (2) and its development is frequently related to chronic inflammation in the liver induced by persistent infection with hepatitis B virus and/or hepatitis C virus (3). Recent epidemiological evidence indicates that the incidence of HCC is rising in developed countries. This is attributed to an increasing prevalence of hepatitis C virus infection and conditions such as non-alcoholic fatty liver disease, which are associated with obesity (4,5). Indeed, the incidence and mortality of HCC in the USA are rapidly increasing (6). Since effective and established chemotherapeutic Abbreviations: ALT, alanine aminotransferase; CYP, cytochrome P450; DEN, diethylnitrosamine; i.p., intraperitoneal; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; KO, knockout; LRAT, lecithin:retinol acyltransferase; MGMT, O6-methylguanine-DNA methyltransferase; mRNA, messenger RNA; PCR, polymerase chain reaction; qRT-PCR, quantitative real-time-polymerase chain reaction; RAR, retinoic acid receptor; RARb, retinoic acid receptor b; WT, wild-type.

agents for HCC are currently unavailable and its recurrence rate is high, the prognosis of HCC is still poor. Diethylnitrosoamine (DEN), also known as N-nitrosodiethylamine, is widely used as a carcinogen in experimental animal models. Upon administration of DEN to mice, either administered orally or by interperitoneal injection, various tumors including ones of the liver, the gastrointestinal tract, skin, the respiratory tract and hematopoietic cells are induced. Many investigators have employed DEN to induce liver tumors in mice by injecting DEN i.p. into weaning mice at 2 weeks after birth, giving rise to hepatic tumors 8 months later (7,8). Since DEN does not itself exert carcinogenicity, it needs to be bioactivated by cytochrome P450 (CYP) enzymes in the liver, resulting in DNA-adducts that form through an alkylation mechanism (9). These alkylation adducts can be removed by a DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), also known as O6-alkylguanine-DNA alkyltransferase (10). Recently, Kang et al. (11) demonstrated that CYP2E1-deficient mice show lower tumor incidence and multiplicity compared with wild-type (WT) mice for DEN-induced hepatocarcinogenesis. This result strongly suggests that CYP2E1 plays an essential role in the activation of DEN, although several other CYP enzymes are proposed to catalyze DEN bioactivation in vivo (9). Retinoids are transcriptional regulators that are essential for mediating cellular proliferation, differentiation and apoptosis (12,13). They are required by the body for maintaining a number of important physiological functions, including normal growth and development, normal vision, healthy immune response, normal reproduction and healthy skin (14). Retinoid metabolism is complex and involves various retinoid forms, including retinyl ester, retinol, retinaldehyde (retinal) and retinoic acid. Retinoid actions within the body are mediated primarily by retinoic acid, which regulates gene expression by acting as a ligand for two distinct nuclear receptor species, the retinoic acid receptors (RARs) and the retinoid X receptors (15). The liver is the main tissue site of retinoid storage in the body and non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver, with .80% of hepatic retinoids and 60% of the entire body’s retinoids being stored in the form of retinyl ester in lipid droplets which are characteristic of HSCs (16). Loss of HSC lipid droplets including their retinyl ester content is one of the first events observed in the development of hepatic disease (16). Lecithin:retinol acyltransferase (LRAT) is the sole enzyme responsible for hepatic retinyl ester synthesis since Lrat knockout (KO) mice completely lack HSC lipid droplets and possess only trace amounts of hepatic retinyl ester (17–19). It has been demonstrated that LRAT protein levels were reduced in various tumors from patients, compared with adjacent normal tissue (20–22). However, Tang et al. (23) reported that overexpression of LRAT in oral basal epithelial cells render them more susceptible to tumorigenesis induced by a carcinogen. An association between LRAT and various malignancies is well documented (20,21), but a specific molecular role for LRAT in carcinogenesis or cancer development remains to be clarified. Because Lrat KO mice have almost no hepatic retinoid stores and since retinoid stores are progressively lost in the development of liver disease (24), these mice represent an attractive model in which to assess the role of hepatic retinoid storage and their loss in HCC development. Thus, we have investigated the effects of endogenous HSC retinoid stores on liver cancer development in WT and Lrat KO mice that had been administered DEN at 15 days of age to induce HCC. Materials and methods Animal maintenance and treatments Animals were housed in a pathogen-free animal facility under a 12 h light/dark cycle at constant temperature and humidity and fed a standard rodent chow and

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water ad libitum. For all of our studies, we employed male WT C57BL/6 mice and Lrat-deficient mice congenic in the C57BL/6 genetic background. Genotypes of mice were determined by a polymerase chain reaction (PCR) protocol described previously (19). Experiments involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Columbia University Institutional Animal Care and Use Committee. For long-term studies of liver tumor development and survival, 15-day-old mice were treated with a single dose of DEN (Sigma–Aldrich) given dissolved in saline at a dose of 25 mg/kg body wt by i.p. injection. Mice in one randomly pre-assigned group were killed 8 months after DEN administration for histological and biochemical analyses. Matched mice in a second group were employed to assess mortality. For short-term studies assessing DEN-induced hepatic injury and compensatory proliferation, mice were treated with DEN through a single i.p. injection and killed at 4, 24 and 48 h after DEN administration. For studies involving 15-day-old mice, a DEN dose of 25 mg/kg body wt was employed. For studies involving 4-month-old mice, a DEN dose of 100 mg/kg body wt was employed. Immunohistochemical and biochemical analyses Liver and tumor tissues were fixed with phosphate-buffered formalin and embedded in paraffin, and the sections were stained with hematoxylin and eosin for histopathological examination. Cell proliferation was assessed by immunohistochemical staining for bromodeoxyrudine (ab6326; Abcam) or Ki-67 (ab15580; Abcam) according to the manufacturer’s instructions. A single i.p. injection of 100 mg/kg bromodeoxyrudine was given to mice 2 h before euthanasia. Hepatic injury was examined by serum alanine aminotransferase (ALT) measurement using an ALT-SL Assay kit (Genzyme), according to the manufacturer’s instructions. Assessment of HCC Immediately after killing, livers were removed, weighed and the numbers of visible tumors on the liver surface were counted macroscopically. The largest lobes were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined microscopically as described previously (7). Protein extraction and western blotting Total hepatic proteins were extracted from 30 mg frozen liver or tumor tissue using 500 ll lysis buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid, 1% NP-40) containing protease inhibitors (Protease Inhibitor Cocktail Set I; Calbiochem) and MiniBeadBeater-1 (BioSpec Products). Proteins (5 lg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nylon membranes (Immobilon-P Transfer Membranes; Millipore). Immunoblots were performed using primary antibodies for p21 (ab7960, 1:1000; Abcam), cytochrome P450 2E1 (ab28146, 1:5000; Abcam), MGMT (ab63930, 1:5000; Abcam) and glyceraldehyde-3-phosphate dehydrogenase (#2118, 1:5000; Cell Signaling Technology). Glyceraldehyde-3-phosphate dehydrogenase was employed as a loading control. Membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare). Each membrane was developed using an enhanced chemiluminescent substrate for the detection of horseradish peroxidase (Thermo Scientific), followed by densitometric scanning using ImageJ. RNA isolation and quantitative real-time-PCR analyses Total RNA was isolated from liver and tumor tissue employing RNeasy Mini Kit (Qiagen) with on-column DNase I-digestion, according to the manufacturer’s instructions. RNA was quantified using a NanoDrop spectrophotometer

(NanoDrop Technologies). Subsequently, complementary DNA synthesis was carried out employing a High-Capacity complementary DNA Reverse Transcription Kit (Applied Biosystems). Sequences of primers used for quantitative real-time-polymerase chain reaction (qRT-PCR) analyses are provided in Table I. For normalization of expression levels, 18S messenger RNA (mRNA) was used. qRT-PCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche) on a LightCycler 480 II instrument (Roche). A dissociation curve program was employed after each reaction in order to verify purity of the PCR products. Statistical analysis Data are expressed as means ± SDs. Differences were analyzed by Student’s t-test and P values ,0.05 were considered significant. Fisher’s exact test was used in comparing incidence of HCC. For overall survival, the log-rank test was used for assessing significance in the Kaplan–Meier analysis.

Results DEN-treated Lrat KO mice show decreased liver tumor development and longer survival than WT mice To investigate whether LRAT and hepatic retinoid storage have roles in liver tumorigenesis, we analyzed the susceptibility of Lrat KO mice to liver carcinogenesis induced by a single i.p. injection of the carcinogen DEN. Macroscopically, nodules were found on the liver surface of both WT and Lrat KO mice 8 months after DEN administration. Representative photographs of the livers of WT and Lrat KO mice are shown in Figure 1A and B, respectively. Histopathological examination revealed that most tumor nodules seen in the livers of the DEN-treated mice were HCC. Representative microscopic images of HCC from the livers of WT and Lrat KO mice are shown in Figure 1C and D, respectively. Upon histological assessment of liver tumors, we found significantly less tumor development in the livers of Lrat KO mice compared with WT mice, characterized by significantly less liver tumor incidence, fewer tumors and smaller tumor size (Figure 1E–G). DEN-treated Lrat KO mice also exhibited significantly less mortality compared with similarly treated WT mice (Figure 1H). Proliferation and apoptosis in liver tumors of 8-month-old DENtreated mice To determine whether LRAT and/or hepatic retinoid storage contribute to liver tumor progression, we studied proliferation and apoptosis in hepatic tumors of WT and Lrat KO mice 8 months after DEN injection. Expression levels of genes for proliferation markers, cyclin D1 and Ki-67; pro-apoptotic markers, caspase-9 and Bax; and antiapoptotic markers, Bcl2 and cFLIP, in liver tumors were analyzed by qRT-PCR 8 months after DEN-induction of hepatic carcinogenesis. We detected no significant differences of the expression levels for any of these genes between tumors present in WT and Lrat KO mice (Figure 2A). In addition, there was no significant difference in the level of proliferation observed between tumors of WT and Lrat KO mice, as assessed by Ki-67 staining (Figure 2B).

Table I. Primers used for qRT-PCR analyses Genes

GenBank accession no.

Forward primers

Reverse primers

Bax Bcl2 Caspase-9 cFLIP Cyclin D1 CYP1A1 CYP1A2 CYP26A1 CYP2E1 Ki-67 MGMT p21 RARb 18S

NM_007527 NM_009741 NM_015733 NM_009805 NM_007631 XM_972645 NM_009993 NM_007811 NM_021282 XM_001000692 NM_008598 NM_007669 NM_011243 NR_003278

5#-CCAGGATGCGTCCACCAAGAA-3# 5#-GTACCTGAACCGGCATCTG-3# 5#-ACACACAGGAGAGAGAGGAGGTTA-3# 5#-TCCAGAAGTACACCCAGTCCA-3# 5#-TCCCAGACGTTCAGAACC-3# 5#-CTCTTTGGAGCTGGGTTTGACAC-3# 5#-TGACACAGTCACCACAGCCATCA-3# 5#-AGAGCAATCAAGACAACAAGTTAG-3# 5#-TGAATATGCCCTACATGGACGCTG-3# 5#-AAACACAGTGCAAACTCCTAA-3# 5#-AGATGGAGCTGTCTGGCTGT-3# 5#-CAAGTTCAAGTGGGAATATAGCAGA-3# 5#-GGTTCCTTGCCACTTCTT-3# 5#-CCATCCAATCGGTAGTAGCG-3#

5#-CTCTGCAGCTCCATATTGCTGT-3# 5#- GGGGCCATATAGTTCCACAA-3# 5#-GGGCCTTCCTGGCCTGATAC-3# 5#-CACTGGCTCCAGACTCACC-3# 5#-AGGGCATCTGTAAATACACT-3# 5#-AGGGTTGGTTACCAGGTACATGAG-3# 5#-CATGGATCTTCCTCTGCACGTT-3# 5#-ATCGCAGGGTCTCCTTAAT-3# 5#-GTGTCTCGGGTTGCTTCGTG-3# 5#-AACTTGCTCACACTCGAT-3# 5#-CCTCTGTGGGGTCAGTGTTT-3# 5#-GAGTCGGGATATTACGGTTG-3# 5#-ACTGACTGACTCCACTGTT-3# 5#-GTAACCCGTTGAACCCCATT-3#

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Fig. 1. Lrat KO mice exhibit decreased liver tumor development and longer survival compared with matched WT mice. (A and B) Livers of 8-month-old DENtreated WT (A) and Lrat KO (B) mice showing differences in tumor development. (C and D) hematoxylin and eosin staining of normal liver and tumor tissue of DEN-treated 8-month-old WT (C) and Lrat KO (D) mice. (Scale bar, 100 lm. T, tumor; N, non-tumorous tissue adjacent to a tumor) (E) Incidence of HCC in WT (n 5 29) and Lrat KO mice (n 5 18) 8 months after DEN (25 mg/kg body wt) administration. The asterisk denotes statistically significant differences (P , 0.05; Fisher’s direct test) relative to WT mice. (F) Number of tumors (.0.5 mm) in livers of WT and Lrat KO mice 8 months after DEN administration. The asterisk denotes statistically significant differences (P , 0.05; Student’s t-test) relative to WT mice. (G) Maximum tumor size (diameter) for WT and Lrat KO mice. (H) Kaplan–Meier survival curve for WT (n 5 13) and Lrat KO mice (n 5 10) injected with DEN at 15 days of age (P 5 0.0048; log-rank test for significance).

Fig. 2. Expression of markers of cell proliferation and apoptosis in liver tumors of 8-month-old DEN-treated WT and Lrat KO mice. (A) Levels of mRNA for cyclin D1, Ki-67, Bax, caspase-9, Bcl2 and cFLIP in liver tumors of WT (n 5 4) and Lrat KO mice (n 5 6) 8 months after DEN administration (25 mg/kg body wt) analyzed by qRT-PCR. (B) Proliferation in adjacent normal liver tissue and liver tumors of 8-month-old DEN-treated WT and Lrat KO mice was assessed by counting the number of positive cells stained immunohistochemically for Ki-67.

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DEN-induced acute hepatic injury and compensatory proliferation We next focused on the tumor initiation stage and investigated shortterm responses elicited by DEN administered to 15-day-old WT or Lrat KO mice. Compared with WT mice, Lrat KO mice showed significantly less hepatic injury after DEN administration, as evidenced by lower serum ALT levels (Figure 3A). Lrat KO mice also displayed lower hepatic mRNA expression of cyclin D1 and fewer Ki67-positive cells after DEN administration compared with WT mice (Figure 3B and C). We examined hematoxylin- and eosin-stained histology for livers from both WT and Lrat KO mice 0, 24 and 48 h after DEN injection but were unable to detect any hepatic lesions at these early times. The literature indicates that initial hepatic injury and the compensatory proliferation that immediately follows injury are key factors for allowing tumor formation in DEN-induced carcinogenesis (7,25). Hence, we also investigated the extent of liver injury and the possibility of increased cell proliferation in the livers of WT and Lrat KO mice in a second short-term study involving the administration of a high-dose of DEN (100 mg/kg body wt) to 4-month-old adult male mice. These mice were killed 24 and 48 h after DEN treatment. This study provided similar results to the one involving 15-day-old mice: Lrat KO mice showed lower serum ALT levels, decreased hepatic mRNA expression of cyclin D1, and significantly fewer proliferating cells positive for bromodeoxyrudine after DEN administration compared with WT mice (data not shown).

DEN-induced effects on expression levels of retinoic acid-responsive genes Since retinoid metabolism is dysregulated in the livers of Lrat KO mice (17), expression levels of retinoic acid-responsive genes were analyzed. Surprisingly, the mRNA levels of several retinoic acid-responsive genes, including CYP26A1, retinoic acid receptor b (RARb) (the probe employed detected all RARb isoforms) and p21, were significantly elevated in the livers of Lrat KO mice, both before and after DEN administration compared with WT mice (Figure 4A–C). Protein levels of p21 in the livers of Lrat KO mice increased after DEN administration and were also significantly higher than those of WT mice for short-term DEN treatment (Figure 4D). Similar results were obtained in a second short-term study using high-dose DEN (100 mg/kg body wt) injected i.p. into 4-month-old adult mice (data not shown). Expression levels of CYP enzymes involved in hepatic DEN bioactivation To gain insight into whether LRAT and hepatic retinoid storage alter DEN metabolism in the liver, we analyzed expression levels for a number of CYP enzymes proposed to be important in catalyzing hepatic DEN bioactivation. For this purpose, 15-day-old WT or Lrat KO mice were given an i.p. injection of DEN and killed 4, 24 and 48 h later. The mRNA levels of CYP1A1, CYP1A2 and CYP2E1 were

Fig. 3. Effect of LRAT deficiency on hepatic injury and cell proliferation. Fifteen-day-old WT and Lrat KO mice (n 5 4–6 for each mouse strain and time point) were injected with DEN (25 mg/kg body wt) and killed 24 and 48 h later. (A) ALT levels in serum of WT and Lrat KO mice. (B) Hepatic levels of cyclin D1 mRNA were analyzed by qRT-PCR for WT and Lrat KO mice. (C) Hepatocyte proliferation in the liver of DEN-injected WT and Lrat KO mice was assessed by counting the number of positive cells upon immunohistochemical staining for Ki-67.  P , 0.05.

Fig. 4. Influence of LRAT deficiency on hepatic expression of retinoic acid-responsive genes in DEN-treated WT and Lrat KO liver. Fifteen-day-old WT and Lrat KO mice (n 5 4–6 for each mouse strain and time point) were injected with DEN (25 mg/kg body wt) and killed 4, 24 and 48 h later. Temporal changes in mRNA expression for the retinoic acid-responsive genes CYP26A1 (A), RARb (B) and p21 (C) were determined for DEN-treated WT and Lrat KO livers by qRT-PCR. (D) p21 protein level was determined by western blotting with protein samples extracted from the liver of mice before and after DEN administration. Upper panel provides a representative western blot where the individual lanes in the blot correspond directly to the bars immediately below the bar graph. The bar graph shows the mean intensities of the protein signals for p21.  P , 0.05.

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determined by qRT-PCR and CYP2E1 protein levels were analyzed by western blotting. In the livers of Lrat KO mice, we detected significantly lower levels of mRNA for CYP1A1 and CYP1A2 at 4 h after DEN injection (data not shown), and for CYP2E1, the predominant CYP responsible for DEN bioactivation (11), at 4, 24 and 48 h after DEN injection (Figure 5A) compared with WT mice. In addition, protein levels of CYP2E1 were significantly lower in the livers of Lrat KO mice compared with WT mice (Figure 5B). Expression levels of MGMT in the liver To investigate whether LRAT and hepatic retinoid storage also may affect DNA repair, we analyzed expression of the DNA repair gene MGMT using 15-day-old WT and Lrat KO mice that were killed in 4, 24 and 48 h after DEN administration by i.p. injection. mRNA and protein levels of MGMT were analyzed by real-time PCR and western blotting, respectively. We detected significantly elevated levels of both MGMT mRNA and protein in the livers of Lrat KO mice relative to that of WT mice at 4 and 24 but not 48 h after DEN injection (Figures 5C and D). Since MGMT protein levels are known to be highly correlated with MGMT enzymatic activity (26), we did not assess MGMT enzymatic activity. Discussion Retinoids are potent modulators of cellular activities, including cell proliferation, differentiation and apoptosis as well as on suppressing development of malignancy in many organs including the liver (12). The relationship between retinoids and cancer has been extensively investigated and many studies have demonstrated that retinoid-deficiency causes an increase in the number of spontaneous and chemically induced tumors in animals (12,27). It has been well established that retinoids exert therapeutic and chemopreventive effects in patients with acute promyelotic leukemia (12,28) and leukoplakia, premalignant lesions of head and neck cancer (12). Clinical studies have demonstrated that administration of acyclic retinoid, a synthetic retinoid, reduces the incidence of post-therapeutic HCC recurrence and thereby improves survival of patients with HCC (29,30). In addition, a progressive

reduction of hepatic retinoid stores has been noted for patients at higher risk for the development of HCC that cannot be accounted for by poor dietary retinoid intake (24). Earlier, we reported preliminary evidence of decreased DEN-induced liver carcinogenesis in Lrat KO mice which totally lack retinoid stores and lipid droplets in HSCs (31). This result, which was contrary to our expectations, showed that lower hepatic retinoid stores are associated with decreased chemically induced hepatocarcinogenesis. This suggested that endogenous retinoid stores allow for the development of DEN-induced hepatic tumors since WT mice had significantly more tumors, both larger tumors and with greater incidence than Lrat KO mice. We were intrigued by this and undertook the present studies to identify the mechanisms that underlie this surprising finding. In the present work, we also found less susceptibility in Lrat KO mice compared with WT mice toward liver tumor development induced by DEN (see Figure 1). Our data indicate that Lrat KO mice experience less hepatic injury, cell proliferation and cancer initiation at very early stages of liver cancer development, within 2 days after DEN administration. Several published studies, which similarly use DEN to induce liver cancer, have shown that acute hepatic injury and compensatory proliferation in the early stages of carcinogenesis are significantly correlated with cell proliferation and development of liver tumors at later times after DEN administration (7,25,32). However, our results showed no significant differences of proliferation and apoptosis in the livers of WT and Lrat KO mice killed 1, 4 and 8 months after DEN administration (data not shown) as well as no significant differences in tumor proliferation and apoptosis among the two mouse strains 8 months after DEN administration. These results imply that LRAT deficiency does not affect tumor progression at later stages. This may be because LRAT expression is established to be very reduced or lost in advanced tumors (20–22). Therefore, we reasoned that only the early stages for cancer development may be affected by the absence of LRAT, suggesting that these early stages of cancer initiation can strongly impact liver tumor load induced by DEN administration. Thus, we assessed the initiation of DEN-induced hepatocarcinogenesis using 15-day-old mice receiving a DEN dose of 25 mg/kg body wt, exactly the same dosage used to initiate HCC

Fig. 5. Expression levels of CYP2E1 and MGMT in the livers of WT and Lrat KO mice. Fifteen-day-old WT and Lrat KO mice (n 5 4–6 for each mouse strain and time point) were injected with DEN (25 mg/kg body wt) and killed 4, 24 and 48 h later. Levels of mRNA for CYP2E1 (A) and MGMT (C) were analyzed by qRTPCR for WT and Lrat KO livers. CYP2E1 (B) and MGMT (D) protein levels were determined by western blotting of hepatic protein obtained from WT and Lrat KO mice before and after DEN administration. The upper panel provides a representative western blot where the individual lanes in the blot correspond directly to the bars immediately below in the bar graph. The bar graph shows the mean intensities of the protein signals.  P , 0.05.

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development in long-term studies. Many published studies have employed young adult (4 months old) mice receiving a DEN dose of 100 mg/kg body wt as an alternative setting for short-term study in order to assess acute hepatic injury and cancer initiation (7,31,32). We undertook both types of short-term studies: one using 15-day-old mice given a DEN dose of 25 mg/kg of body wt and another using 4-month-old mice given a DEN dose of 100 mg/kg of body wt. Both studies showed similar results with regard to a number of parameters, specifically measures of hepatic injury, proliferation and retinoic acidresponsive gene expression. But other parameters were different between the two studies, specifically levels of CYP expression and MGMT levels. Unlike DEN-treated 15-day-old mice, 4-month-old mice do not demonstrate significant differences of CYP enzymes, including CYP1A1, 1A2 and 2E1 (both mRNA and protein) and MGMT levels between WT and Lrat KO mice for any time after DEN injection (data not shown). These findings suggest that it may not be appropriate to employ adult mice (4-month-old mice) and a higher dose of DEN for short-term studies of the factors/mechanisms underlying DENinduced HCC since the hepatic responses of 15-day-old and 4month-old mice to these i.p. injections of DEN are not identical. Several key findings from this study could account for our observations of lessened hepatocarcinogenesis in Lrat KO mice compared with WT mice. First, Lrat KO mice showed increased retinoid-responsive signaling in the liver and this could have contributed to suppressing hepatocarcinogenesis. Higher levels of retinoic acid-responsive genes, such as CYP26A1, RARb and p21, indicate increased retinoid signaling. Although other investigators have previously reported elevated expression of CYP26A1 in Lrat-deficient mice (31,33), we are the first to demonstrate enhanced retinoid signaling indicated by upregulation of CYP26A1, RARb and p21 both before and after DEN treatment of 15-day-old Lrat KO mice, specifically for mice in the C57BL/6 genetic background. A reason that may be put forward to explain why Lrat KO mice might possess increased retinoid signaling is because these mice may have higher levels of bioactive retinoid forms, like retinoic acid, owing to the lack of conversion of retinol into retinyl esters in the absence of LRAT and enhanced conversion of retinol to retinoic acid (17,31,33). For suppressing liver tumorigenesis in Lrat KO mice, p21 and RARb have critical roles among various retinoic acid-responsive genes. The cyclin-dependent kinase inhibitor p21, also known as p21WAF1/Cip1, is regulated by retinoic acid (34–36) and exerts anti-proliferative effects by promoting cell cycle arrest (37). RARb is also retinoic acid inducible and RARb2 is recognized to act as a tumor suppressor, being the predominant retinoid receptor, which mediates the inhibitory effects of retinoic acid on cell proliferation (38,39). A second explanation as to why we observed lessened hepatocarcinogenesis in Lrat KO mice is that Lrat KO mice exhibit decreased expression levels of CYP enzymes for DEN bioactivation (see Figure 5). In order for DEN to exert its full carcinogenic potency, DEN needs to be activated by CYP enzymes (9). This gives rise to production of DNA-adducts and cancer initiation. The specific CYP enzymes implicated in DEN bioactivation include CYP1A1, CYP1A2 and CYP2E1 (40). CYP2E1 is thought to be the predominant enzyme involved in the bioactivation of DEN in vivo since CYP2E1-deficient mice show significantly lower tumor incidence and multiplicity compared with WT mice upon DEN-induced hepatocarcinogenesis (11). Our data obtained from Lrat KO mice show lower levels of these DEN-activating enzymes (see Figure 5). This indicates that the livers of Lrat KO mice undergo less DEN activation compared with that of WT mice. This results in less DNA-adduct formation and suppressed liver cancer initiation. CYP2E1 is proposed to be the principal enzyme responsible for alcohol-enhanced catabolism of retinoic acid in alcoholic liver (33). Our data establish that the increased retinoid signaling observed in Lrat KO mice liver does not affect CYP2E1 level at any time after treatment (see Figure 5). Moreover, CYP2E1 expression was not induced by DEN administration per se. Our data do not allow us to understand why CYP2E1 expression is diminished in the livers of Lrat KO mice. To assess this, more detailed experiments are required if we are to show

a relation among retinoid metabolism, CYP2E1 expression and DEN metabolism. In addition to less DEN bioactivation, we also observed increased hepatic levels of the DNA repair enzyme MGMT in Lrat KO mice. This increased expression of MGMT probably also contributes to the observed attenuated initiation of liver cancer induced by DEN in Lrat KO mice. MGMT is established to be a DNA repair protein, which protects the cellular genome and critical oncogenic genes from the mutagenic action of endogenous and exogenous alkylating agents such as DEN (41,42). The enzyme removes alkyl adducts, such as the ethyl adducts generated from bioactivation of DEN, from the O6-position of guanine in DNA (43,44). Epigenetic inactivation of MGMT by promoter CpG island hypermethylation is implicated in various human cancers, including HCC (10,45). A relation between retinoid signaling and epigenetic gene silencing has been well documented (46,47). There also is a published report showing that MGMT expression is increased by treatment with a synthetic retinoic acid derivative N-(4-hydroxyphenyl) retinamide (also known as fenretinide) (43). This suggests an association between retinoid signaling and MGMT levels. Altered epigenetic regulation by retinoids evidently plays a role in determining expression levels of MGMT. Paradoxically, it has been established from clinical observations that LRAT expression is very reduced or lost in advanced tumors and based on these observations, it has been suggested that LRAT loss may contribute to cancer development (20–22). Our studies focus on LRAT actions early in the cancer initiation stage, not on later stages reflected in human tumor tissues, and we observed a significant beneficial effect of LRAT absence on hepatocarcinogenesis. We found that absence of LRAT plays a pivotal role for inhibiting hepatocarcinogenesis mainly by enhancing retinoic acid signaling, which is evidenced by an upregulation in CYP26A1 and RARb expression. Both these genes are known to be directly responsive to retinoic acid transcriptional regulation and both are normally taken as markers for enhanced retinoic acid signaling. Thus, retinoic acid signaling in normal tissue is enhanced due to LRAT absence. However, in advanced tumor tissue, LRAT expression is reduced due to diminished retinoid signaling. It is well established in the literature that LRAT expression is regulated by retinoic acid transcriptional activation (48). Several reports have shown that dominant-negative retinoid X receptoralpha expression impairs retinoid signaling in liver tumors (49) and that expression of a dominant-negative form of retinoic acid receptor-alpha is associated with hepatic tumorigenesis (50). Thus, tumors display diminished retinoic acid signaling. This difference in responses between healthy and cancerous liver can also be observed for other parameters. For instance, cyclin D1 levels are significantly reduced in livers of 15-day-old Lrat KO mice shortly after DEN administration (Figure 3B), whereas cyclin D1 levels are elevated 8 months after DEN treatment (Figure 2A). We propose that reduced LRAT expression in advanced tumors is a consequence of decreased retinoic acid signaling arising from reduced hepatic levels of retinoic acid and retinoic acid signaling that are secondary to tumor development. In conclusion, despite an absence of retinoid storage in the liver, Lrat KO mice exhibited suppressed DEN-induced liver tumor development. The mechanisms that underlie this involve: (i) higher hepatic levels of p21 and RARb which suppress cell proliferation and carcinogenesis; (ii) less conversion of DEN to its active metabolites owing to decreased levels of CYP enzymes needed for the bioactivation of DEN resulting in less DNA-adduct formation and cancer initiation and (iii) increased hepatic levels of MGMT giving rise to decreased DNA-adduct formation and less carcinogenesis. Funding National Institutes of Health (RC2 AA019413, R01 DK68437, R01 DK079221). Conflict of Interest Statement: None declared.

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