Roxithromycin Inhibits Constitutive Activation of Nuclear Factor κB by Diminishing Oxidative Stress in a Rat Model of Hepatocellular Carcinoma Free

Hepatocellular carcinoma is one of the most common malignancies in the world, especially in Asia and Africa. The vast majority of patients have preexisting cirrhosis at the time they develop hepatocellular carcinoma. Despite many treatments for this disease in recent decades, its long-term therapeutic outcome remains poor. Prevention seems to be the best strategy in lowering the present incidence of the disease. A preventive effect of an acyclic retinoid on the development of a second tumor after ablation of an original tumor has been shown by one group (1, 2). Conflicting results on the potential preventive effect of IFN have also been reported (3–5). To validate and expand the concept of chemoprevention to other therapeutics, the molecular events that contribute to hepatocarcinogenesis in the liver with cirrhosis need to be identified and targeted.

It was reported that tumor growth is angiogenesis dependent by the discovery of angiostatin or endostatin, both of which specifically inhibit endothelial proliferation (6, 7). There are several drugs that have angiogenesis inhibitory properties; however, most of these are restricted by excessive toxicity and limited efficacy. Roxithromycin is a new 14-member macrolide antibiotic that has a wide antibacterial spectrum against pathogens and an immunomodulatory effect. Interestingly, on tumor angiogenesis, Yatsunami et al. (8) reported that roxithromycin at concentrations >20 μmol/L inhibited endothelial cell migration and tube formation. Moreover, we previously reported the strong inhibitory effect of roxithromycin on tumor necrosis factor α, which induces vascular endothelial growth factor expression in human periodontal ligament cells in culture (9). In this mechanism, roxithromycin suppressed activation of transcription factors activator protein 1 and specificity protein 1, which are critical factors in vascular endothelial growth factor transcription. We also showed that roxithromycin inhibited angiogenesis through inhibition of vascular endothelial growth factor production from human hepatoma cells (HepG2) using an air sac assay model (10). Compared with TNP-470, which is a well-known angiogeneic inhibitor, the inhibitory effect of treatment with roxithromycin (100 mg/kg/d) was equal to, if not greater than, the inhibitory effect of TNP-470 (50 mg/kg/d).

Recently, Mauriz et al. (11) reported that treatment with angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor κB (NF-κB) activation, and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis induced by diethylnitrosamine. It has been known that NF-κB is a transcription factor that plays important roles in cell proliferation and apoptosis (12, 13). Several researchers revealed that constitutive activation of NF-κB was accompanied by hepatocarcinogenesis, and IFN-α may have a role in blocking the NF-κB activating pathway triggered by the hepatitis B virus (14–16). From the viewpoint of the inhibitory effect of oxidative stress and NF-κB activation, we herein tested the potential antitumoral effect of roxithromycin in a rat model of hepatocellular carcinoma induced by diethylnitrosamine.

Animal. Male Wistar rats weighing 200 to 230 g (10-12 weeks of age) were used in this study. The animals were housed in compliance with good laboratory practice for the protection of experimental animals (constant temperature: 22 ± 2°C; relative humidity: 55 ± 10%; regular alternation light-dark: light 7:00 a.m.-5:00 p.m.; water and food ad libitum). The rats had free access to food and diethylnitrosamine solution (100 mg/L; Sigma-Aldrich, St. Lois, MO) throughout the study. The protocol was approved by the University of Medicine of Kagoshima Animal Care Committee. At the time of sacrifice, animals were anesthesized with an i.p. injection of thiopental.

Experimental design: Protocol 1. To verify the progression of experimental hepatocarcinoma, a total of 10 rats were killed at weeks 10 and 17 (n = 5, each) after the initiation of hepatocarcinogenesis by oral diethylnitrosamine administration. Pathomorphologic observation and histochemical assay using placental glutathione S-transferase (GST-P) staining were done (17).

Protocol 2. The 60 rats that were submitted to the 17-week diethylnitrosamine regimen were divided into four groups and received i.p. injections of either normal saline for controls (n = 15), roxithromycin (40 mg/kg, n = 15, or 100 mg/kg, n = 15), or TNP-470 (50 mg/kg, n = 15) thrice per week between weeks 10 and 17. The animals were sacrificed at 17 weeks after the diethylnitrosamine exposure. Samples of both tumoral and nontumoral liver tissue were frozen and stored at −80°C or fixed in 4% buffered formalin and embedded in paraffin following macroscopic analysis.

Placental glutathione S-transferase staining. Using the liver tissue at 10 weeks after the diethylnitrosamine exposure, immnunohistochemical staining for GST-P was done by a standard method using the LSAB universal kit (DAKO, Glostrup, Denmark). The deparaffinized sections were immersed and washed in 0.05 mol/L Tris-HCl (pH 7.6). Following incubation with albumin (Wako Pure Chemicals, Osaka, Japan) for 40 minutes, the sections were incubated with primary antibodies overnight in a humidified chamber at 4°C. The primary antibodies used were rabbit polyclonal anti–GST-P antibody (1:1,000 dilution; MBL, Nagoya, Japan). After incubation, the sections were washed with Tris-HCl and processed for immunohistochemistry according to the instructions of the manufacturer.

Macroscopic findings of tumoral lesions. The liver from each subject from all groups (n = 9-10 per group) was removed and weighed. Furthermore, the liver was sliced into 4 mm thickness and photographed together and scaled. Using volumetric analysis (18), the volumes of tumoral and nontumoral liver tissues were analyzed based on image analysis computer software.

Nuclear extracts. All solutions used for the preparation of nuclear extracts contained protease inhibitors (19). After macroscopic analysis, liver tissue was homogenized. Nuclear extracts were prepared as previously described (19). Nuclear extracts were frozen and stored at −80°C until use. Protein concentrations were measured by the Bradford method with γ globulin as a standard.

Electrophoretic mobility shift assays: Probes. The NF-κB binding sequence from the class 1 major histocompatibility enhancer element (H2K) was used as double-stranded DNA probes as previously described (19). The NF-κB probes were prepared by annealing the complementary oligonucleotides in a thermal cycler in 50 mmol/L Tris (pH 8.0), 1 mmol/L EDTA. Annealed oligonucleotides were purified by PAGE. Both oligonucleotide probes were prepared by end labeling with [³²P]ATP by using T4 polynucleotide kinase.

Assays. Ten micrograms of nuclear protein were used in each assay and incubated with 0.2 ng of ³²P-end-labeled double-stranded oligonucleotide probe (19). The mixture was incubated for 30 minutes at room temperature and electrophoresed through 5% polyacrylamide Tris-glycine-EDTA gels. For antibody supershift assays, 1 μL of the antibody (1 μg/μL; all from Santa Cruz Biotechnology, Santa Cruz, CA) was added to respective samples after 30 minutes of incubation with the labeled probe. Samples were incubated at room temperature for an additional 30 minutes before electrophoresis. Gels were dried and exposed to Kodak X-AR film (Kodak, Rochester, NY) from 2 hours to 2 days.

Nitric oxide synthase activity. Liver tissue of 1 cm³, taken by scraping with a glass slide, was weighed and homogenized in 3 volumes of ice-cold buffer [50 mmol/L Tris-Cl (pH 7), 300 mmol/L sucrose, 1 mmol/L DTT, 100 μg/mL phenylmethylsulfonylfluoride, 10 μg/mL leupeptin, 10 μg/mL soybean trypsin inhibitor, and 2 g/mL aprotinin]. The liver lysate was subjected to ultracentrifugation at 10,000 × g, at 4°C for 20 minutes, and the supernatant was removed and stored at −80°C before protein extraction and measurement of nitric oxide synthase (NOS) activity (as described below). Protein concentrations within each homogenate were determined by the Bradford assay with bovine serum albumin as the standard (20). NOS activity was measured according to Chen et al. (21). The NOS activity in the supernatant (as described above) was measured by adding 30 μL of the supernatant to 70 μL of incubation buffer containing 50 mmol/L Bis-Tris propane (pH 7.2), 2 mmol/L l-arginine, 3 mmol/L DTT, 2 mmol/L NADPH, 4 μmol/L tetrahydro-l-biopterin, and 1 mmol/L CaCl₂. Samples were incubated at 37°C for 90 minutes before termination of the reaction by removing NADPH. The NADPH was removed by incubation with 10 units of lactate dehydrogenase and 5 mmol/L sodium pyruvate at 37°C for 10 minutes. Nitrite levels in the reaction mixtures were spectrophotometrically determined using Griess reagent. The reaction mixture and Griess reagent were incubated in microtiter plates at room temperature for 20 minutes in the dark. Absorbance was read at 540 and 620 nm with a multiwell plate reader. Serial dilutions of sodium nitrite were used to generate standards. The NOS activity in the liver homogenate was further characterized by incubation with 1 mmol/L EDTA instead of 1 mmol/L CaCl₂ to determine the Ca²⁺ dependence of the enzyme activity. The Ca²⁺-dependent activity under these assay conditions was taken as the total NOS activity identified by constitutive NOS activity plus inducible nitric oxide synthase (iNOS) activity. The amount that was not inhibited by the EDTA incubation was taken as the activity of the Ca²⁺-independent iNOS. The constitutive NOS activity was determined by subtracting iNOS activity from total NOS activity.

Assay of lipid peroxidation in liver tissue. Lipid peroxidation in the liver was determined by measuring the levels of malondialdehyde, which is an end product of lipid metabolism. Each liver from all of the groups (n = 5 per group) was flushed with ice-cold 0.9% NaCl via the portal vein and rapidly excised. A portion of the liver was removed and immediately frozen and stored at −80°C until homogenization. Homogenatizations were done after combining a ratio of 0.5 g of wet tissue with 5 mL of 50 mmol/L phosphate buffer (pH 7.4; homogenizer, Iuchi Seieidou, Osaka, Japan). The content of malondialdehyde in the homogenate was determined using a colorimetric reaction with thiobarbituric acid, as described by Bieri et al. (22). The protein concentration was calculated according to the method of Bradford et al. (20) by using Bio-Rad Protein Assay Kits (Bio-Rad Laboratories, Hercules, CA).

Hepatocarcinogenesis (protocol 1). In all rats that were sacrificed 10 weeks after diethylnitrosamine exposure, hepatocellular carcinoma nodules were detected at histologic analysis using GST-P staining (Fig. 1). Furthermore, macroscopic malignant nodules arose from livers with cirrhosis after the 17-week administration (Fig. 2). All nodules were identified as hepatocellular carcinoma at histologic examination.

Inhibition of neoplastic lesions by roxithromycin and TNP-470 (protocol 2). Of a total of 60 rats, two rats died during the regimen. One rat was from the roxithromycin 100 mg/kg group and one was from the TNP-470 50 mg/kg group.

Morphology of the tumor and nontumor lesions. When comparing the whole liver weight and the volume of hepatocellular carcinoma lesions by volumetric analysis after the initiation of 17-week diethylnitrosamine exposure (Table 1), the average weight of the liver was significantly decreased in the group given roxithromycin 100 mg/kg (17.7 ± 2.3 g) or TNP-470 50 mg/kg (14.8 ± 4.0 g) than in the group given normal saline only (control; 33.5 ± 8.5 g) or roxithromycin 40 mg/kg (23.4 ± 9.0 g). The liver weight in the group given roxithromycin 40 mg/kg was intermediate between the control and roxithromycin 100 mg/kg groups, although the differences were significant. Similarly, the average volume of hepatocellular carcinoma lesions was significantly lower in the three groups (roxithromycin 40 and 100 mg/kg and TNP-470 50 mg/kg) than in the control group. The hepatocellular carcinoma volume in the group given roxithromycin 100 mg/kg (1.38 ± 1.91 cm³) was almost the same as that in the TNP-470 group (1.15 ± 1.35 cm³).

Binding of transcription factors. We analyzed by electrophoretic mobility shift assay the binding of transcription factors NF-κB in liver nuclear extracts in the four groups. The gel shift analysis for NF-κB and the supershift analysis are presented in Fig. 3. The increase of NF-κB was noticed only in liver tissue of the control group; however, the activities were inhibited in the three groups given roxithromycin or TNP-470.

Nitric oxide production. The levels of constitutive NOS activity were not significantly different among any of the groups after saline, roxithromycin, or TNP-470 challenge (data not shown). The iNOS activities were significantly higher in the control group than in the other three groups. Roxithromycin 100 mg/kg and TNP-470 50 mg/kg significantly reduced iNOS activity after the 17-week diethylnitrosamine regimen (Fig. 4).

Lipid peroxidation of liver tissue. The malondialdehyde level 17 weeks after diethylnitrosamine administration was clearly increased in the normal saline (control) group compared with groups given roxithromycin 100 mg/kg or TNP-470 50 mg/kg (Fig. 5). In addition, the effect of roxithromycin 40 mg/kg was smaller than that of the higher dose for the tissue malondialdehyde level.

The protocol of diethylnitrosamine-induced liver injury used in the present study caused the sequential formation of cirrhosis and hepatocellular carcinoma. In this model, we show that roxithromycin (100 mg/kg) administration inhibits oxidative stress, NF-κB activation, and iNOS activity, and reduces tumor formation in the liver. These beneficial effects were very similar to that of TNP-470 (50 mg/kg). All these effects were absent in animals receiving normal saline only. It has been shown that TNP-470 and roxithromycin had an inhibitory effect against tumor growth by inhibition of tumor angiogenesis (8, 10). Furthermore, this study indicates that roxithromycin could have an additional antitumor effect similar to that of TNP-470 reported by Mauriz et al. (11).

Using the Mdr2-knockout mice that develop cholestatic hepatitis and hepatocellular carcinoma, Pikarsky et al. (23) reported that NF-κB is essential for promoting inflammation-associated cancer, and is therefore a potential target for cancer prevention in chronic inflammatory diseases. Furthermore, Futakuchi et al. (24) showed that NF-κB inhibitors (e.g., pentoxifylline) have the potential to inhibit distant metastasis from rat hepatocellular carcinomas in vivo and its mechanism may involve suppression of vascular cell adhesion molecule 1 and vascular endothelial growth factor A188. Chan et al. (25) also indicated that NF-κB dysregulation and overexpression of urokinase plasminogen activator (one of the downstream target genes of NF-κB) may lead to a more aggressive tumor behavior in human hepatocellular carcinomas. It is known that reactive oxygen species produced by several mechanisms (e.g., NADPH oxidase, mitochondrial electron transport, and NOS) act as small second messenger molecules and key elements to activate NF-κB in the early events of inflammation. Thus, to prevent hepatocarcinogenesis correlating with inflammation, it may be important to mediate oxidative stress and the following NF-κB activation, as shown by the present study.

In several human gastrointestinal neoplasms including hepatitis C virus–associated hepatocellular carcinoma, the immunologic (type II) isoform of NOS (iNOS) generates nitric oxide from l-arginine in inflamed tissues and is elevated (26). Nitric oxide causes DNA breaks and enhances DNA mutations, especially in hepatitis C virus–associated hepatocellular carcinoma (27). Because cloning and functional analysis of the human iNOS gene promoter have identified several copies of NF-κB response elements and several copies of activator protein 1 binding sites, roxithromycin could affect the inactivation of iNOS, as reported in the present study and our former studies (9). Furthermore, iNOS and NADPH oxidase are major sources of reactive oxygen species in experimental hepatocarcinogenesis (28). The produced reactive oxygen species could again secondarily lead to NF-κB activation (see Fig. 6).

The mechanisms of action for the anti-inflammatory properties of macrolides such as roxithromycin are still being investigated, but they are clearly multifactorial (29). Many researchers have shown that an important aspect of inflammation is extravasation of neutrophils into tissues (30–32). Macrolides inhibit the production of many proinflammatory cytokines such as interleukin 1, interleukin 6, interleukin 8, and tumor necrosis factor α, perhaps by suppressing the transcription factor NF-κB or activator protein 1 (9). Inhibition of cytokine production has been observed in vitro and also in bronchoalveolar lavage fluid, which contains less interleukin 8 and fewer neutrophils after treatment with macrolides. Thus, at least, inactivation of neutrophils may also be one of the possible mechanisms to explain the effectiveness of roxithromycin on oxidative stress and NF-κB inactivation.

In conclusion, the present study indicates that roxithromycin inhibits oxidative stress, nitric oxide production, and NF-κB activation induced by experimental hepatocarcinogenesis. Because roxithromycin is widely used in clinical practice, it may represent an effective new strategy for human hepatoma therapy.