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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Transforming growth factor beta (TGF-β) signaling involves both tumor-suppression and oncogenesis. TGF-β activates the TGF-β type I receptor (TβRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TβRI-dependent pSmad3C transmits a tumor-suppressive TGF-β signal, while JNK-dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain-specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N-diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125-treated rats than those in the vehicle-treated rats (7.9 ± 0.8 versus 17.7 ± 0.9: P < 0.001; 6.3 ± 1.2 versus 7.1 ± 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN-induced HCC (113 versus 97 days: log-rank P = 0.0018). JNK/pSmad3L/c-Myc was enhanced in the rat hepatocytes exposed to DEN. However, TβRI/pSmad3C/p21WAF1 was impaired as DEN-induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c-Myc in the damaged hepatocytes and enhanced pSmad3C/p21WAF1, acting as a tumor suppressor in normal hepatocytes. Conclusion: Administration of SP600125 to DEN-treated rats shifted hepatocytic Smad3-mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression. (HEPATOLOGY 2009.)

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, accounting for 600,000 deaths annually.1 HCC is characterized as a highly chemoresistant cancer with no effective systemic therapy.2, 3 Despite surgical or locoregional therapies, the prognosis remains poor because of high tumor recurrence or tumor progression and there are no well-established effective adjuvant therapies.4, 5 Recently, sorafenib, a multitargeted tyrosine kinase inhibitor, was found to improve survival of patients with advanced-stage HCC.6 The molecular events that affect hepatocarcinogenesis need to be identified and targeted to validate and expand the concept of chemoprevention to other therapeutics.

Inflammatory conditions are present in human hepatocarcinogenesis before a malignant change occurs. A hepatitis viral infection triggers chronic inflammation, increasing the risk of HCC development. Tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) are multifunctional cytokines largely responsible for the hepatic response to chronic inflammation.7-9 The concentrations of the cytokines in serum are increased in chronic liver inflammation including hepatitis viral infection and steatohepatitis.10 The c-Jun NH2-terminal kinase (JNK) is a key signal transducer of inflammatory cytokines and has emerged as an important endogenous tumor promoter.11-13 Recent studies have shown involvement of JNK activation and c-Jun phosphorylation in hepatic carcinogenesis,14-16 even though the molecular mechanisms remain largely unknown. Therefore, JNK is a potential therapeutic target for liver cancer. In fact, recent in vitro studies indicated that a JNK inhibitor (SP600125) had antitumor activity by inducing growth arrest and by sensitization to CD95-mediated apoptosis, in hepatoma cell lines.17, 18 However; it remains to be elucidated as to whether SP600125 has an antitumor activity for liver cancer in vivo.

Transforming growth factor beta (TGF-β) signaling involves both tumor-suppression and oncogenesis.19-22 TGF-β activates TGF-β type I receptor (TβRI) and JNK, which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L).23 Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that TβRI-dependent pSmad3C transmits a tumor-suppressive TGF-β signal, whereas JNK-dependent pSmad3L promotes its oncogenic activities such as cell growth and invasion.24 JNK-dependent pSmad3L accelerates cancer development in human liver and colorectal tumors.25, 26 Although JNK could directly phosphorylate the linker region of Smad3 in vitro,23 the involvement of JNK in pSmad3L-mediated signaling in vivo has not yet been fully elucidated.

It is unclear whether chronic inflammation was sufficient for the development of human HCC. That is, could chronic inflammation cause human HCC in the absence of an exogenous carcinogen such as hepatitis B and C virus? Several lines of evidence suggest that it can. In a rat model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis, chronic inflammation reproduces the progression of cirrhosis toward HCC.27 The rat model of DEN-induced HCC has a histological and genetic signature similar to human HCC and involves chronic inflammation as in human HCC.28 Accordingly, the animal models of DEN-induced HCC were used as a tool to test preventive treatments for HCC.28-30 Successfully arresting the development of rat HCC by chemotherapeutic reagents may thus lead to a potentially useful treatment for human HCC. Based on these clues, this study clarified how a JNK inhibitor, SP600125, affected DEN-induced rat HCC, focusing on oncogenic and tumor-suppressive Smad3 signaling.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Experimental Design and Animal Procedures.

All animal care and experiments were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Wistar rats (4-week-old male, weighing 70 ± 10 g; SLC, Hamamatsu, Japan) were fed with 100 ppm DEN (Sigma, St. Louis, MO) in drinking water for 56 days and kept for an additional 28 days without DEN. Seven days after DEN administration, 45 rats were randomly assigned to the three groups (n = 15/group): the DEN alone group (DEN), JNK inhibitor group (DEN+SP), or Vehicle control group (DEN+V). The 15 rats of the DEN+SP or DEN+V groups received subcutaneous injections of either SP600125 (Tocris) at 2 mg/kg body weight or of vehicle (30% PEG-400/20% polypropylene glycol/15% Cremophor EL/5% ethanol/30% saline) 11 times weekly. All rats were sacrificed for evaluation of HCC development 84 days after initial DEN administration (Fig. 1). Blood was collected from the vena cava and serum was separated for measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The number of all liver nodules with a diameter of 3 mm or more, detectable upon examination of the liver surface, were counted. Samples of both tumoral and nontumoral liver tissue were frozen immediately in liquid nitrogen and stored at −80°C or fixed in 10% buffered formalin and embedded in paraffin.

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Figure 1. Experimental design. The horizontal bar represents N-diethylnitrosamine (DEN) in drinking water (100 ppm) administration. Vertical downward arrows represent the weekly subcutaneous injections of SP600125 at 2 mg/kg in JNK inhibitor group (DEN+SP), which was replaced by vehicle (Vehicle control group; DEN+V). DEN indicates DEN alone group. DEN, N-diethylnitrosamine.

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Western Blot Analysis.

A western blot analysis was performed as described previously31 while using the following primary antibodies and dilutions: anti-phosphorylated c-Jun (p-c-Jun) antibody (#sc-16312-R; Santa Cruz Biotechnology, Santa Cruz, CA) at 1 μg/mL; anti-phosphorylated p38 mitogen activated protein kinase (MAPK) antibody (#9211; Cell Signaling Technology, Danvers, MA) at 1 μg/mL; anti-phosphorylated p44/42 MAPK (extracellular signal-related kinase 1/2 [ERK 1/2]) antibody (#9101; Cell Signaling Technology) at 1 μg/mL; anti-phosphorylated Akt antibody (#9271; Cell Signaling Technology) at 1 μg/mL; anti-p21WAF1 antibody (#ab7960; Abcam, Cambridge, UK) at 5 μg/mL; anti-p53 antibody (#9282; Cell Signaling Technology) at 1 μg/mL; anti-c-Myc antibody (#sc-764; Santa Cruz Biotechnology) at 1 μg/mL; anti-proliferating cell nuclear antigen (PCNA) antibody (#sc-56; Santa Cruz Biotechnology) at 0.5 μg/mL; and anti-α-tubulin antibody (#CP06; Calbiochem, San Diego, CA) at 0.5 μg/mL.

The intensity of the bands was quantified using an imaging analysis software program called Quantity One (Bio-Rad Laboratories, Hercules, CA) and they were normalized using α-tubulin as an internal control.

Immunoprecipitation and Immunoblotting.

Frozen tissue specimens were extracted with TNE buffer (10 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L ethylene diamine tetraacetic acid, 1% Nonidet P-40, and 100 mmol/L phenylmethylsulfonyl fluoride). Protein concentration was determined using the BCA protein assay kit (Pierce). Cell extracts were subjected to immunoprecipitation with monoclonal anti-Smad2/3 antibody (#610843; BD Transduction Laboratories, Lexington, KY) followed by adsorption to protein G–sepharose (Pharmacia, Peapack, NJ) for 1 hour. After washing three times with TNE buffer, the immunoprecipitates were separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Immobilon membranes (Millipore, Billerica, MA). Phosphorylation of Smad3 was analyzed using rabbit polyclonal anti-pSmad3C antibody (4 μg/mL) and anti-pSmad3L (4 μg/mL).32

Liver Histology and Immunohistochemical Analyses.

Formalin-fixed, paraffin-embedded sections were cut into 4-μm thicknesses and mounted on silanized glass slides. The slides were stained with hematoxylin and eosin for standard histology.33 Immunohistochemical analyses were performed as described previously.31

The primary antibodies used in this study included rabbit polyclonal anti-phosphorylated c-Jun (p-c-Jun) antibody (10 μg/mL, #sc-16312-R; Santa Cruz Biotechnology), rabbit polyclonal anti-pSmad3C (2 μg/mL), rabbit polyclonal anti-pSmad3L (5 μg/mL), and mouse monoclonal anti-PCNA antibody (5 μg/mL, #sc-56; Santa Cruz Biotechnology). Anti-pSmad3C antibody was cross-reacted weakly with COOH-terminally phosphorylated Smad2. To block the binding of anti-pSmad3C antibody to phosphorylated domains in Smad2, anti-pSmad3C antibody was adsorbed with 1 μg/mL COOH-terminally phosphorylated Smad2 peptide.

Survival Analysis.

Forty-five rats were randomly assigned to three groups (DEN, DEN+V, and DEN+SP; n = 15/group) and each of the three groups was treated as described above. The observation period was 120 days after the initial DEN administration.

Statistical Analysis.

All data were expressed as the means ± standard error of the mean (SEM). The statistical significance of a difference between groups was assessed using the Mann-Whitney U-test. A Kaplan-Meier analysis and log-rank test were performed to determine survival time between groups. P values < 0.05 were regarded as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

SP600125 Suppresses JNK Phosphorylation in DEN-Induced Nontumor Liver Tissues.

The DEN-induced HCC model is a good model for investigating HCC tumorigenesis.34 Compensatory proliferation appears to have a critical role in DEN-induced hepatocarcinogenesis,15 and inflammatory cytokines are necessary for normal liver regeneration.35 DEN-induced hepatocarcinogenesis depends on an inflammatory response, triggered by hepatocyte necrosis, that leads to activation of JNK. Moreover, JNK activation is closely associated with hepatic carcinogenesis.15, 16, 36 Accordingly, the JNK activity of noncancerous and cancerous tissues were initially analyzed in DEN-treated rat livers.37 JNK activity was evaluated by western blot analysis of c-Jun, the target of JNK.38, 39 After DEN treatment, c-Jun was markedly phosphorylated in the rat livers. A representative western blot analysis and quantification by a photographic densitometer are shown in Fig. 2A. The levels of phosphorylated c-Jun were clearly increased in both the nontumor and tumor liver tissues on day 84, whereas the phosphorylation levels of other MAPKs such as ERK1/2 and p38 and Akt were unchanged (data not shown). The levels of phosphorylated c-Jun were already observed to have increased about two-fold in the damaged livers on day 7 after DEN administration in comparison to normal livers, whereas c-Jun phosphorylation was maintained at the high level until day 84 (Fig. 2A-C). SP600125 specifically inhibited c-Jun phosphorylation in the DEN-treated livers for 7 days after subcutaneous injection (Fig. 2B,C). These results indicated that the JNK activation was up-regulated at an early stage of the chemical carcinogenic steps.

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Figure 2. The JNK activity increased in DEN-induced HCCs while it was inhibited by SP600125. (A) A western blot analysis of p-c-Jun was performed 84 days after DEN administration (left side) and the western blot was analyzed densitometrically (right side). (B) To analyze the effects of SP600125 on JNK activity, rats were treated with SP600125 (2 mg/kg) 7 days after DEN administration (day 7) and killed 1, 4, or 7 days later (day 8, 11, or 14). Western blot analysis of p-c-Jun on day 0, 7, 8, 11, and 14 was performed in DEN+V (left side, upper panel) and DEN+SP (left side, lower panel) and the western blot was analyzed densitometrically (right side); the intensity of the bands was quantified with imaging analysis software Quantity One (Bio-Rad Laboratories, Hercules, CA) and normalized using α-tubulin as an internal control. Data are shown as the means ± SEM (n = 3). (C) Representative paraffin-embedded liver sections collected on day 0, day 7, and day 14 of the Vehicle control group (DEN+V) and the JNK inhibitor group (DEN+SP) after DEN administration were immunostained with anti-p-c-Jun antibodies (×200). p-c-Jun, phosphorylated c-Jun; N, normal liver tissues; T, tumoral liver tissues; NT, nontumoral liver tissues.

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SP600125 Suppresses HCC Development from DEN-Induced Nontumor Liver Tissues.

The above results led us to examine whether disruption of the JNK signaling pathway could have preventive effects on carcinogenesis in DEN-treated rat livers (n = 15/group). To test this possibility, a JNK inhibitor, SP600125, was frequently administered to DEN-treated rats. Multiple tumors were observed in the damaged livers on day 84 after DEN administration (Fig. 3A). A histological examination showed that tumors larger than 3 mm in diameter were definitely HCCs. Every animal carried at least one typical HCC. HCCs were moderately or well differentiated, with various patterns (trabecular, pseudoglandular, or massive) from one tumor to another in the same liver (Fig. 3B).

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Figure 3. SP600125 suppressed DEN-induced HCC and prolonged survival. (A) Macroscopic views of livers; Vehicle control group (DEN + V) (left panel) and JNK inhibitor group (DEN + SP) (right panel) on day 84. Arrowheads indicate tumors (≥3 mm). (B) Photomicrographs of an HCC nodule (arrowhead) surrounded by liver tissue with cirrhosis are shown at low (40×, left panel) and high (400×, right panel) magnifications. (C) In each group of animals (n = 15/group), HCC tumors were counted on the surface of the liver according to predefined criteria (a diameter ≥3 mm; left panel). In each group of animals (n = 15/group), liver weight/body weight ratio served as an index of tumor mass (right panel). Data are shown as the means ± SEM. *P < 0.001, **P < 0.05. (D) Survival analysis. Three rats of the JNK inhibitor group (DEN + SP; 20.0%) were alive until 120 days after DEN administration, whereas all of the rats of the DEN-alone group (DEN) and Vehicle control group (DEN + V) died by day 119 (n = 15/group). The 120-day survival rate was compared among the three groups (Kaplan-Meier analysis and log-rank test). Heavy solid line indicates the JNK inhibitor group (DEN+SP). Solid line and dashed line show the DEN-alone group (DEN) and the Vehicle control group (DEN+V), respectively.

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In the JNK inhibitor group (DEN+SP), the number of tumors (≥3 mm) on the surface of the liver was significantly lower (7.9 ± 0.8) than in the DEN-only group (DEN: 19.1 ± 1.0) and Vehicle control group (DEN+V: 17.7 ± 0.9; P < 0.001, Fig. 3C, left panel). Furthermore, in the DEN+SP group, the number of tumors (≥5 mm) on the surface of the liver was significantly lower (1.1 ± 0.3) than in the DEN (3.1 ± 0.7) and DEN+V (2.4 ± 0.4: P < 0.05) groups. The number of all liver nodules with a diameter of 3 mm or more, which are detectable upon examination of the cross-section of fresh left lobe liver cut into 5-mm slices, were counted for the purpose of evaluating the multiplicity of tumor nodules developing inside the liver (n = 10/group). In the DEN+SP group, the number of tumors (≥3 mm) on the cross-section of fresh left lobe was significantly lower (1.0 ± 0.3) than in the DEN (2.7 ± 0.2) and DEN+V (2.8 ± 0.3: P < 0.01, Supporting Fig. 1) groups. The liver weight/body weight ratio was measured as another marker of primary tumor mass, which was significantly lower in the DEN+SP (6.3% ± 1.2%) than in either the DEN (7.3 ± 0.2%) or DEN+V (7.1 ± 0.2% P < 0.05, Fig. 3C, right panel) groups. Three of the 15 rats in the DEN+SP group were alive at day 120, while all rats of both the DEN and DEN+V groups died within 120 days after DEN administration (Fig. 3D). The 120-day survival rate did not differ significantly between DEN and DEN+V groups. However, the 120-day survival rate was significantly higher for the DEN+SP than for the DEN+V group (20.0 versus 0.0%, log-rank P = 0.0018). Moreover, the median survival time in the DEN+SP group (113 days) was significantly longer than that in DEN (90 days) or DEN+V (97 days) groups. These results indicate that the JNK inhibitor suppresses HCC development in DEN-treated rats.

In comparison with the DEN-treated rats (DEN and DEN+V), a histological examination did not show obvious drug-induced toxicities in the SP600125-treated rat livers. No significant difference in the plasma AST and ALT levels was found between the DEN+SP and DEN+V groups (AST 173.8 ± 18.8 IU/L versus 197.7 ± 14.3 IU/L; P = 0.3; ALT 156.4 ± 16.7 IU/L versus 168.5 ± 10.1 IU/L; P = 0.5). In addition, the body weight was not significantly different between DEN+SP and DEN+V groups (234.5 ± 6.2 g versus 230.7 ± 4.5 g, P = 0.4). Taken together, SP600125 administration was therefore not found to cause any serious adverse reactions.

SP600125 Administration to DEN-Treated Rats Shifts Hepatocytic Smad3 Signaling from Oncogenic pSmad3L/c-Myc to Tumor-Suppressive pSmad3C/p21WAF1 Pathway.

The linker segment in Smad3 acts as a substrate beside c-Jun for JNK.23, 24, 26 Moreover, JNK-dependent pSmad3L promotes human hepatic carcinogenesis,24, 25 whereas the pSmad3C pathway involves tumor suppression.23, 25, 26 Accordingly, the number of pSmad3C-immunoreactive and pSmad3L-immunoreactive hepatocytes per 1000 hepatocytes were counted in the SP600125-treated and DEN-treated liver specimens (Fig. 4A). The representative pSmad3C and pSmad3L distribution on day 28 and day 84 is shown in Fig. 4B. Immunostaining with antibody against Smad3 at the C-terminal region indicated that Smad3 in normal hepatocytes was slightly phosphorylated at the C-terminal region (data not shown). In DEN-treated livers (DEN and DEN+V), the phosphorylation level of Smad3C was elevated in the hepatocytic nuclei on day 28 and declined thereafter (Fig. 4B, left side). In contrast to the moderately phosphorylated state of Smad3C, Smad3L showed little phosphorylation in normal hepatocytes. As the DEN-treated liver damage developed, Smad3 became phosphorylated at the linker region (Fig. 4A, right side). In the DEN and DEN+V groups, pSmad3L was located predominantly in the hepatocytic nuclei (Fig. 4B, lower side in DEN and DEN+V panels). Collectively, the damaged hepatocytes show a reciprocal change of pSmad3L and pSmad3C in the cell nuclei during DEN-induced hepatocarcinogenesis.

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Figure 4. SP600125 restored Smad3 signaling from pSmad3L to pSmad3C pathway in noncancerous hepatocytes. (A) On day 0, 28, 56, and 84, each liver section of the three groups was immunostained with anti-pSmad3C and anti-pSmad3L and the positive cells were identified. The number of pSmad3C-immunoreactive and pSmad3L-immunoreactive hepatocytes per 1000 hepatocytes at 200× magnification in five nonoverlapping regions selected at random was evaluated. Data are shown as the means ± SEM (n = 5 per time point). (B) Representative paraffin-embedded liver sections collected on day 28 and day 84 after DEN administration were immunostained with anti-pSmad3C or anti-pSmad3L antibodies (×200). (C) After immunoprecipitation (IP) of cell lysates with anti-Smad2/3 antibody, degree of Smad3 phosphorylation was analyzed in immunoblot (IB) using each anti-phospho-Smad3 antibody (upper panels). The relative amounts of endogenous Smad2/3 were determined by IB using anti-Smad2/3 antibody (bottom panel). pSmad3C, C-terminally phosphorylated Smad3; pSmad3L, linker-phosphorylated Smad3; NT, nontumoral liver tissues.

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When SP600125 was additively administered in DEN-treated rats (DEN+SP), Smad3 in the hepatocytic nuclei was remarkably phosphorylated at C-terminal region on day 28 (Fig. 4A, left side; Fig. 4B, upper side in DEN+SP panel). In contrast, its linker phosphorylation was gradually suppressed in the hepatocytic nuclei as DEN-treated livers progressed (Fig. 4A, right side; Fig. 4B, lower side in DEN+SP panel). Therefore, on day 28, the number of pSmad3C-immunoreactive hepatocytes in the DEN+SP group was 83.7% higher than those in the Vehicle control group (DEN+V: 585.8 ± 36.6 versus 319.2 ± 15.8 per 1000 hepatocytes, P = 0.009), whereas the number of pSmad3L-immunoreactive hepatocytes on day 84 in the DEN+SP group was 62.8% lower than those in the DEN+V group (262.4 ± 29.6 versus 705.0 ± 45.5 per 1000 hepatocytes, P = 0.009).

The extent of phosphorylation at Smad3C and Smad3L was then quantified by immunoblotting. In the DEN+SP group, hepatocytic phosphorylation at Smad3C on day 28 after DEN administration was higher than that in the other two groups, whereas hepatocytic phosphorylation at Smad3L on day 84 was lower than that in the other two groups (Fig. 4C). Considering the immunoblotting findings together with the results obtained from immunohistochemical analyses (Fig. 4A,B), the JNK inhibitor SP600125 could restore oncogenic pSmad3L to the basal pSmad3C pathway in DEN-treated rat livers.

Reversible Smad3-dependent signaling in hyperactive Ras-expressing epithelial cells suggests that JNK-dependent pSmad3L promotes cell growth via the up-regulation of c-Myc transcription, whereas pSmad3C transmits a tumor-suppressive TGF-β signal by up-regulating p21WAF1.24 Therefore; the expression of p21WAF1 and c-Myc proteins were investigated in SP600125 and DEN-treated liver tissues by western blot analyses (Fig. 5A,B). The expression of p21WAF1 was low in the normal hepatocytes. In the DEN-treated livers (DEN and DEN+V), p21WAF1 expression was slightly increased during early carcinogenesis (Fig. 5A). Normal hepatocytes showed little c-Myc expression, whereas the c-Myc expression significantly increased in the DEN-treated livers (Fig. 5B). To assess these proliferative states, the nuclear expression of PCNA was examined immunohistochemically. The number of PCNA-immunoreactive hepatocytes increased as the chronically damaged livers progressed (Fig. 5C). To support this notion, hepatocyte expression of PCNA protein increased in DEN-treated livers (DEN and DEN+V in Fig. 5D) as c-Myc expression increased (Fig. 5B).

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Figure 5. SP600125 restored Smad3 signaling from oncogenesis to tumor suppression in noncancerous hepatocytes. (A) A western blot analysis of p21WAF1 was performed among the three groups on day 28 (left side) and the western blot was analyzed densitometrically (right side). (B) A western blot analysis of c-Myc was performed among the three groups on day 84 (left side) and the western blots were analyzed densitometrically (right side). (C) On day 0, 28, 56, and 84, each liver section of the three groups was immunostained with anti-PCNA and the positive cells were identified. The number of PCNA-immunoreactive hepatocytes per 1000 hepatocytes at 400× magnification in 10 nonoverlapping regions selected at random was evaluated. Data are shown as means ± SEM (n = 10 per time point). (D) A western blot analysis of PCNA was performed among the three groups on day 84 (left side) and the western blot was analyzed densitometrically (right side). Data are shown as the means ± SEM (n = 4). *P < 0.05. NT, nontumoral liver tissues; PCNA, proliferating cell nuclear antigen.

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In parallel with the increase in pSmad3C in the hepatocytic nuclei after the SP600125 administration (DEN+SP), the expression of p21WAF1 protein was elevated on day 28 (Fig. 5A). Similar to the suppressive effect of SP600125 on hepatocytic pSmad3L in DEN-treated livers (Fig. 4), the c-Myc expression was significantly suppressed in the DEN+SP group (Fig. 5B). SP600125 administration led to a decrease in the PCNA expression similar to the profile of c-Myc expression. Therefore, the number of PCNA-immunoreactive hepatocytes and expression of PCNA protein were remarkably suppressed by SP600125 administration to the DEN-treated livers (DEN+SP; Fig. 5C,D).

The specific function of JNK in human HCC cells was further analyzed in a human HCC cell line, Huh7. These cells were infected with adenoviral vectors encoding dominant negative JNK1 (Ad-dnJnk1)40 and green fluorescent protein (Ad-GFP) as a control. The efficient JNK inactivation was confirmed by western blot analyses (Supporting Fig. 2A). JNK inactivation significantly attenuated the proliferation of cultured Huh7 cells (Supporting Fig. 2B). These results indicate that the inhibition of JNK activation attenuates the proliferation of human Huh7 HCC cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Epidemiological studies have shown that chronic inflammation in the liver predisposes individuals to HCC. The hallmarks of inflammation-related hepatocarcinogenesis include the presence of inflammatory cells and inflammatory mediators (TNF-α, IL-1β, and IL-6) in preneoplastic liver tissues such as chronic hepatitis and cirrhosis. Several studies have discussed how chronic inflammation affects the proliferation and survival of hepatocytes.41, 42 The rodent model of DEN-induced HCC has a histology and genetic signature similar to that of human HCCs with poor prognosis15 and recapitulates a dependence on chronic inflammation seen in human HCC.37 Whether TNF-α, IL-1β, and IL-6 are causal or contributory to HCC is unknown. However, the cytokines are thought to contribute to hepatocyte proliferation,37 and DEN administration to mice results in production of the cytokines from Kupffer cells.43 In this study, we observed that DEN increased the messenger RNA levels of inflammatory cytokines such as IL-1α, IL-1β, and IL-6 with TGF-β in the liver (data not shown). JNK is a key signal transducer of inflammatory cytokines including TNF-α and IL-1β, and has emerged as an important endogenous tumor promoter. Support for the connection between HCC occurrence and chronic inflammation is strengthened by the current study of the role of JNK during DEN-induced hepatocarcinogenesis. This study showed that hepatocytes affected by chronic inflammation undergo transition from the tumor-suppressive TGF-β type I receptor (TβRI)/pSmad3C/p21WAF1 pathway to the oncogenic JNK/pSmad3L/c-Myc pathway (Fig. 6A).

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Figure 6. Proposed model how SP600125 inhibits HCC development. (A) As DEN induces hepatocarcinogenesis from preneoplastic regions to HCC, the pSmad3L/c-Myc pathway shows increasing prominence, while the pSmad3C/p21WAF1 pathway decreases in hepatocytes. TGF-β activates TβRI, further leading to direct phosphorylation of Smad3C. The TβRI/pSmad3C pathway involves TGF-β-mediated growth inhibition via p21WAF1. On the other hand, proinflammatory cytokines can transmit the signals through JNK-dependent pSmad3L, thus participating in hepatocytic growth by stimulating transcriptional activity of c-Myc gene. Linker phosphorylation of Smad3 indirectly prevents Smad3C phosphorylation, pSmad3C-mediated p21WAF1 transcription and consequently the antiproliferative effect of TGF-β on hepatocytes. (B) A JNK inhibitor SP600125 restores the lost tumor-suppressive pSmad3C signaling observed in normal hepatocytes by eliminating oncogenic pSmad3L-mediated signaling. Selective blockade of JNK by SP600125 abolishes pSmad3L-mediated oncogenic properties via c-Myc gene and restores the TGF-β-dependent tumor-suppressive function via p21WAF1 involving pSmad3C that is shown by normal hepatocytes. DEN, N-diethylnitrosamine; JNK, c-Jun N-terminal kinase; TGF-β, transforming growth factor-β; TβRI, TGF-β type I receptor; pSmad3C, C-terminally phosphorylated Smad3; pSmad3L, linker-phosphorylated Smad3.

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JNK, which interacts with such non-Smad pathways as c-Jun, plays an important role in pSmad3L-dependent signaling in the development of human HCC, because pSmad3L is identified as a substrate for JNK.24, 25 The proliferative capacity of hepatocytes was elevated along with the transition, indicating high rates of HCC in liver tissues.44, 45 This study demonstrated that SP600125, a JNK inhibitor, suppressed chemical-induced HCC and prolonged the median survival time. SP600125 enhanced the tumor-suppressive pSmad3C/p21WAF1 pathway in hepatocytic nuclei, while suppressing the oncogenic JNK/pSmad3L/c-Myc pathway (Fig. 6B). Therefore, SP600125 could restore a diminished tumor-suppressor function in normal hepatocytes, thus eliminating the effects that promote hepatocarcinogenesis in the early phase, and thereafter persistently attenuating tumor development in the later phase. It is well known SP600125 is a inhibitor of JNK that is known to strongly induce apoptosis and block cell cycle progression.17, 46 Meanwhile, a recent study has shown SP600125 to not be a specific inhibitor of JNK.47 To assess the influence of other protein kinases that may be inhibited by SP600125, the expression of Aurora kinases, which was recently highlighted in the field of HCC,48 were examined. However, SP600125 did not affect the messenger RNA levels of Aurora B or C in either cancerous or noncancerous lesions (data not shown). Although SP600125 might not be a specific inhibitor of JNK, the finding that this compound suppresses rat chemical hepatocarcinogenesis is significant.

Among the three mammalian JNK genes, JNK1 and JNK2 are ubiquitously expressed, while the JNK3 expression is restricted to the brain, heart, and testis. However, it remains to be determined by which molecular mechanism both JNK1 and/or JNK2 regulate hepatocarcinogenesis. A most recent study has shown the proliferation of human HCC cells and chemically induced mouse liver cancers to require JNK1.49 Our findings using an adenovirus vector encoding dominant negative JNK1 also demonstrated JNK1 inactivation to attenuate the proliferation of HCC cells. Therefore, these results suggested that JNK, especially JNK1, might thus play an important role in tumor development.

Due to numerous advances in surgical techniques and perioperative management, the short-term outcome of liver resection has dramatically improved over the last decade.50 The long-term prognosis, however, remains guarded because of the frequent development of locoregional tumor recurrence. Recurrence in the liver remnant may originate from multicentric new primaries in a cirrhotic liver.51 However, attempts to improve the long-term outcome with adjuvant therapy after the potentially curative treatment of HCC have so far produced only limited success.52, 53 The current study revealed that JNK activation was involved in HCC development, because SP600125 could block either tumorigenesis or tumor progression. Knowledge about JNK/pSmad3L-induced HCC development is therefore expected to be useful for developing new approaches for the diagnoses and prevention of human HCC. Because inhibition of JNK activity by SP600125 suppressed rat HCC without any serious drug toxicity, this may be useful for either the chemoprevention or therapy for HCC.

A key therapeutic aim in chronic liver disorders is the restoration of the lost tumor-suppressive function observed in mature hepatocytes at the expense of effects promoting hepatic carcinogenesis. These results strongly suggest that JNK is an important target for the development of chemopreventive and therapeutic measures to reduce the emergence of HCC in the context of chronic liver injury and to slow progression of pre-existing tumors. In addition, strategies designed to enhance tumor suppressor activities will provide novel approaches for treating a broad range of malignancies. The current data may provide a strong rationale for pursuing additional studies of JNK as potential therapeutic targets for HCC and possibly other types of malignancy associated with chronic inflammation. In molecularly targeted therapy for human cancer, pSmad3L and pSmad3C may therefore be potentially useful biomarkers for evaluating the benefits obtained by the specific inhibition of the JNK pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Associate Prof. Hiroki Aoki (Cardiovascular Research Institute, Kurume University) for his kind gift of the adenoviral vector of dominant negative JNK1.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_22860_sm_SuppFig1.TIF78KSupporting Fig. 1. The number of liver nodules on the cross-section of fresh left lobe (?ge;3mm). In each group of animals (n±10/group), the HCC tumors were counted on the cross-section of the fresh left lobe according to the predefined criteria (a diameter ?ge;3mm). Data are shown as the means ± SEM. *P < 0.01.
HEP_22860_sm_SuppFig2.TIF78KSupporting Fig. 2. The inhibition of JNK1 by an adenoviral vector encoding dominant negative JNK1 (Ad-dnJnk1) in a human hepatocellular carcinoma cell line, Huh7. (A) Huh7 cells were infected with adenoviral vectors encoding dominant negative JNK1 (Ad-dnJnk1) and green fluorescent protein (Ad-GFP) as a control. JNK and phosphorylated c-Jun (p-c-Jun) protein levels were detected by Western blot analyses at 2 days after the Ad-JNK1 or Ad-GFP infection. (B) The proliferation of cells was quantified at 2 days after either Ad-JNK1 or Ad-GFP infection using a cell counting kit (CCK) assay. Data are shown as the means ± SEM of four wells. *P < 0.05.

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