Thyroid hormone receptor ligands induce regression of rat preneoplastic liver lesions causing their reversion to a differentiated phenotype

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Triiodothyronine (T3), through interaction with its intracellular thyroid hormone receptors (TRs), influences various physiological functions, including metabolism, development, and growth. We investigated the effect of T3 and the selective TR-β agonist GC-1 in two models of hepatocarcinogenesis. Preneoplastic lesions were induced in F-344 rats via a single dose of diethylnitrosamine, followed by a choline-deficient (CD) diet for 10 weeks. Rat subgroups were then fed the CD diet or a CD diet containing either 4 mg/kg T3 or 5 mg/kg GC-1 for another week. Rats fed a CD diet alone showed a large number (65/cm2) of preneoplastic lesions positive for the placental form of glutathione S-transferase (GSTP). Coadministration of T3 for the last week caused an almost complete disappearance of the foci (3/cm2). A reduction of GSTP-positive foci was also observed in rats fed a CD + GC-1 diet (28/cm2 versus 75/cm2 of rats fed a CD diet alone) in the absence of significant differences in labeling or apoptotic index of preneoplastic hepatocytes between the two groups. An antitumoral effect of GC-1 was also observed with the resistant hepatocyte model of hepatocarcinogenesis. Nodule regression was associated with a return to a fully differentiated phenotype, indicated by the loss of the fetal markers GSTP and gamma glutamyl transpeptidase, and reacquisition of the activity of glucose 6-phosphatase and adenosine triphosphatase, two enzymes expressed in normal hepatocytes. Conclusion: Our results indicate that activated TRs negatively influence the carcinogenic process through induction of a differentiation program of preneoplastic hepatocytes. The results also suggest that TRs could be a meaningful target in liver cancer therapy. (HEPATOLOGY 2009.)

The adult liver is a quiescent organ characterized by an extremely low replicative activity, mitosis being observed only in 1/20,000 hepatocytes.1 However, hepatocytes begin to proliferate in a relatively synchronized manner in response to a reduction in liver mass caused by partial surgical removal of the liver or after chemical, nutritional, vascular, and viruses-induced liver injury (compensatory regeneration or compensatory hyperplasia). Several lines of evidence suggest that conditions characterized by liver damage and enhanced hepatocyte turnover are associated with an increased incidence of hepatocellular carcinoma (HCC) in humans as well as experimental animals.2–4 Risk factors include chronic infection with hepatitis B virus and hepatitis C virus, aflatoxin B1, hemochromatosis, alcohol, and so forth.5–7 All these evidences have led to the hypothesis,8 although controversial,9 that cell proliferation per se may be carcinogenic, and carcinogens that increase cell proliferation may be operating exclusively by this mechanism. On the other hand, triiodothyronine (T3), a potent hepatomitogen that causes liver hyperplasia in the absence of cell death,10, 11 unlike other proliferative stimuli, induces a rapid regression of carcinogen-induced hepatic nodules, and reduces the incidence of HCC and lung metastasis.12 Notably, the antitumoral property of T3 appears to be specific and not shared by other liver mitogens, such as ciprofibrate, a member of the class of peroxisome proliferators.13 These findings raise the important issue as to whether activation of thyroid hormone receptor (TR) may play a critical role in the inhibition of liver cancer progression. Therefore, in this study we examined the effect of T3 on the fate of preneoplastic lesions induced in rats by a nutritional model consisting of a choline-deficient (CD) diet. Chronic feeding of a CD diet in rodents is known to cause fatty change, hepatocyte injury, fibrosis, cirrhosis, oxidative DNA damage via 8-hydroxydeoxyguanosine generation, and HCC.14 Moreover, feeding a CD diet to rats following a single initiating dose of a chemical carcinogen enhances both the induction of enzyme-altered preneoplastic foci and the subsequent progression of the foci to HCC.15 Notably, the CD model, similar to that seen in humans, is characterized by an initial phase consisting of hepatic steatosis that can progress to steatohepatitis, a state characterized by necroinflammatory changes consisting of ballooning degeneration and apoptosis of hepatocytes; this in turn elicits inflammatory and eventually fibrogenic responses that can lead to development of cirrhosis and HCC, which has similarities in its histopathological sequence to human HCC development with cirrhosis.16

Because T3-based therapies often result in undesired side effects, particularly cardiac dysfunctions such as tachycardia, arrhythmias, and precipitation of ischemic episodes or heart failure,17 another important issue raised in the present study was whether the possible antitumoral effect of T3 could be achieved also by new thyroid hormone analogs devoid of the cardiac effects of thyroid hormones, such as GC-1.18

Abbreviations

2-AAF, 2-acetylaminofluorene; AI, apoptotic index; ATPase, adenosine triphosphatase; BrdU, bromodeoxyuridine; CD, choline-deficient; CS, choline-supplemented; DENA, diethylnitrosamine; G6Pase, glucose 6-phosphatase; GGT, gamma glutamyl transpeptidase; GSTP, placental glutathione S-transferase; HCC, hepatocellular carcinoma; PH, partial hepatectomy; R-H, resistant hepatocyte; T3, triiodothyronine; TR, thyroid hormone receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Materials and Methods

Animals.

Male Fischer F-344 rats (100-125 g) purchased from Charles River (Milan, Italy) were maintained on a laboratory diet (Ditta Mucedola, Milan, Italy). The animals were given food and water ad libitum with a 12-hour light/dark cycle and were acclimatized for 1 week before the start of the experiment. Guidelines for the Care and Use of Laboratory Animals were followed during the investigation. Diethylnitrosamine, 3,5,3'-triiodo-L-thyronine and 2-acetylaminofluorene (2-AAF) were purchased from Sigma (Sigma Chemical Co., St Louis, MO). The CD diet was prepared according to the original recipe.19 A choline-supplemented (CS) diet, which was identical to the CD diet but supplemented with a normal content of choline, was used as a control diet.19 GC-1 was synthesized according to Chiellini et al.18

Experimental Protocols.

In Experiment I (Fig. 1), rats were injected intraperitoneally with a single dose of diethylnitrosamine (DENA), dissolved in saline, at the dose of 150 mg/kg body weight. Following a 2-week recovery period, rats were fed a CD diet for 10 weeks and then divided into three groups: (1) group 1 was maintained on a CD diet for an extra week; (2) group 2 was fed a CD diet containing 4 mg/kg T3 for 1 week and sacrificed on day 7; (3) group 3 was fed a CD diet for an extra week and then placed on a CS diet for another week. An additional group of DENA-treated rats was fed a CS diet throughout the experimental period. In Experiment II (Fig. 1), rats were treated essentially as in Experiment I, except that group 2 was cofed a CD diet supplemented with GC-1 (5 mg/kg diet) instead of T3. In Experiment III (Fig. 1), 2 weeks after DENA administration, rats were treated according to the resistant hepatocyte (R-H) model,20 consisting of feeding a basal diet containing 0.02% 2-AAF for 1 week followed by a standard two-thirds partial hepatectomy (PH), and feeding of the 2-AAF–containing diet for an additional week. The rats were then returned to the basal diet for 5 weeks and divided into two groups; group 1 was continued on the basal diet for 7 and 14 days, whereas group 2 was fed a diet containing 5 mg/kg of GC-1 for 7 or 14 days.

Figure 1.

Schematic representation of the experimental protocol (see Materials and Methods for details). CD + GC-1, CD supplemented with GC-1 (5 mg/kg diet); CD + T3, CD diet supplemented with T3 (4 mg/kg diet).

Histology, Histochemistry, and Immunohistochemistry.

Liver sections were fixed in 10% formalin, embedded in paraffin, and routinely stained with hematoxylin-eosin or for immunohistochemical detection of bromodeoxyuridine (BrdU) and the placental form of glutathione S-transferase (GSTP). Other liver slices were immediately frozen in liquid nitrogen precooled isopentane at approximately −140°C and stored at −80°C. Serial sections were cut in a cryostat (Leitz 1720), mounted onto slides, and stained for gamma glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose 6-phosphatase (G6Pase), and GSTP.21, 22

Double-Labeling of BrdU and GSTP-Positive Hepatocytes in DENA-Initiated Rat Liver.

BrdU incorporation into nuclei was determined in formalin-fixed sections using an anti-BrdU monoclonal antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) and Dako Envision peroxidase mouse (K4001; Dako Corporation, Carpinteria, CA). Briefly, tissue sections were exposed to 0.3% hydrogen peroxide in ethanol for 10 minutes to block endogenous peroxidase, treated with 2N HCl, incubated with trypsin 0.1% for 20 minutes, and then with normal goat serum for 20 minutes at room temperature. The sections were then incubated for 2 hours with an anti-BrdU monoclonal antibody, followed by peroxidase goat anti-mouse immunoglobulin G. The sites of peroxidase binding were detected with diaminobenzidine. A segment of the duodenum, an organ with a high rate of cell proliferation, was included from each rat to confirm delivery of the DNA precursor.

The location of GSTP in the liver was determined by using an anti-rat GSTP polyclonal antibody (MBL, Nagoya, Japan) and Dako Envision alkaline phosphatase goat anti-rabbit (K4018, DAKO Corporation, Carpinteria, CA). The sites of phosphatase binding were detected with BCIP/NBT substrate system (K598, Dako Corporation).

Measurement of GSTP-Positive Foci.

GSTP-positive foci were measured with a computer-assisted image processor, programmed for the three-dimensional calculation according to Abramoff et al.23 Only foci greater than 76 μm in diameter were measured.

Determination of Labeling Index and Acidophilic Body Index in GSTP-Positive Preneoplastic Foci.

The incidence of BrdU-labeled hepatocytes was determined within randomly selected GSTP-positive foci. The labeling index was calculated as BrdU-positive hepatocyte nuclei/100 hepatocyte nuclei. At least 3,500 hepatocyte nuclei per rat were scored. The incidence of acidophilic bodies within randomly selected preneoplastic lesions was determined in hematoxylin-eosin–stained sections by scoring 3,000 to 4,000 preneoplastic hepatocytes as described.12 Only acidophilic bodies containing nuclear fragments were recorded. The acidophilic body index was calculated as the number of acidophilic bodies per 100 hepatocytes.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Assay.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit–POD (cod. 817 910; Roche Diagnostics, Mannheim, Germany).

Western Blot Analysis.

Total cell extracts were prepared from frozen livers and kidneys as described.11 For immunoblot analysis, equal amounts (from 100 to 150 μg/lane) of proteins were electrophoresed on 12% or 8% sodium dodecyl sulfate polyacrylamide gels (Sigma Chemical Co.). After gels electrotransfer onto nitrocellulose membranes (Osmonics, Westborough, MA) to ensure equivalent protein loading and transfer in all lanes, the membranes and the gels were stained with 0.5% (wt/vol) Ponceau S red (ICN Biomedicals, Irvine, CA) in 1% acetic acid, and with Coomassie blue (ICN Biomedical) in 10% acetic acid, respectively. After blocking in TBS-T + 5% nonfat dried milk, membranes were washed in TBS-T and then incubated with the appropriate primary antibodies. Depending on the origin of primary antibody, filters were incubated with anti-mouse or anti-rabbit horseradish peroxidase–conjugated immunoglobulin G (Santa Cruz Biotechnology, CA). Immunoreactive bands were identified with a chemiluminescence detection system (Supersignal Substrate; Pierce, Rockford, IL).

Antibodies.

Primary antibodies used in this study were: mouse monoclonal antibodies against caspase-9 (C-9) (Cell Signalling Technology, MA); actin (clone AC40; Sigma Chemical Co.), Bax (B-9) and Bcl-xL (H-5) (Santa Cruz Biotechnology); and rabbit polyclonal antibody against GSTP (MBL, Nagoya, Japan).

Statistical Analysis.

Instant software (GraphPad Instat, San Diego, CA) was used to analyze the data. The results of multiple observations were presented as the mean ± standard error of at least two separate experiments. A P value of < 0.05 was regarded as a significant difference between groups.

Results

Effect of Treatment with T3 on DENA + CD-Induced GSTP-Positive Foci.

Feeding a CD diet to rats following administration of a single initiating dose of a chemical carcinogen promotes the development of foci of enzyme-altered preneoplastic hepatocytes and their subsequent progression to HCC.12 Accordingly, whereas an insignificant number of GSTP-positive foci (1.8/cm2) developed in rats fed a CS diet for 11 weeks, a CD diet for the same length of time resulted in a high number of such foci (65/cm2), with approximately 5% of the liver being positive for GSTP staining (Fig. 2A,B). Cofeeding T3 with the CD diet for the last week of treatment dramatically reduced the number of preneoplastic foci (3.2/cm2 ± 1.7). T3 cofeeding also caused a striking reduction in the percentage of the area occupied by GSTP-positive hepatocytes (0.25% versus 5.1% of CD alone), with no change in the mean area of GSTP-positive foci (Fig. 2A). According to our previous results,24 whereas livers from CD diet–fed rats exhibited extensive steatosis compared with CS-fed animals (Fig. 2B), cofeeding T3 for 1 week following 10 weeks of a CD diet caused an almost complete disappearance of fat accumulation, which was associated with a normal ratio liver weight/body weight (Fig. 2A,B). To investigate the possibility that the disappearance of preneoplastic hepatocytes could be a consequence of T3-induced accelerated removal of triglycerides and the associated liver damage, DENA-initiated rats were fed a CD diet for 11 weeks and subsequently placed on a CS diet for an extra week. The results (Fig. 2A,B) revealed that whereas shifting the CD diet–fed rats to a CS diet for 1 week resulted in a rapid regression of fat accumulation, no decrease occurred in the number of GSTP-positive foci (65.2 in CD-fed rats versus 63.3 of rats fed a CD diet for 11 weeks and then placed on a CS diet for 1 week). The respective mean GSTP-positive areas were 0.07 versus 0.06, and the percentage of GSTP-positive areas were 5.10 versus 4.13. These results clearly indicate that the disappearance of GSTP-positive foci caused by T3 is not the consequence of accelerated fat removal from the liver.

Figure 2.

(A) Effect of T3 on the number and size of preneoplastic GSTP-positive foci in the liver of rats initiated with DENA. (B) Photomicrographs of liver from rats treated with DENA + CS, DENA + CD, DENA + CD + T3, and DENA + CD + CS. Rats were injected intraperitoneally with a single dose of DENA (150 mg/kg). After 2 weeks, rats were placed on a CD diet for 10 weeks. The animals were then divided into three groups: group 1 continued on a CD diet for an extra week; group 2 was fed a CD diet containing 4 mg/kg of T3 for 1 week; and group 3 was fed a CD diet for 11 weeks, then placed on a CS diet for an extra week. Controls received DENA and were fed a CS diet for 11 weeks. Sections were stained for GSTP and counterstained with hematoxylin (magnification ×4). (C) Effect of GC-1 on the number and size of preneoplastic GSTP-positive foci in the liver of rats initiated with DENA. (D) Photomicrographs of liver from rats treated with DENA + CS, DENA + CD, and DENA + CD + GC-1. Rats were injected intraperitoneally with a single dose of DENA (150 mg/kg). After 2 weeks, rats were placed on a CD diet for 10 weeks. The animals were then divided into two groups: group 1 continued on a CD diet for an extra week, while group 2 was shifted to a CD diet containing 5 mg/kg of GC-1 and sacrificed 1 week later. Controls received DENA and were fed a CS diet for 11 weeks. Sections were stained for GSTP and counterstained with hematoxylin (magnification ×4).

Effect of Treatment with GC-1 on DENA + CD-Induced GSTP-Positive Foci.

The above results, together with previous findings,12 suggest that T3 exerts an anticarcinogenic effect. Because T3-based therapies often result in undesired side effects, particularly cardiac dysfunction (tachycardia, arrhythmias, and precipitation of ischemic episodes or heart failure),17 we felt it was important to investigate whether the anticarcinogenic effect observed with T3 could also be achieved using new thyroid hormone analogs devoid of the cardiac effects of thyroid hormones, such as GC-1.18 Therefore, experiments were performed in which DENA-initiated rats were fed a CD diet for 10 weeks followed by feeding for an additional week a CD diet supplemented with 5 mg/kg of GC-1. Similar to that shown in the previous protocol, administration of a single initiating dose of DENA alone did not induce a significant number of GSTP-positive foci (Fig. 2C,D). On the other hand, subsequent feeding of a CD diet for 11 weeks resulted in a much higher number of such foci (74.6/cm2 in the liver of CD-fed rats versus 1.3/cm2 of rats fed a CS diet), with 4.4% of the liver being positive for GSTP staining. Cofeeding GC-1 with the CD diet for the last week of treatment strongly reduced the number of preneoplastic foci (27.8/cm2) (Fig. 2C,D). GC-1 cofeeding also reduced the percentage of the area occupied by GSTP-positive hepatocytes (2.8% versus 4.4%), though the difference was not statistically significant. These results suggest that activation of thyroid hormone receptors (TRs) by natural or synthetic ligands may exert an antitumoral effect.

The loss of preneoplastic foci caused by T3 and GC-1 could be the consequence of a direct transcriptional repression of GSTP expression by TRs ligands. However, this possibility appears to be highly unlikely in view of the following: (1) treatment with T3 for 1 week showed no inhibitory effect on GSTP expression of bile ducts, as shown by immunohistochemical staining of livers from CD + T3–fed rats (Fig. 3A), and, (2) T3 treatment for 1 week did not modify the expression of GSTP in rat kidney, an organ that expresses a large amount of this protein (Fig. 3B). Finally, feeding a CD + GC-1 diet caused a striking decrease in the number of preneoplastic lesions identified by their positivity for GGT, the second-best marker for preneoplasia,25 compared with the animals fed the CD diet alone (11.6 ± 2 foci/cm2 versus 37.8 ± 4 foci/cm2). Taken together, these findings seem to rule out the possibility that the decrease in the number of GSTP-positive preneoplastic foci caused by TR ligands is merely a consequence of a loss of staining due to their inhibitory effect on GSTP gene expression or protein degradation.

Figure 3.

(A) Immunohistochemical positivity for GSTP of ducts in livers of rats cofed a CD + T3 diet for 1 week (magnification ×20). (B) Western blot analysis of GSTP from the kidney of untreated rats; Western analysis was performed as described in Materials and Methods. Actin was used as a loading control.

Effect of GC-1 on Labeling Index and Apoptotic Index of GSTP-Positive Foci.

Next, we investigated whether the TR ligand-induced disappearance of preneoplastic foci could have occurred as a consequence of an initial inhibition of hepatocyte proliferation within the foci. To address this question, the proliferative activity of preneoplastic hepatocytes was determined in rats cofed GC-1 for only 3 days after 10 weeks of a CD diet, a time point when no major variation in the number of the foci was observed between this group and the control group (Fig. 4A). The labeling index was determined by analyzing the number of BrdU-positive hepatocyte nuclei within the GSTP-positive foci using a double immunohistochemical technique. As shown in Fig. 4A,B, feeding of GC-1 for 3 days caused an increase, although not statistically significant, of the labeling index of GSTP-positive foci compared with that of foci from rats maintained on a CD diet (66.3% versus 42.2%, respectively). The range of the labeling index was 44% to 91% in foci from GC-1–treated rats versus 20% to 87% of controls. No inhibition of preneoplastic hepatocyte proliferation could be observed when the labeling index was determined 7 days after GC-1 cotreatment (82% in foci from CD-fed rats versus 74% in CD + GC-1–treated animals).

Figure 4.

(A) Number of GSTP foci, labeling index, and apoptotic index of GSTP-positive hepatocytes in the liver of rats given a single injection of DENA and then fed a CD diet for 11 weeks with or without GC-1 for the last 3 days. For labeling index, at least 3,000 GSTP-positive hepatocyte nuclei were scored. The labeling index was expressed as the number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as the mean ± standard error of 5 to 6 rats per group. The incidence of apoptotic bodies within randomly selected nodules was determined in hematoxylin-eosin–stained sections by scoring at least 3,500 hepatocytes. Only apoptotic bodies containing nuclear fragments were recorded. The apoptotic index was calculated as the number of apoptotic bodies/100 hepatocytes. (B) Photomicrographs of liver sections double-stained for GSTP and BrdU from rats treated with DENA + CD or DENA + CD + GC-1. All rats were given BrdU (1 mg/mL) in drinking water for the last 3 days. Several labeled hepatocytes are present in GSTP-positive hepatocytes (magnification ×20). (C) Left: Liver focus from a rat subjected to DENA + CD. Note the absence of fat accumulation in the preneoplastic hepatocytes compared with the surrounding liver; the inset shows the presence of several acidophilic bodies with remnants of fragmented nuclei (hematoxylin-eosin, magnification ×40; inset, magnification ×100). Right: Photomicrograph from liver sections showing TUNEL-positive acidophilic bodies within a preneoplastic lesion from rats fed a CD diet. (D) Western blot analysis of Bax, pro-caspase 9, cleaved-caspase 9, and Bcl-XL. Western analysis was performed as described in Materials and Methods. Actin was used as a loading control. Each lane represents a single sample.

We next investigated whether selective apoptosis of preneoplastic hepatocytes could be responsible for the reduction in the number of the foci caused by GC-1–cotreatment. As shown in Fig. 4C, apoptosis identified by the presence of acidophilic bodies containing nuclear fragments was easily detected in preneoplastic foci generated by DENA + a CD diet. When the incidence of apoptosis within preneoplastic foci was quantitated, it was found that treatment of DENA-initiated rats fed a CD diet alone induced an acidophilic body index of 2.45% ± 0.79% (Fig. 4A). Cofeeding GC-1 for 3 days caused no increase of the acidophilic body index, but rather a reduction of it, though statistically not significant (1.2% ± 0.46). TUNEL assay was performed to confirm that acidophilic bodies are truly representative of apoptosis. As shown in Fig. 4C, acidophilic bodies were indeed found to be TUNEL-positive. These results therefore show that apoptosis is not responsible for the reduction of the foci observed after GC-1 cofeeding. Notably, an inhibitory role of GC-1 on cell death was also observed in the whole liver, as indicated by the decreased expression of the proapoptotic proteins Bax and caspase-9 (Fig. 4D) in the absence of any change in the liver expression of the antiapoptotic protein Bcl-xL.

Effect of Treatment with GC-1 on Preneoplastic Nodules Generated by the R-H Model.

The effect of GC-1 on preneoplastic lesions was also investigated in rats treated according to the R-H model, which causes no fat accumulation.20 In agreement with previous data,12 livers from rats exposed to DENA + AAF + PH and sacrificed 6 weeks after release of 2-AAF showed the presence of several macroscopically evident, white nodules, merging from the surface. Immunohistochemically, two types of nodules could be easily identified: (1) those characterized by a uniform GSTP staining, and therefore classified as persistent nodules, and (2) those showing a progressive loss of GSTP staining (remodeling nodules). The total number of GSTP-positive nodules was 39.4/cm2 (Table 1). Feeding GC-1 for 2 weeks did cause a dramatic change in the macroscopic appearance of the liver. Indeed, most GC-1–fed rats exhibited a liver with a smooth surface and only a few protruding nodules. Accordingly, the number of GSTP-positive nodules in GC-1–treated rats revealed a two-fold reduction in their number (from 39.4 GSTP-positive foci/cm2 to 20.4 GSTP-positive foci/cm2). Reduction of the number of GSTP-positive nodules was accompanied by a dramatic decrease in the percentage of the area occupied by GSTP-positive hepatocytes (40.5% versus 6.3%). No inhibition of hepatocyte proliferation was associated with the decreased number of GSTP-positive nodules caused by GC-1. Indeed, 7 days after GC-1 treatment, the labeling index of preneoplastic lesions was 85% versus 42%. Similar results were obtained when the extent of BrdU incorporation was measured at the end of the second week (labeling index, 91% versus 49%).

Table 1. Effect of GC-1 on the Number and Size of Preneoplastic GSTP-Positive Foci Generated by the R-H Model
TreatmentBWLW/BW %GSTP-Positive Foci/cm2 (No./cm2)Mean GSTP-Positive Area (mm2)GSTP-Positive Area (%)
  • Values are expressed as the mean ± standard error of 5 to 6 animals per group.

  • Abbreviations: BW, body weight; LW, liver weight.

  • **, *

    Significantly different from DENA + 2-AAF-PH (P < 0.05).

  • **

    Significantly different from DENA + 2-AAF-PH (P < 0.01).

DENA + 2-AAF-PH270 ± 85.9 ± 0.239.4 ± 2.81.00 ± 0.1640.5 ± 9.0
DENA + 2-AAF-PH + GC-1 (2 weeks)227 ± 7**5.2 ± 0.420.4 ± 2.3**0.76 ± 0.1616.3 ± 4.4*

The biochemical phenotype of preneoplastic hepatocytes induced by genotoxic carcinogens closely resembles that of fetal or neonatal hepatocytes in that they lack enzymes normally expressed by differentiated hepatocytes (P-450, ATPase, G6Pase). On the other hand, they have high levels of products of genes whose expression is greatly reduced or absent in fully differentiated hepatocytes (GGT, GSTP, G6PDH, alpha-fetoprotein).26–28 This fact, together with the observation that most of the preneoplastic lesions are able to remodel to adult appearing liver,29, 30 suggests that these lesions are susceptible to a differentiation program. It also raises the question of whether TRs may be able to induce a differentiation program involving a general loss of preneoplastic markers, a program that could be the cause of the observed disappearance of preneoplastic lesions. We tested the possibility in rats subjected to the R-H model and treated with GC-1. We determined the expression of various markers of preneoplasia, such as GSTP, GGT, G6Pase, and ATPase, 1 week after GC-1 feeding, a time point when no significant difference in the number of GSTP-positive lesions was observed between the groups (data not shown). The results showed that a progressive loss of fetal markers such as GSTP and GGT and reacquisition of G6Pase and ATPase, two proteins expressed in normal differentiated liver (Fig. 5), occurred in preneoplastic lesions following GC-1 treatment. This shift toward a fully differentiated phenotype preceded the loss of the preneoplastic lesions.

Figure 5.

Immunohistochemistry and histochemistry of serial sections through nodules induced in rat liver after treatment with the R-H model followed by feeding (A) a basal diet or (B) a GC-1–supplemented diet. (A) Persistent nodule uniformly stained for GSTP and GGT and completely negative for ATPase and G6Pase. (B) Nodule showing a progressive loss of preneoplastic markers in the liver of rats fed a CD + GC-1 diet (dashed lines). Note how the loss of positive markers (GSTP and GGT) occurs in the same areas of the nodule, which shows a reacquisition of negative markers (G6Pase and ATPase). A persistent nodule is also observed (solid lines) (magnification ×20).

Discussion

This study demonstrates that a short-term treatment with T3 and the TR-β agonist GC-1 strongly reduces the number of preneoplastic foci generated in two different experimental models of liver carcinogenesis. Interestingly, T3 exerted a stronger effect than the TR-β–selective agonist GC-1 in accelerating the disappearance of preneoplastic foci (compare Fig. 2A and Fig. 2C), suggesting the involvement of both TR isoforms.

Notably, loss of preneoplastic lesions occurred despite a striking preneoplastic hepatocyte proliferation, ruling out the possibility that the mechanism responsible for the disappearance of the foci by TR agonists is due to inhibition of the growth of preneoplastic hepatocytes by these ligands.

Another factor influencing growth of preneoplastic lesions is the rate of cell death. Indeed, promoting agents such as phenobarbital31 and dioxin32 are thought to act, at least in part, by inhibiting apoptosis; however, dietary restriction and S-adenosyl-methionine, potent inhibitors of carcinogenesis, appear to exert their anti-carcinogenic effect by inducing an increased apoptotic incidence in preneoplastic lesions.33, 34 Our results, however, did not support a significant role for apoptosis in the regression of GSTP-positive foci induced by TRs ligands. Indeed, no significant difference in acidophilic body index between CD + GC-1 and CD rats was observed.

Our results also appear to rule out the possibility that the loss of GSTP-positive foci is due to a specific inhibition of GSTP expression by TRs ligands, similar to that proposed for peroxisome proliferators35; indeed, T3 administration did not reduce the normal GSTP expression exhibited by rat kidney, nor did it inhibit GSTP expression of bile ductular cells. Moreover, a reduction in the number of preneoplastic lesions was observed even 7 days after T3 withdrawal.

The biochemical phenotype of preneoplastic hepatocytes induced by genotoxic carcinogens closely resembles that of fetal or neonatal hepatocytes in that they lack enzymes normally expressed by differentiated hepatocytes (P-450, ATPase, G6Pase); they have instead high levels of products of genes whose expression is greatly reduced or absent in fully differentiated hepatocytes (GGT, GSTP, glucose-6-phosphate dehydrogenase, alpha-fetoprotein).26–28 It is also known that, during the carcinogenic process, a slow but continuous regression of putative preneoplastic lesions occurs that is characterized by a progressive loss of the less-differentiated phenotype and a return to a normal one (remodeling),29, 30 suggesting that the remodeling process is a genetically programmed phenomenon. However, the exact nature of this process, remains elusive. Our results show loss of several markers associated with preneoplasia after treatment with T3 and GC-1. They indicate, therefore, that TRs ligands strongly accelerate remodeling of preneoplastic lesions. Our findings, along with the notion that TRs are involved in differentiation and metamorphosis,36, 37 make it possible to postulate that the mitogenic activity mediated by activated TRs is associated with or followed by a process of differentiation. The acceleration of remodeling of preneoplastic hepatocytes exerted by T3 and GC-1 offers a unique opportunity in that a normally long-term process (several months), occurs in a very short time (1 week). This feature could allow us to determine whether TR ligands exert their effects by stimulating genes responsible for regulation and maintenance of the differentiated state of hepatocytes (C/EBPβ, HNF4) and/or by inhibiting genes that code for proteins expressed in less-differentiated hepatocytes (alpha-fetoprotein, cytokeratins). Among the molecular mechanisms whereby TRs could induce a differentiation of preneoplastic lesions, remodeling/regression induced through a redifferentiation program appears to be the most likely explanation for the loss of hepatic nodules caused by T3 and GC-1. TRs are members of the same superfamily of nuclear receptors of PPARs, RARs, and RXRs, and these receptors have been shown to have profound effects on cellular proliferation and differentiation.38, 39 The finding that a strong enhancement of preneoplastic hepatocyte proliferation accompanies changes of preneoplastic markers (GSTP, GGT, G6Pase, ATPase) suggests that a redifferentiation program may require cell replication. How TR-induced cell proliferation is associated with a differentiation program is not known; however, it should be stressed that hepatocyte proliferation induced by TR agonists and ligands of other nuclear receptors differs in several aspects from that occurring during liver regeneration after two-thirds PH or necrogenic agents.40 Indeed, hepatocyte proliferation induced by ligands of nuclear receptors occurs in the absence of activation of transcription factors (AP-1, NF-κB, STAT3) and is not associated with an increased expression of immediate early genes (c-fos, c-jun, early growth response-1).11, 41, 42 The finding that an inhibitory effect on preneoplastic hepatocytes occurred despite the strong mitogenic activity of T3 suggests that enhanced cell proliferation per se is not necessarily critical in the progression of carcinogen-altered cells to HCC, because the nature of the proliferative stimuli is the important determinant in the process of HCC development. Notably, the inhibitory effect exerted by TR ligands appears to be unique, because ligands of PPAR or RAR showing hepatomitogenic activity, unlike T3, promote rodent liver cancer.43, 44

In conclusion, the present results, together with our previous findings13 and those of others reporting that short-term treatment with KAT-681 (a liver selective thyromimetic) also inhibits the carcinogenic process in the liver,45 suggest that TRs may represent a meaningful target for liver cancer therapy. In this context it is important to note that somatic mutations in both TR-α and TR-β are detected in human HCC.46, 47 Moreover, an additional mutant TR-α allele, first identified as the v-erb A oncogene in an avian retrovirus, can cause HCC when expressed as a transgene in mice.48 The precise contributions of these mutant TRs to oncogenesis remain incompletely understood; however, their high frequency in these neoplasias (65% of HCC samples displayed TR-α1 mutations; 76% displayed TR-β1 mutations, with both loci mutated in some samples) is highly suggestive of their critical role. Because these alterations in transcriptional regulation and DNA recognition appear likely to contribute to oncogenesis by reprogramming the differentiation and proliferative properties of the hepatocytes, in which the mutant TRs are expressed, further studies on the exact role of TRs in the carcinogenic process are needed

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