- Email: [email protected]
Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, inflammation, cellular gene products and cell-cycle-related proteins in rats
Food and Chemical Toxicology 38 (2000) 991±995
www.elsevier.com/locate/foodchemtox
Research Section Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, in¯ammation, cellular gene products and cell-cycle-related proteins in rats SHUANG-EN CHUANG 1,*, ANN-LII CHENG 2, JEN-KUN LIN 3 and MIN-LIANG KUO 4 1 Division of Cancer Research, National Health Research Institute, Taipei, 2Department of Internal Medicine, National Taiwan University Hospital, Taipei, 3Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, and 4Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan
(Accepted 1 June 2000) AbstractÐCurcumin (CCM), a major yellow pigment of turmeric obtained from powdered rhizomes of the plant Curcuma longa Linn, is commonly used as coloring agent in foods, drugs and cosmetics. In this study we report that gavage administration of 200 mg/kg or 600 mg/kg CCM eectively suppressed diethylnitrosamine (DEN)-induced liver in¯ammation and hyperplasia in rats, as evidenced by histopathological examination. Immunoblotting analysis showed that CCM strongly inhibited DEN-mediated the increased expression of oncogenic p21ras and p53 proteins in liver tissues of rats. In cell-cycle-related proteins, CCM selectively reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin E and p34cdc2, but not Cdk2 or cyclin D1. Moreover, CCM also inhibited the DEN-induced increase of transcriptional factor NFkappa B. However, CCM failed to aect DEN-induced c-Jun and c-Fos expression. It has become widely recognized that the development of human hepatocellular carcinoma (HCC) is predominantly due to the chronic in¯ammation by virus, bacteria or chemical. Our results suggest a potential role for CCM in the prevention of HCC. # 2000 Elsevier Science Ltd. All rights reserved
Abbreviations: CCM=curcumin (diferuloylmethane); DEN=diethylnitrosamine; HCC=human hepatocellular carcinoma; PCNA=proliferating cell nuclear antigen; TPA=12-O-tetradecanoylphorbol-13-acetate.
INTRODUCTION
The role in carcinogenesis of prolonged cellular damage such as viral-, bacterial- or chemical-related chronic in¯ammations has become widely recognized (Higaki et al., 1999; Ohshima and Bartsch, 1994). It has been proposed that active oxygen species and some cytokines generated in in¯amed tissues can cause injury to DNA of target cells and result in survival of damaged cells that ultimately leading to carcinogenesis (Calmels et al., 1997). Epidemiological *Corresponding author: Laboratory of Molecular & Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, No.1, Sec., 1, Jen-Ai Road, Taipei, Taiwan.
and laboratory studies indicate that chronic in¯ammation and hyperplasia induced by viral hepatitis B and C, or nitroso compounds are closely associated with the development of HCC (Ahn et al., 1999; Beasley et al., 1981). HCC is the most important and leading cause of cancer death in Taiwan (Yu et al., 1991). Many people who carried hepatitis B or C virus developed chronic hepatitis and became a high risk for HCC (Chen and Chen, 1999). Changes in expression of several cellular gene products such as p21ras, p53, proliferating cell nuclear antigen (PCNA) and some cell-cycle-related proteins have been found to express the in¯ammatory and hyperplasia processes of certain cancers in human and rats (Higekis et al., 1999; Nardone et al., 1999; Tsunematsu et al., 1997). Interestingly, these genes were also highly
0278-6915/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(00)00101-0
992
S.-E. Chuang et al.
expressed in fully malignant tumors (Heinze et al., 1999; Ferreira-Gonzalez et al., 1995). It suggests that these oncogenic products and cell-cycle-related proteins could play an important role in the in¯ammationrelated cancers. Curcumin (diferuloylmethane; CCM), a major component of the food ¯avoring turmeric, is isolated from the rhizomes of Curcuma longa. CCM has been widely used for the treatment of a variety of in¯ammatory conditions and other diseases (Ammon and Wahl, 1991; Nadkarani et al., 1976). CCM is also a potent inhibitor of the initiation and promotion of chemical carcinogen-induced tumor formation in animals (Huang et al., 1992b, 1998). Huang et al. have shown that topical application of CCM to the backs of CD-1 mice markedly inhibited 12-O-tetradecanoylphorbol13-acetate (TPA)-induced in¯ammation, ornithine decarboxylase activity, DNA synthesis, hyperplasia, hydrogen peroxide formation in mouse epidermis (Huang et al., 1992a). CCM blocked cytokineinduced NF-kappa B activation and the subsequent downstream gene such as iNOS in macrophages and other immune cells (Brouet and Ohshima, 1995; Chan et al., 1998). These studies suggest that CCM prevents the carcinogenic process possibly by ameliorating the in¯ammation-related events. Based on these observations, we wished to know whether CCM could aect hepatic in¯ammation in animals. In the present study we report the inhibitory eects of CCM on DEN-induced hepatic in¯ammation and hyperplasia. We also monitored the levels of several cellular targets possibly in¯uenced by CCM treatment including oncogenic and cell-cycle-related proteins, which have been found to be associated with the development of hepatocellular carcinoma.
MATERIALS AND METHODS
Animal and treatment 4-wk-old male Wistar rats were kept on a standard rat chow diet. Groups of six or eight rats were given either corn oil vehicle (2 mg/kg) or 150 mg/kg DEN by ip injection once a week for 4 wk. 1 day before DEN treatment, two dierent doses, 200 mg/kg or 600 mg/kg, of CCM were given to two of the groups of rats by gavage. One group was given 600 mg/kg CCM only. On day 40, animals were fasted overnight before sacri®ce. Animals were killed, after CO2 anesthesia, by decapitation and the livers were immediately removed, weighed and placed in ice-cold phosphate buered saline. Parts of the livers were homogenized in RIPA lysis buer, the other parts were ®xed in formalin solution for pathological examination. Western blotting Total cellular proteins were prepared as described previously (Kuo et al., 1996). A 50-mg sample of each
lysate was subjected to electrophoresis on 10% SDSpolyacrylamide gels for detection of following cellular proteins: P21ras, p53, PCNA, c-Jun, c-Fos, CDC2, Cdk1, NF-kB and a-tubulin. The samples were then electrotransferred onto nitrocellulose paper. After blocking, blots were incubated with antibodies in PBST for 1 hr, followed by two washes in PBST (PBS containing 0.1% Tween 20), and then incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 30 min. After washing, the blots were incubated for 1 min with Western blotting reagent ECL, and chemiluminescence was detected by exposure of the ®lters to Kodak X-omat ®lms for 10 sec to 10 min. Histopathologic examination After treatment, the liver tissues were removed and ®xed in a formalin solution. Paran embedding of the tissues was then conducted following dehydration. Liver sections were stained with hematoxylin and eosin solution for light microscopic examinations to evaluate hepatic in¯ammation and hyperplasia.
RESULTS
The average body weight, liver weight and relative liver weight in each group of rats at the end of the experiment are shown in Table 1. There was no signi®cant dierence in average body weight between groups. The liver weight and relative liver weight of DEN-treated rats were increased by 33% and 35%, respectively, when compared to that of control rats. This increase may be due to the development of hepatic regeneration and hyperplasia. This DENinduced-hepatomegaly was almost completely prevented by CCM. Further examination of the histopathologic patterns of liver tissues from DEN-treated rats revealed a markedly cellular proliferation and dilation of bile ducts with neutrophilic in®ltration into the parenchyma and periductal regions (Plate 1B) when compared to livers of control rats (Plate 1A). The DEN-induced hepatic pathological alterations were Table 1. Eect of curcumin on liver and relative liver weights of male Wistar rats Group treatment
No treatment DEN DEN+CCM (L)b DEN+CCM (H)b CCM (H) a
Relative liver Liver No. of rats Body examined wtÿ1 (g)a wtÿ1 (g) wtÿ1 (g/1000 g body wt)a 6 8 8 8 6
19018 8.51.2 18715 11.31.7 19512 8.81.4 18917 8.42.1 18617 8.42.1
4.20.23 6.040.85 4.510.71c 4.440.84c 4.520.86
Mean SD. (L) Low curcumin dose treatment, 200 mg/g body weight. (H) High curcumin dose treatment, 600 mg/kg body weight. Signi®cantly dierent from DEN group by Student's t-test, P<0.01.
b c
Anti-in¯ammatory eect of curcumin
993
Plate 1. Representative liver sections from rats treating with (A) control (corn oil vehicle), (B) 150 mg/kg DEN, (C) DEN plus 200 mg/kg CCM or (D) DEN plus 600 mg/kg CCM. Liver sections from DENtreated rats revealed a remarkable hyperplasia and dilation of bile ducts with neutrophilic in®ltration into the parenchymal and periductal regions. H&E staining.
eectively inhibited in the rats fed either with the low (200 mg/kg) or high dose (600 mg/kg) of CCM (Plate 1C, D). There were no signi®cant pathological changes of liver tissue observed in rats fed CCM alone (data not shown). To determine precisely these hepatic alterations induced by DEN, we examined whether some oncogenic and cell-cycle-related proteins would be aected during the experiment. The representative data of immunoblotting showed that the oncogenic p21ras and p53 proteins were induced in liver tissues of rats treated with DEN (Plate 2, lanes 3 and 4). Administration of 200 mg/kg or 600 mg/kg CCM all eectively reduced DEN-induced increase in both p21ras and p53 proteins in rat livers (Plate 2, lanes 5±10). In the analysis of cell-cycle-related proteins, we found that PCNA, p34cdc2 and cyclin E were prominently
Plate 2. CCM inhibits DEN-induced the expression of p53 and p21ras proteins. Immunoblot analysis of p53 and p21ras proteins in rats treated with vehicle only (lanes 1 and 2), 150 mg/kg DEN (lanes 3 and 4), DEN plus 600 mg/kg CCM (lanes 5,6, and 7), or DEN plus 200 mg/kg (lanes 8,9, and 10). Tissue lysates were prepared and immunoblotting performed as described in Materials and Methods.
induced in DEN-treated rat livers; however, the administration of CCM strongly inhibited the DENinduced increased expression of cell-cycle-related proteins (Plate 3). In contrast, other cell-cycle-related proteins such as Cdk2 and cyclin D1 were not altered in livers of rats given DEN, CCM, or DEN plus CCM (Plate 3). Transcriptional factors NF-kB, cJun and c-Fos were all elevated in DEN-treated rats, but only the expression of NF-kB was diminished by CCM (Plate 4).
Plate 3. Eect of CCM on the DEN-induced the expression of cell-cycle regulators. Rats were treated with vehicle only (lanes 1 and 2), DEN (lanes 3 and 4 ), DEN plus high-dose CCM (lanes 5 and 6 ) or DEN plus low-dose CCM (lanes 7 and 8). The expression of cell-cycle regulators was determined by immunoblotting as described in Materials and Methods.
994
S.-E. Chuang et al.
Plate 4. Eect of CCM on the level of transcriptional factors in DEN-fed rats. Rats were treated similarly to Plate 3. Immunoblotting was performed as described in Materials and Methods. Rats were treated with vehicle only (lanes 1 and 2), DEN (lanes 3 and 4), DEN plus high-dose CCM (lanes 5 and 6) or DEN plus low-dose CCM (lanes 7 and 8). DISCUSSION
The results in the present study demonstrate that the induction of liver hyperplasia and in¯ammation with a high dose of DEN was inhibited by CCM. To the best of our knowledge this is the ®rst report to demonstrate the inhibitory activity of CCM on the chemical induction of hepatic in¯ammation and hyperplasia in rats. During the DEN-induced hepatic in¯ammation and hyperplasia, expression of Ras and p53 proteins were evidently elevated. Consistent with this study, Goodwin and Grizzle (1994) have found that Ras and p53 were up-regulated during hepatic in¯ammation and regeneration in DEN-exposed ®sh (Goodwin and Grizzle, 1994). Similar observations have been made in other studies (Herbst et al., 1991; Servais et al., 1990), revealing that many oncogenic proteins including p53 and p21ras were expressed in rat livers after CCl4 administration. Increased p21ras and p53 levels during the in¯ammatory process may indicate their important roles in in¯ammation-related carcinogenesis (Puig et al., 1992). It is probable that either in¯ammation per se, or DNA damage caused by DEN, or both, can induce expression of these genes. It has been demonstrated repeatedly that DNA damage indeed can stimulate transcription of the p53 gene, as well as increase the stability of the p53 protein (Chehab et al., 1999). Oxidative stress is also known to play a key role in the expression of p21ras and p53 as well as in the recruitment of neutrophils to the in¯ammatory regions (Bertini et al., 1999; Deora et al., 1998). Here we demonstrate that CCM at concentrations of 200 mg/kg or 600 mg/kg could eectively inhibit DEN-induced p53 and p21ras expression. These eects probably are mediated through CCM's anti-in¯ammatory and anti-oxidant activity. Cell-cycle-related proteins are closely associated with the hyperplasia in liver of rats (Song et al., 1999). CCM appears to reduce selectively the expression of PCNA, p34cdc2 and cyclin E, but not Cdk2 and cyclin D1. PCNA and p34cdc2 functions at S and G1, G2 phase, respectively, suggesting that CCM interferes with the cell-cycle progression, and
that this may be mediated through reducing the function or level of some speci®c cell-cycle regulators. Exposure of rats to DEN can stimulate the expression of some immediate early response genes such as c-Jun and c-Fos (Giri and Das, 1996). In agreement with other studies, we also found here that DEN treatment could elicit a prominent expression of c-Jun and c-Fos proteins in liver tissues of rats. However, CCM did not aect DEN-induced c-Jun and c-Fos expression. This ®nding apparently diered from our previous studies showing that CCM was capable of inhibiting phorbol ester-mediated c-Jun and c-Fos expression in NIH 3T3 cells (Huang et al., 1991). This dierence is possibly due to (1) dierent induction mechanisms of c-Jun and c-Fos by TPA and DEN; or (2) inherently dierent properties between the in vitro and in vivo system. Recently, it has become clear that chronic hepatocellular injury caused by chemicals or hepatitis virus can lead to an in¯ammatory response and subsequent regenerative hyperplasia, and eventually to the development of HCC. Thus, our present study suggests that CCM, proven to be eective in antiin¯ammation and anti-hyperplasia, may be of potential use in the prevention of HCC. AcknowledgementÐThis study was supported by the grant of National Science Council, NSC 85-B-002201, Taipei, Taiwan. REFERENCES
Ahn B., Han B. S., Kim D. J. and Ohshima H. (1999) Immunohistochemical localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors iduced by Nnitrosodiethylamine. Carcinogenesis 20, 1337±1344. Ammon H. P. T. and Wahl M. A. (1991) Pharmacology of Curcoma longa. Planta Medica 57, 1±7. Beasley R. P., Lin C. C., Hwang L. Y. and Chen C. S. (1981) Hepatocellular carcinoma and hepatitis B virus: a prospective study of 22,707 men in Taiwan. Lancet 2, 1129±1133. Bertini R., Howard O. M., Dong H. F., Oppenheim J. J. and Ghezzi P. (1999) Thioredoxin, a redox enzyme released in infection and in¯ammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. Journal of Experimental Medicine 189, 1783±1789. Brouet I. and Oshima H. (1995) Curcumin, an anti-tumor promotor and anti-in¯ammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochemical and Biophysical Research Communications 206, 533±540. Calmels S., Hainaunt P. and Ohshima H. (1997) Nitric oxide induces conformational and functional modi®cations of wild-type p53 tumor suppressor protein. Cancer Research 57, 3365±3369. Chan M. M., Huang H. I., Fenton M. R. and Fong D. (1998) In vitro inhibition of nitric oxide synthase gene expressions by curcumin, a cancer preventive natural product with anti-in¯ammatory properties. Biochemical Pharnacology 55, 1955±1962. Chehab N. H., Malikzay A., Stavridi E. S. and Halazonetis T. D. (1999) Phosphorylation of Ser-20 mediates stabili-
Anti-in¯ammatory eect of curcumin zation of human p53 in response to DNA damage. Proceedings of the National Academy of Sciences of the U.S.A. 96, 13777±13782. Chen P. J. and Chen D. S. (1999) Hepatitis B virus infection and hepatocelluar carcinoma: molecular genetics and clinical perspectives. Seminars in Liver Disease 19, 253± 262. Deora A. A., Win T., Vanhaesebroeds B. and Lander H. M. (1998) A redox-triggered ras-eector interaction. Recruitment of phosphatidylinositol-30 -kinase to Ras by redox stress. Journal of Biological Chemistry 273, 29923±29928. Ferreira-Gonzalez A., DeAngelo A. B., Nasim S. and Garrett C. T. (1995) Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids. Carcinogenesis 16, 495±500. Giri R. K. and Das B. R. (1996) Dierential expression of cjun and c-myc in N-nitrosodiethylamine-induced hepatic oncogenesis in ARK mice. Cancer Letters 109, 121±127. Goodwin A. E. and Grizzle J. M. (1994) Oncogene expression in hepatocytes of the ®sh Rivulus ocellatus marmoratus during the necrotic and regenerative phases of dimethylnitrosamine toxicity. Carcinogenesis 15, 1985±1992. Heinze T., Jonas S., Karsten A. and Neuhaus P. (1999) Determination of the oncogenes p53 and c-erbB2 in the tumor cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time. Anticancer Research 19, 2501±2503. Herbst H., Milani S., Schuppan D. and Stein H. (1991) Temporal and spatial patterns of proto-oncogene expression at early stages of toxic liver injury in the rat. Laboratory Investigation 65, 324±333. Higaki S., Akazawa A., Nakamura H., Yauai H., Yoshida T. and Okita K. (1999) Metaplastic polyp of the colon develops in response to in¯ammation. Journal of Gastroenterology and Hepatology 14, 709±714. Huang M. T., Lysz T., Ferraro T. and Conney A. H. (1992a) Inhibitory eects of curcumin on tumor promotion and arachidonic acid metabolism in mouse epidermis. In Cancer Prevention, ed. L. Watternberg, M. Lipkin, C. W. Boone and G. J. Kello, pp. 375±391. CRC Press, Boca Raton, FL. Huang M. T., Smart R. C., Wang G. Q. and Conney A. H. (1998) Inhibitory eect of curcuma, chlorogenic acid, caeric acid, and ferulic acid on tumor promotion in mouse skin by 12-0-tetradecanoylphorbol-13-acetate. Cancer Research 48, 5941±5946. Huang M. T., Wang Z. R., Georgiadis C. A., Laskin J. D. and Comey A. H. (1992b) Inhibitory eect of curcuma on
995
tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. Carcinogenesis 13, 2183±2186. Huang T. S., Lee S. C. and Lin J. K. (1991) Suppression of the c-Jun/AP-1 activation by an inhibitor of tumor formation in mouse ®broblast cells. Proceedings of the National Academy of Sciences of the U.S.A. 88, 5292± 5296. Kuo M. L., Huang T. S. and Lin J. K. (1996) Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in hman leukemia cells. Biochimica et Biophysica Acta 1317, 95±100. Nadkarani K. M. (1976) Curcuma longa. In India Materia Medica, ed. K.M. Nadkarani, pp. 414±416. Popular Praleasham Publishing Co., Bombay. Nardone G., Staibano S., Rocco A., Mezza E., D'armiento F. P., Unsabato l., Coppla A., Salvatore G., Lucariello A., Figura N., DeRosa G. and Budillon G. (1999) Eect of helicobacter pylori infection and its eradication on cell proliferation, DNA safety, and oncogene expression in patients with chronic gastritis. Gut 44, 789±799. Ohshima H. and Bartsch H. (1994) Chronic infectious and in¯ammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutation Research 305, 253±264. Puig P. L., Flejou J. F., Fabre M., Bedossa P., Belghiti J., Gayral F. and Franco D. (1992) Overexpression of p53: a rare event in a large series of white patients with hepatocelluar carcinoma. Hepatology 16, 1171±1175. Servais P., Taton G. and Galand P. (1990) Modulation by indomethacin of dithylnitrosamine-induced early changes in c-myc and c-ras expression and late incidence of preneoplastic lesions in rat liver. Carcinogenesis 11, 1433± 1436. Song K. Y., Lin I. K., Park S. C., Lee S. O., Park H. S., Choi Y. K. and Hyun B. H. (1999) Eect of modulation on the expression of glutathione s-transferase placental form and proliferating cell nuclear antigen in N-nitrosodiethylamine initiated hepatocarcinogenesis in the male Fischer 344 rat. Carcinogenesis 20, 1541±1548. Tsunematsu S., Satio H., Tada S., Ebinuma H., Tsuchiya M., Kumagai N., Morizane T., Nomura T. and Ischii H. (1997) Susceptibility of experimental autoimmune hepatitis in transgenic mice overexpressing the C-H-ras gene. Journal of Gastroenterology and Hepatology 12, 319±324. Yu M. W., You S. L., Chang A. S., Lu S. N., Liaw Y. F. and Chen C. J. (1991) Association between hepatitis C virus antibodies and hepatocelluar carcinoma in Taiwan. Cancer Research 51, 5621±5625.
www.elsevier.com/locate/foodchemtox
Research Section Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, in¯ammation, cellular gene products and cell-cycle-related proteins in rats SHUANG-EN CHUANG 1,*, ANN-LII CHENG 2, JEN-KUN LIN 3 and MIN-LIANG KUO 4 1 Division of Cancer Research, National Health Research Institute, Taipei, 2Department of Internal Medicine, National Taiwan University Hospital, Taipei, 3Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, and 4Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan
(Accepted 1 June 2000) AbstractÐCurcumin (CCM), a major yellow pigment of turmeric obtained from powdered rhizomes of the plant Curcuma longa Linn, is commonly used as coloring agent in foods, drugs and cosmetics. In this study we report that gavage administration of 200 mg/kg or 600 mg/kg CCM eectively suppressed diethylnitrosamine (DEN)-induced liver in¯ammation and hyperplasia in rats, as evidenced by histopathological examination. Immunoblotting analysis showed that CCM strongly inhibited DEN-mediated the increased expression of oncogenic p21ras and p53 proteins in liver tissues of rats. In cell-cycle-related proteins, CCM selectively reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin E and p34cdc2, but not Cdk2 or cyclin D1. Moreover, CCM also inhibited the DEN-induced increase of transcriptional factor NFkappa B. However, CCM failed to aect DEN-induced c-Jun and c-Fos expression. It has become widely recognized that the development of human hepatocellular carcinoma (HCC) is predominantly due to the chronic in¯ammation by virus, bacteria or chemical. Our results suggest a potential role for CCM in the prevention of HCC. # 2000 Elsevier Science Ltd. All rights reserved
Abbreviations: CCM=curcumin (diferuloylmethane); DEN=diethylnitrosamine; HCC=human hepatocellular carcinoma; PCNA=proliferating cell nuclear antigen; TPA=12-O-tetradecanoylphorbol-13-acetate.
INTRODUCTION
The role in carcinogenesis of prolonged cellular damage such as viral-, bacterial- or chemical-related chronic in¯ammations has become widely recognized (Higaki et al., 1999; Ohshima and Bartsch, 1994). It has been proposed that active oxygen species and some cytokines generated in in¯amed tissues can cause injury to DNA of target cells and result in survival of damaged cells that ultimately leading to carcinogenesis (Calmels et al., 1997). Epidemiological *Corresponding author: Laboratory of Molecular & Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, No.1, Sec., 1, Jen-Ai Road, Taipei, Taiwan.
and laboratory studies indicate that chronic in¯ammation and hyperplasia induced by viral hepatitis B and C, or nitroso compounds are closely associated with the development of HCC (Ahn et al., 1999; Beasley et al., 1981). HCC is the most important and leading cause of cancer death in Taiwan (Yu et al., 1991). Many people who carried hepatitis B or C virus developed chronic hepatitis and became a high risk for HCC (Chen and Chen, 1999). Changes in expression of several cellular gene products such as p21ras, p53, proliferating cell nuclear antigen (PCNA) and some cell-cycle-related proteins have been found to express the in¯ammatory and hyperplasia processes of certain cancers in human and rats (Higekis et al., 1999; Nardone et al., 1999; Tsunematsu et al., 1997). Interestingly, these genes were also highly
0278-6915/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(00)00101-0
992
S.-E. Chuang et al.
expressed in fully malignant tumors (Heinze et al., 1999; Ferreira-Gonzalez et al., 1995). It suggests that these oncogenic products and cell-cycle-related proteins could play an important role in the in¯ammationrelated cancers. Curcumin (diferuloylmethane; CCM), a major component of the food ¯avoring turmeric, is isolated from the rhizomes of Curcuma longa. CCM has been widely used for the treatment of a variety of in¯ammatory conditions and other diseases (Ammon and Wahl, 1991; Nadkarani et al., 1976). CCM is also a potent inhibitor of the initiation and promotion of chemical carcinogen-induced tumor formation in animals (Huang et al., 1992b, 1998). Huang et al. have shown that topical application of CCM to the backs of CD-1 mice markedly inhibited 12-O-tetradecanoylphorbol13-acetate (TPA)-induced in¯ammation, ornithine decarboxylase activity, DNA synthesis, hyperplasia, hydrogen peroxide formation in mouse epidermis (Huang et al., 1992a). CCM blocked cytokineinduced NF-kappa B activation and the subsequent downstream gene such as iNOS in macrophages and other immune cells (Brouet and Ohshima, 1995; Chan et al., 1998). These studies suggest that CCM prevents the carcinogenic process possibly by ameliorating the in¯ammation-related events. Based on these observations, we wished to know whether CCM could aect hepatic in¯ammation in animals. In the present study we report the inhibitory eects of CCM on DEN-induced hepatic in¯ammation and hyperplasia. We also monitored the levels of several cellular targets possibly in¯uenced by CCM treatment including oncogenic and cell-cycle-related proteins, which have been found to be associated with the development of hepatocellular carcinoma.
MATERIALS AND METHODS
Animal and treatment 4-wk-old male Wistar rats were kept on a standard rat chow diet. Groups of six or eight rats were given either corn oil vehicle (2 mg/kg) or 150 mg/kg DEN by ip injection once a week for 4 wk. 1 day before DEN treatment, two dierent doses, 200 mg/kg or 600 mg/kg, of CCM were given to two of the groups of rats by gavage. One group was given 600 mg/kg CCM only. On day 40, animals were fasted overnight before sacri®ce. Animals were killed, after CO2 anesthesia, by decapitation and the livers were immediately removed, weighed and placed in ice-cold phosphate buered saline. Parts of the livers were homogenized in RIPA lysis buer, the other parts were ®xed in formalin solution for pathological examination. Western blotting Total cellular proteins were prepared as described previously (Kuo et al., 1996). A 50-mg sample of each
lysate was subjected to electrophoresis on 10% SDSpolyacrylamide gels for detection of following cellular proteins: P21ras, p53, PCNA, c-Jun, c-Fos, CDC2, Cdk1, NF-kB and a-tubulin. The samples were then electrotransferred onto nitrocellulose paper. After blocking, blots were incubated with antibodies in PBST for 1 hr, followed by two washes in PBST (PBS containing 0.1% Tween 20), and then incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 30 min. After washing, the blots were incubated for 1 min with Western blotting reagent ECL, and chemiluminescence was detected by exposure of the ®lters to Kodak X-omat ®lms for 10 sec to 10 min. Histopathologic examination After treatment, the liver tissues were removed and ®xed in a formalin solution. Paran embedding of the tissues was then conducted following dehydration. Liver sections were stained with hematoxylin and eosin solution for light microscopic examinations to evaluate hepatic in¯ammation and hyperplasia.
RESULTS
The average body weight, liver weight and relative liver weight in each group of rats at the end of the experiment are shown in Table 1. There was no signi®cant dierence in average body weight between groups. The liver weight and relative liver weight of DEN-treated rats were increased by 33% and 35%, respectively, when compared to that of control rats. This increase may be due to the development of hepatic regeneration and hyperplasia. This DENinduced-hepatomegaly was almost completely prevented by CCM. Further examination of the histopathologic patterns of liver tissues from DEN-treated rats revealed a markedly cellular proliferation and dilation of bile ducts with neutrophilic in®ltration into the parenchyma and periductal regions (Plate 1B) when compared to livers of control rats (Plate 1A). The DEN-induced hepatic pathological alterations were Table 1. Eect of curcumin on liver and relative liver weights of male Wistar rats Group treatment
No treatment DEN DEN+CCM (L)b DEN+CCM (H)b CCM (H) a
Relative liver Liver No. of rats Body examined wtÿ1 (g)a wtÿ1 (g) wtÿ1 (g/1000 g body wt)a 6 8 8 8 6
19018 8.51.2 18715 11.31.7 19512 8.81.4 18917 8.42.1 18617 8.42.1
4.20.23 6.040.85 4.510.71c 4.440.84c 4.520.86
Mean SD. (L) Low curcumin dose treatment, 200 mg/g body weight. (H) High curcumin dose treatment, 600 mg/kg body weight. Signi®cantly dierent from DEN group by Student's t-test, P<0.01.
b c
Anti-in¯ammatory eect of curcumin
993
Plate 1. Representative liver sections from rats treating with (A) control (corn oil vehicle), (B) 150 mg/kg DEN, (C) DEN plus 200 mg/kg CCM or (D) DEN plus 600 mg/kg CCM. Liver sections from DENtreated rats revealed a remarkable hyperplasia and dilation of bile ducts with neutrophilic in®ltration into the parenchymal and periductal regions. H&E staining.
eectively inhibited in the rats fed either with the low (200 mg/kg) or high dose (600 mg/kg) of CCM (Plate 1C, D). There were no signi®cant pathological changes of liver tissue observed in rats fed CCM alone (data not shown). To determine precisely these hepatic alterations induced by DEN, we examined whether some oncogenic and cell-cycle-related proteins would be aected during the experiment. The representative data of immunoblotting showed that the oncogenic p21ras and p53 proteins were induced in liver tissues of rats treated with DEN (Plate 2, lanes 3 and 4). Administration of 200 mg/kg or 600 mg/kg CCM all eectively reduced DEN-induced increase in both p21ras and p53 proteins in rat livers (Plate 2, lanes 5±10). In the analysis of cell-cycle-related proteins, we found that PCNA, p34cdc2 and cyclin E were prominently
Plate 2. CCM inhibits DEN-induced the expression of p53 and p21ras proteins. Immunoblot analysis of p53 and p21ras proteins in rats treated with vehicle only (lanes 1 and 2), 150 mg/kg DEN (lanes 3 and 4), DEN plus 600 mg/kg CCM (lanes 5,6, and 7), or DEN plus 200 mg/kg (lanes 8,9, and 10). Tissue lysates were prepared and immunoblotting performed as described in Materials and Methods.
induced in DEN-treated rat livers; however, the administration of CCM strongly inhibited the DENinduced increased expression of cell-cycle-related proteins (Plate 3). In contrast, other cell-cycle-related proteins such as Cdk2 and cyclin D1 were not altered in livers of rats given DEN, CCM, or DEN plus CCM (Plate 3). Transcriptional factors NF-kB, cJun and c-Fos were all elevated in DEN-treated rats, but only the expression of NF-kB was diminished by CCM (Plate 4).
Plate 3. Eect of CCM on the DEN-induced the expression of cell-cycle regulators. Rats were treated with vehicle only (lanes 1 and 2), DEN (lanes 3 and 4 ), DEN plus high-dose CCM (lanes 5 and 6 ) or DEN plus low-dose CCM (lanes 7 and 8). The expression of cell-cycle regulators was determined by immunoblotting as described in Materials and Methods.
994
S.-E. Chuang et al.
Plate 4. Eect of CCM on the level of transcriptional factors in DEN-fed rats. Rats were treated similarly to Plate 3. Immunoblotting was performed as described in Materials and Methods. Rats were treated with vehicle only (lanes 1 and 2), DEN (lanes 3 and 4), DEN plus high-dose CCM (lanes 5 and 6) or DEN plus low-dose CCM (lanes 7 and 8). DISCUSSION
The results in the present study demonstrate that the induction of liver hyperplasia and in¯ammation with a high dose of DEN was inhibited by CCM. To the best of our knowledge this is the ®rst report to demonstrate the inhibitory activity of CCM on the chemical induction of hepatic in¯ammation and hyperplasia in rats. During the DEN-induced hepatic in¯ammation and hyperplasia, expression of Ras and p53 proteins were evidently elevated. Consistent with this study, Goodwin and Grizzle (1994) have found that Ras and p53 were up-regulated during hepatic in¯ammation and regeneration in DEN-exposed ®sh (Goodwin and Grizzle, 1994). Similar observations have been made in other studies (Herbst et al., 1991; Servais et al., 1990), revealing that many oncogenic proteins including p53 and p21ras were expressed in rat livers after CCl4 administration. Increased p21ras and p53 levels during the in¯ammatory process may indicate their important roles in in¯ammation-related carcinogenesis (Puig et al., 1992). It is probable that either in¯ammation per se, or DNA damage caused by DEN, or both, can induce expression of these genes. It has been demonstrated repeatedly that DNA damage indeed can stimulate transcription of the p53 gene, as well as increase the stability of the p53 protein (Chehab et al., 1999). Oxidative stress is also known to play a key role in the expression of p21ras and p53 as well as in the recruitment of neutrophils to the in¯ammatory regions (Bertini et al., 1999; Deora et al., 1998). Here we demonstrate that CCM at concentrations of 200 mg/kg or 600 mg/kg could eectively inhibit DEN-induced p53 and p21ras expression. These eects probably are mediated through CCM's anti-in¯ammatory and anti-oxidant activity. Cell-cycle-related proteins are closely associated with the hyperplasia in liver of rats (Song et al., 1999). CCM appears to reduce selectively the expression of PCNA, p34cdc2 and cyclin E, but not Cdk2 and cyclin D1. PCNA and p34cdc2 functions at S and G1, G2 phase, respectively, suggesting that CCM interferes with the cell-cycle progression, and
that this may be mediated through reducing the function or level of some speci®c cell-cycle regulators. Exposure of rats to DEN can stimulate the expression of some immediate early response genes such as c-Jun and c-Fos (Giri and Das, 1996). In agreement with other studies, we also found here that DEN treatment could elicit a prominent expression of c-Jun and c-Fos proteins in liver tissues of rats. However, CCM did not aect DEN-induced c-Jun and c-Fos expression. This ®nding apparently diered from our previous studies showing that CCM was capable of inhibiting phorbol ester-mediated c-Jun and c-Fos expression in NIH 3T3 cells (Huang et al., 1991). This dierence is possibly due to (1) dierent induction mechanisms of c-Jun and c-Fos by TPA and DEN; or (2) inherently dierent properties between the in vitro and in vivo system. Recently, it has become clear that chronic hepatocellular injury caused by chemicals or hepatitis virus can lead to an in¯ammatory response and subsequent regenerative hyperplasia, and eventually to the development of HCC. Thus, our present study suggests that CCM, proven to be eective in antiin¯ammation and anti-hyperplasia, may be of potential use in the prevention of HCC. AcknowledgementÐThis study was supported by the grant of National Science Council, NSC 85-B-002201, Taipei, Taiwan. REFERENCES
Ahn B., Han B. S., Kim D. J. and Ohshima H. (1999) Immunohistochemical localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors iduced by Nnitrosodiethylamine. Carcinogenesis 20, 1337±1344. Ammon H. P. T. and Wahl M. A. (1991) Pharmacology of Curcoma longa. Planta Medica 57, 1±7. Beasley R. P., Lin C. C., Hwang L. Y. and Chen C. S. (1981) Hepatocellular carcinoma and hepatitis B virus: a prospective study of 22,707 men in Taiwan. Lancet 2, 1129±1133. Bertini R., Howard O. M., Dong H. F., Oppenheim J. J. and Ghezzi P. (1999) Thioredoxin, a redox enzyme released in infection and in¯ammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. Journal of Experimental Medicine 189, 1783±1789. Brouet I. and Oshima H. (1995) Curcumin, an anti-tumor promotor and anti-in¯ammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochemical and Biophysical Research Communications 206, 533±540. Calmels S., Hainaunt P. and Ohshima H. (1997) Nitric oxide induces conformational and functional modi®cations of wild-type p53 tumor suppressor protein. Cancer Research 57, 3365±3369. Chan M. M., Huang H. I., Fenton M. R. and Fong D. (1998) In vitro inhibition of nitric oxide synthase gene expressions by curcumin, a cancer preventive natural product with anti-in¯ammatory properties. Biochemical Pharnacology 55, 1955±1962. Chehab N. H., Malikzay A., Stavridi E. S. and Halazonetis T. D. (1999) Phosphorylation of Ser-20 mediates stabili-
Anti-in¯ammatory eect of curcumin zation of human p53 in response to DNA damage. Proceedings of the National Academy of Sciences of the U.S.A. 96, 13777±13782. Chen P. J. and Chen D. S. (1999) Hepatitis B virus infection and hepatocelluar carcinoma: molecular genetics and clinical perspectives. Seminars in Liver Disease 19, 253± 262. Deora A. A., Win T., Vanhaesebroeds B. and Lander H. M. (1998) A redox-triggered ras-eector interaction. Recruitment of phosphatidylinositol-30 -kinase to Ras by redox stress. Journal of Biological Chemistry 273, 29923±29928. Ferreira-Gonzalez A., DeAngelo A. B., Nasim S. and Garrett C. T. (1995) Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids. Carcinogenesis 16, 495±500. Giri R. K. and Das B. R. (1996) Dierential expression of cjun and c-myc in N-nitrosodiethylamine-induced hepatic oncogenesis in ARK mice. Cancer Letters 109, 121±127. Goodwin A. E. and Grizzle J. M. (1994) Oncogene expression in hepatocytes of the ®sh Rivulus ocellatus marmoratus during the necrotic and regenerative phases of dimethylnitrosamine toxicity. Carcinogenesis 15, 1985±1992. Heinze T., Jonas S., Karsten A. and Neuhaus P. (1999) Determination of the oncogenes p53 and c-erbB2 in the tumor cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time. Anticancer Research 19, 2501±2503. Herbst H., Milani S., Schuppan D. and Stein H. (1991) Temporal and spatial patterns of proto-oncogene expression at early stages of toxic liver injury in the rat. Laboratory Investigation 65, 324±333. Higaki S., Akazawa A., Nakamura H., Yauai H., Yoshida T. and Okita K. (1999) Metaplastic polyp of the colon develops in response to in¯ammation. Journal of Gastroenterology and Hepatology 14, 709±714. Huang M. T., Lysz T., Ferraro T. and Conney A. H. (1992a) Inhibitory eects of curcumin on tumor promotion and arachidonic acid metabolism in mouse epidermis. In Cancer Prevention, ed. L. Watternberg, M. Lipkin, C. W. Boone and G. J. Kello, pp. 375±391. CRC Press, Boca Raton, FL. Huang M. T., Smart R. C., Wang G. Q. and Conney A. H. (1998) Inhibitory eect of curcuma, chlorogenic acid, caeric acid, and ferulic acid on tumor promotion in mouse skin by 12-0-tetradecanoylphorbol-13-acetate. Cancer Research 48, 5941±5946. Huang M. T., Wang Z. R., Georgiadis C. A., Laskin J. D. and Comey A. H. (1992b) Inhibitory eect of curcuma on
995
tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. Carcinogenesis 13, 2183±2186. Huang T. S., Lee S. C. and Lin J. K. (1991) Suppression of the c-Jun/AP-1 activation by an inhibitor of tumor formation in mouse ®broblast cells. Proceedings of the National Academy of Sciences of the U.S.A. 88, 5292± 5296. Kuo M. L., Huang T. S. and Lin J. K. (1996) Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in hman leukemia cells. Biochimica et Biophysica Acta 1317, 95±100. Nadkarani K. M. (1976) Curcuma longa. In India Materia Medica, ed. K.M. Nadkarani, pp. 414±416. Popular Praleasham Publishing Co., Bombay. Nardone G., Staibano S., Rocco A., Mezza E., D'armiento F. P., Unsabato l., Coppla A., Salvatore G., Lucariello A., Figura N., DeRosa G. and Budillon G. (1999) Eect of helicobacter pylori infection and its eradication on cell proliferation, DNA safety, and oncogene expression in patients with chronic gastritis. Gut 44, 789±799. Ohshima H. and Bartsch H. (1994) Chronic infectious and in¯ammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutation Research 305, 253±264. Puig P. L., Flejou J. F., Fabre M., Bedossa P., Belghiti J., Gayral F. and Franco D. (1992) Overexpression of p53: a rare event in a large series of white patients with hepatocelluar carcinoma. Hepatology 16, 1171±1175. Servais P., Taton G. and Galand P. (1990) Modulation by indomethacin of dithylnitrosamine-induced early changes in c-myc and c-ras expression and late incidence of preneoplastic lesions in rat liver. Carcinogenesis 11, 1433± 1436. Song K. Y., Lin I. K., Park S. C., Lee S. O., Park H. S., Choi Y. K. and Hyun B. H. (1999) Eect of modulation on the expression of glutathione s-transferase placental form and proliferating cell nuclear antigen in N-nitrosodiethylamine initiated hepatocarcinogenesis in the male Fischer 344 rat. Carcinogenesis 20, 1541±1548. Tsunematsu S., Satio H., Tada S., Ebinuma H., Tsuchiya M., Kumagai N., Morizane T., Nomura T. and Ischii H. (1997) Susceptibility of experimental autoimmune hepatitis in transgenic mice overexpressing the C-H-ras gene. Journal of Gastroenterology and Hepatology 12, 319±324. Yu M. W., You S. L., Chang A. S., Lu S. N., Liaw Y. F. and Chen C. J. (1991) Association between hepatitis C virus antibodies and hepatocelluar carcinoma in Taiwan. Cancer Research 51, 5621±5625.