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Expression of P-glycoprotein in rat hepatocarcinogenesis by diethylnitrosamine and the modulation by anticancer drugs
Hepatology Research 22 (2002) 107– 118
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Expression of P-glycoprotein in rat hepatocarcinogenesis by diethylnitrosamine and the modulation by anticancer drugs Yasuo Takeuchi a, Motonobu Sugimoto a,*, Kaori Ochiai a, Kinji Ito b a
Second Department of Internal Medicine, Toho Uni6ersity School of Medicine, 6 -11 -1 Omori-nishi, Ota-ku, Tokyo 143 -8541, Japan b Second Department of Pathology, Toho Uni6ersity School of Medicine, 5 -21 -16 Omori-nishi, Ota-ku, Tokyo 143 -8541, Japan Received 31 October 2000; received in revised form 15 May 2001; accepted 15 June 2001
Abstract P-glycoprotein (P-GP) is known to be a multidrug resistant 1 gene product and to exhibit resistance to a broad range of drugs including anticancer drugs such as epirubicin. Its overexpression is reported in human hepatocellular carcinoma and in adenomatous hyperplasia of the liver as well. In order to clarify the evolution of P-GP expression during hepatocarcinogenesis and its modulation by anticancer drugs, we performed an immunohistochemical study in male Wistar rat livers exposed to diethylnitrosamine (DEN) for 12 weeks. Some rats were pretreated with cisplatin or epirubicin 1 week prior to the exposure, and some rats were treated with them at the 10th week after the exposure. While there was no P-GP expression in the liver of the control, cisplatin, and epirubicin (DEN-free) rats, expression was confirmed in the hepatocytes of DEN-treated rats. The immunostaining of hyperplastic nodules was significantly more intense than in well-differentiated hepatocellular carcinomas, and no staining was observed in poorly-differentiated carcinomas. Markedly intense staining was observed in the early hyperplastic nodules of cisplatin-pretreated rats, as well as in epirubicin-pretreated rats. Plasma a-fetoprotein levels were markedly elevated in DEN-treated rats, while tumor necrosis factor-a levels were not. In conclusion, the results suggest that P-GP confers a protective effect against anticancer drugs and provides a great advantage to the initiated cells. Furthermore, in addition to epirubicin, cisplatin also promotes the induction of P-GP in the initiated cell. © 2002 Elsevier Science B.V. All rights reserved. Keywords: P-glycoprotein; Hepatocarcinogenesis; Hepatocellular carcinoma; Hyperplastic nodule; Diethylnitrosamine; Cisplatin; Epirubicin
1. Introduction P-glycoprotein (P-GP) is known to be a multidrug resistant 1 (MDR1) gene product and to * Corresponding author. Tel.: + 81-3-3762-4151; fax: + 813-3763-8542. E-mail address: [email protected] (M. Sugimoto).
exhibit resistance to a broad range of drugs including anticancer drugs [1–3]. Although it is also known to normally express in the surface of epithelial cells of various organs such as liver, kidney, adrenal gland, jejunum and colon [4–6], its overexpression is obviously seen in human hepatocellular carcinoma (HCC) [7,8]. Recently it was reported that P-GP expression inversely correlate
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with chemotherapeutic response in human HCC treated with VP-16 or doxorubicine [9]. Since epirubicine (EPIR) is a derivative of doxorubicine, EPIR may be effective in tumors without P-GP expression. However, there have been no reports regarding cisplatin (CDDP) despite its worldwide utilization. On the other hand, P-GP was found to overexpress in adenomatous hyperplasia of the liver and its immunostaining grade to be stronger than HCC [10]. Some human HCCs are etiologically linked to adenomatous hyperplasia [11,12], which mimics hyperplastic nodules (HPN) in rat and mouse hepatocarcinogenesis [13]. Several workers found increased MDR1 mRNA levels or P-GP expression in HPN induced by chemical hepatocarcinogenesis [14–17]. However, few studies have been performed regarding basic experiments using anticancer drugs. In the present study, in order to clarify the evolution of P-GP expression during hepatocarcinogenesis and the modulation by anticancer drugs, we performed immunohistochemical study in rat livers treated with diethylnitrosamine (DEN), with or without CDDP or EPIR pre- and post-treatment. The experimental schedule was made under the hypothesis that EPIR pretreatment may induce P-GP expression in the DENtreated rat liver, whereas CDDP pretreatment may not.
2. Materials and methods Male Wistar rats weighing approximately 200 g were employed. Eighty rats were once treated intraperitoneally with DEN (Wako Pure Chemical Industries, Osaka, Japan) at a dose of 200 mg/kg, and subsequently fed water containing 0.05% DEN for 12 weeks (DEN-treated group). Within this group, 1 week prior to DEN treatment, 24 rats were pretreated intraperitoneally once with CDDP (Nippon Kayaku Co, Tokyo, Japan) at a dose of 2 mg/kg (CDDP +DEN group), and another 24 rats were pretreated intraperitoneally once with EPIR (Pharmacia Upjohn Co, Tokyo, Japan) at a dose of 1.5 mg/kg (EPIR +DEN group). Eight rats of DEN group
were prepared for post-treatment with CDDP (1 mg/kg) (DEN+ CDDP group), and another eight rats were prepared for post-treatment with EPIR (0.75 mg/kg) (DEN + EPIR group) at the 10th week after DEN treatment. Remaining 16 rats were fed with water containing DEN, without any anticancer drugs (DEN group). Forty-eight rats were simultaneously fed without DEN treatment (DEN-free group). Within this group, 16 rats were treated with single injection of CDDP (CDDP group), and another 16 rats were treated with EPIR (EPIR group). Remaining 16 rats were normally fed without any drugs (control group). Every 4 weeks after DEN treatment, three rats were randomly chosen from each group, and under the ether anesthesia livers were removed and blood samples were drown from cardiac cavity. Intact rats and rats with single injection of CDDP or EPIR were also sacrificed as controls. Rats of DEN + CDDP and DEN +EPIR groups were sacrificed only at the 12th week (Fig. 1). Two parts of each liver seemed to be representative lesions were utilized for the immunohistochemical study. The liver tissue was fixed in 4% paraformaldehyde, dehydrated with ethanol and embedded in paraffin, then cut into 5 mm thick section. The sections were stained with hematoxylin and eosin (HE). For immunohistochemistry, the sections were deparaffinized, blocked with 10% anti-goat serum (Wako Pure Chemical Industries, Osaka, Japan), and then reacted with primary antibody, anti-P-GP (Oncogene Research Products, Cambridge, MA, USA) diluted with phosphatebuffered saline (PBS) (1:400) overnight. After being rinsed in PBS, sections were then reacted with secondary antibody, biotin-conjugated antirabbit goat IgG (Wako Pure Chemical Industries, Osaka, Japan) for 3 h. Subsequently they were stained with streptavidin– biotin–peroxidase complex (Wako Pure Chemical Industries, Osaka, Japan) and 3,3%-diamino-benzidine tetrachloride (Wako Pure Chemical Industries, Osaka, Japan). Histological evaluation was performed for two liver tissues from each liver with HE staining. Immunohistochemical evaluation was performed for same two liver tissues from each liver with
Y. Takeuchi et al. / Hepatology Research 22 (2002) 107–118
immunostaining. The negative immunostaining (no P-GP expression) was rated as − ; positive staining (P-GP expression) was rated as + ; intense staining (P-GP overexpression) was rated as + + + ; and intermediate staining between + and + + + was rated as + +. For the objective evaluation, 1000 hepatocytes were examined and the number of positively stained hepatocytes (PGP positive cell count) was used to grade P-GP expression. The blood samples were immediately centrifuged and plasma fractions were obtained. They were stored at − 80 °C until use. Alpha-fetoprotein (AFP) and tumor necrosis factor (TNF)-a were assayed with enzyme linked immuno-solvent assay (ELISA). Statistical analysis was performed using Student’s t-test.
Fig. 1. Experimental schedule. DEN, diethylnitrosamine; CDDP, cisplatin; EPIR: epirubicin. Male Wistar rats were once treated intraperitoneally with DEN at a dose of 200 mg/kg, and subsequently fed water containing 0.05% DEN for 12 weeks (DEN-treated group). One week prior to the DEN treatment, some rats were pretreated intraperitoneally once with CDDP at a dose of 2 mg/kg (CDDP+ DEN group), and some were pretreated intraperitoneally once with EPIR at a dose of 1.5 mg/kg (EPIR+DEN group). Ten weeks after the DEN treatment, some rats were treated with CDDP (1 mg/kg) (DEN+ CDDP group), and some were treated with EPIR (0.75 mg/kg) (DEN +EPIR group). Remaining rats were fed with water containing DEN, without any anticancer drugs (DEN group). Rats without DEN treatment were simultaneously fed as controls (DEN-free group). Some rats were once treated with CDDP (CDDP group), and some were once treated with EPIR (EPIR group). Remaining rats were normally fed without any drugs (control group). Every 4 weeks after the experiment, three rats were sacrificed for the evaluation.
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3. Results Out of 16 rats of DEN group, one rat died by the 4th week, two by the 8th week, and three by the 12th week. Out of 24 rats of CDDP+ DEN group, nine rats died by the 4th week, five by the 8th week, and four by the 12th week. Out of 24 rats of EPIR+ DEN group, four rats died by the 4th week, two by the 8th week, and three by the 12th week. Out of eight rats of DEN+ CDDP group, two rats died by the 8th week, and one by the 12th week. Out of eight rats of DEN+ EPIR group, two rats died by the 8th week, and three by the 12th week. Out of 16 rats each of CDDP group, EPIR group and control group, none died through experimental periods. Since every 4 weeks after DEN treatment, three rats were sacrificed for experiments, none of CDDP+ DEN group rats were left at the 12th week. Therefore, no evaluation was performed on rats at the 12th week of this group. Macroscopically, most rats at the 8th week of DEN-treated groups showed hepatomegaly with uneven liver surface. Most rats at the 12th week of DEN-treated groups revealed jaundice, ascites, and liver tumor. On the other hand, no rats of control, CDDP and EPIR (DEN-free) groups showed remarkable changes through experimental periods. Microscopically, while DEN-free group rats showed no pathological changes until 12th week (Fig. 2A), rats at the 4th week of the DEN group showed degeneration, spotty necrosis and regenerative foci in centrilobular areas (Fig. 3A). DENtreated group rats revealed scattered hyperplastic nodules by 8 weeks after DEN treatment (Fig. 4A). Twelve weeks after DEN treatment, hyperplastic nodules were evident and well-differentiated, and partly poorly-differentiated HCCs were also found (Fig. 5A). Rats of DEN+ CDDP and DEN + EPIR group showed no tumor regression. Immunohistochemically, there was no P-GP expression in the livers of DEN-free groups through 12 weeks (Fig. 2B). In the liver of DEN-treated group rats, the expression of P-GP was confirmed correspondingly with the hepatocellular membrane of the bile canalicular side at the 4th week (Fig. 3B). The overexpression of P-GP was ob-
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Fig. 2. Microscopic finding of a control group rat liver at the 12th week of the experiment. (A) Hematoxylin and eosin (HE) staining shows no pathological changes ( × 20). (B) Immunostaining shows no P-GP expression in the liver ( × 20).
served in the hepatocytes of hyperplastic nodules at the 8th week after DEN treatment (Fig. 4B). However, there was sparse expression in the well-
differentiated HCCs, and no expression in the poorly-differentiated HCCs (Fig. 5B). In the CDDP + DEN group (Fig. 6A), as well as
Y. Takeuchi et al. / Hepatology Research 22 (2002) 107–118
EPIR + DEN group (Fig. 6B), immunostaining was rather stronger than DEN group at the 4th week after DEN treatment. In the DEN+ CDDP group, as well as DEN+EPIR group, immunostaining was negative for HCCs (Table 1).
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P-GP positive cell counts in DEN group rats at the 4th, 8th, and 12th week were 2329 123, 6759 128, and 2099 58, respectively. The 8th week value was significantly higher than the 4th and 12th week values. At the 4th week, the values
Fig. 3. Microscopic finding of a DEN group rat liver at the 4th week of the experiment. (A) HE staining shows a degeneration, spotty necrosis and regenerative foci in centrilobular areas ( × 20). (B) Immunostaining shows the expression of P-GP correspondingly with the hepatocellular membrane of the bile canalicular side ( ×20).
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Fig. 4. Microscopic finding of a DEN group rat liver at the 8th week of the experiment. (A) HE staining shows a hyperplastic nodule ( × 20). (B) Immunohistostaining shows overexpression of P-GP in hepatocytes of the hyperplastic nodule ( × 20).
were high in CDDP+ DEN group (6429 118), EPIR +DEN group (4419219), and DEN group (2329 123) in that order. Differences between
them were statistically significant. At the 8th week, the values were high in DEN group (6759 128), EPIR+ DEN group (4369 160), and
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CDDP + DEN group (4369150), in that order. There were statistically significant differences between the DEN and CDDP +DEN groups, and
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the DEN and EPIR + DEN groups. P-GP positive cells were mainly in hyperplastic nodules. At the 12th week, the values were high in the
Fig. 5. Microscopic finding of a DEN group rat liver at the 12th week of the experiment. (A) HE staining shows a well-differentiated, and partly poorly-differentiated HCC ( × 20). (B) Immunostaining shows sparse expression of P-GP in the well-differentiated HCC, and no expression in the poorly-differentiated HCC ( × 20).
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Fig. 6. Immunohistochemical finding of a CDDP+ DEN and EPIR+ DEN group rat liver at the 4th week of the experiment. (A) In a CDDP+ DEN group rat liver, the expression of P-GP is much stronger than in DEN group rat liver ( × 20). (B) In a EPIR+ DEN group rat liver, the expression of P-GP is rather stronger than DEN group ( ×20).
EPIR + DEN group (4179 190) and DEN group (209958), in that order. There was a statistically significant difference between the DEN and
EPIR + DEN groups. P-GP positive cells were mainly from well-differentiated HCCs. The values of DEN-free group rats, as well as those in the
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DEN +CDDP and DEN + EPIR groups were too low to be calculated (Fig. 7). Plasma AFP and TNF-a values were not always assayed for three rats each time, because some rats died or some blood samples were inappropriate for the assays. Plasma AFP levels of DEN-free group rats showed no elevation until 12th week. Mean value of control rats was 2995 ng/ml. On the other hand, those of DEN-treated group rats showed marked elevation. Mean values of DEN group rats at the 4th, 8th, and 12th week were 3430, 11 300,
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and 290 000 ng/ml, respectively. The values of DEN + CDDP group and DEN + EPIR group rats were apparently lower than those of DEN group rats (Fig. 8). Statistical analyses among them were not performed because of their small samples. Plasma TNF-a values of control group and CDDP group rats were less than 16 ng/ml until the 12th week. The value of one rat of ERIR group at the 4th week showed 22 ng/ml. Those of DEN group rats were less than 16 ng/ml until the 8th week, though one rat of this group at the 12th week showed 29 ng/ml. The value of one rat each
Table 1 Common histological findings observed in the present study 4W
8W
12W
HE
P-GP
HE
P-GP
HE
P-GP
DEN-free Control CDDP EPIR
Normal Normal Normal
− − −
Normal Normal Normal
− − −
Normal Normal Normal
− − −
DEN-treated DEN CDDP+DEN EPIR+DEN DEN+CDDP DEN+EPIR
Degeneration HPN HPN – –
+ +++ ++
HPN HPN HPN – –
+++ ++ ++
HCC (Dead) HCC HCC HCC
well +
poor −
well ++ poor − well − poor − well − poor −
HE, hematoxylin and eosin staining; P-GP, P-glycoprotein immunostaining; HPN, hyperplastic nodule; HCC, hepatocellular carcinoma; well, well-differentiated; poor, poorly-differentiated.
Fig. 7. P-GP positive cell count in each rat group. The value presents the number of P-GP positive cells among 1000 hepatocytes. *, Under estimation.
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Fig. 8. Plasma a-fetoprotein (AFP) level in each rat group.
Fig. 9. Plasma tumor necrosis factor (TNF)-a level in each rat group.
of CDDP +DEN group and EPIR + DEN group at the 8th week showed 32 and 26 ng/ml, respectively. The value of two rats of DEN+ CDDP group were 21 and 26 ng/ml, and the value of one rat DEN +EPIR group was 18 ng/ml (Fig. 9).
4. Discussion P-GP is normally expressed in the bile canalicular membrane of the hepatocyte [4–6], though the present immunohistochemical study showed no P-GP expression in the liver of the control, CDDP, or EPIR groups, probably due to the poor sensitivity of the primary antibody utilized in our study. However, overexpression of P-GP
was observed in the hyperplastic nodules in the liver at the 8th week after DEN treatment. There was sparse expression in the well-differentiated HCCs and no expression in the poorly-differentiated HCCs. These findings are consistent with earlier human studies showing expression of P-GP in adenomatous hyperplasias of the liver more extensive than HCC [10]. Recent pathological study of 101 cases of HCC revealed that P-GP positive immunostaining was seen in 92% of the tumors, but the expression was less extensive in the tumors than in adjacent nontumorous liver tissue [18]. The results of the present study suggest that P-GP confers a protective effect against multiple substances, including anticancer drugs, and provides a great advantage to the initiated cells.
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Moreover, in the present study, the CDDP+ DEN group, as well as the EPIR+ DEN group, showed more intense immunostaining than the DEN group at the 4th week after DEN treatment. This suggests that not only EPIR, which is a known substrate of P-GP [19,20], but also CDDP, is a substrate-like substance of P-GP. CDDP and EPIR may be effective in tumors without P-GP expression. Furthermore, inhibition of P-GP should be considered to enhance the effectiveness of anticancer treatment. Although varapamil [20] and cyclosporin-A [21] have been used as P-GP inhibitors in overcoming anticancer drug-resistant neoplasms, newer compounds with fewer side effects should be utilized. AFP is a well known representative tumor marker of human HCC. The profile of rat plasma AFP in the current study revealed a prominent increase, which correlated with DEN-induced tumor progression. However, plasma AFP elevation was seen even at the 4th week after DEN treatment. While high levels of plasma AFP are believed to be strongly suggestive of HCC [22], some cases of chronic hepatitis and liver cirrhosis in which AFP exceeds 400 ng/ml, without co-existing of HCC, have been reported. Earlier, we encountered a case of cirrhosis with transitory AFP elevation as high as 7190 ng/ml [23]. It is possible that regeneration after acute exacerbation of chronic liver disease might have caused the increase in AFP production. In the present experimental study, the same mechanism might function during the DEN-induced liver injury, adding to the chemical hepatocarcinogenesis. However, although high levels of plasma TNFa have been reported in human HCC patients [24], it remains controversial as a tumor marker of human HCC. TNF-a is thought to be an indicator of apoptosis of hepatocytes [25]. Recently, it was reported that inhibition of transcription factor NF-kB through the adenoviral delivery of a modified form of IkBa-an inhibitor of NF-kBsensitizes chemoresistant tumors to the apoptotic potential of TNF-a, resulting in tumor regression [26]. However, the profile of rat plasma TNF-a in the present study showed no correlation with DEN-induced tumor progression.
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In conclusion, our results show that P-GP may confer a protective effect against anticancer drugs, and that both EPIR and CDDP increase P-GP expression when given before DEN exposure. However, the mechanism responsible and the significance of the findings will require further investigation.
Acknowledgements The present study was supported in part by a grant from the Japanese Ministry of Education, Science and Culture (1996–1998). The authors are grateful to Professor Neil Kaplowitz (University of Southern California School of Medicine, Los Angeles, USA) for his critical comments and advice in preparing the manuscript.
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[10] Nagasue N, Dhar DK, Makino Y, Yoshimura H, Nakamura T. Overexpreion of P-glycoprotein in adenomatous hyperplasia of human liver with cirrhosis. J Hepatol 1995;22:197 – 201. [11] Nagasue N, Yukaya H, Chang YC, Kimura N, Ota N, Nakamura T. Hepatocellular pseudotumor (regenerating nodule) in the cirrhotic liver mimicking hepatocellular carcinoma. Br J Surg 1988;75:1124 –8. [12] Takayama T, Makuuchi M, Hirohashi S, et al. Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 1990;336:1150 –3. [13] Farber E. Cellular biochemistry of the stepwise development of cancer with chemicals: G.H.A. Clowes Memorial Lecture. Cancer Res 1984;44:5463 –74. [14] Thorgeirsson SS, Huber BE, Sorrell S, Fojo A, Pastan I, Gottesman MM. Expression of the multidrug-resistance gene in hepatocarcinogenesis and regenerating rat liver. Science 1987;236:1120 –2. [15] Fairchild CR, Ivy SP, Rushmore T, et al. Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular carcinoma. Proc Natl Acad Sci USA 1987;84:7701 – 5. [16] Nakatsukasa H, Evarts RP, Burt RK, Nagy P, Thorgeirsson SS. Cellurar pattern of multidrug-resistance gene expression during chemical hepatocarcinogenesis in the rat. Mol Carcinog 1992;6:190 –8. [17] Bradley G, Sharma R, Rajalakshmi S, Ling V. P-glycoprotein expression during tumor progression in the rat liver. Cancer Res 1992;52:5154 –61.
[18] Ng IOL, Liu CL, Fan ST. Expression of P-glycoprotein in hepatocellular carcinoma: a determinant of chemotherapy response. Am J Clin Pathol 2000;113:355 – 63. [19] Naito M, Hamada H, Tsuruo T. ATP/Mg2 + -dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells. J Biol Chem 1988;263:11887 – 91. [20] Naito M, Tsuruo T. Competitive inhibition by verapamil of ATP-dependent high affinity vincristine binding to the plasma membrane of multidrug-resistant K562 cells without calcium ion involvement. Cancer Res 1989;49:1452 – 5. [21] Kashani-Sabet M, Wang W, Scanlon KJ. Cyclosporine A suppresses cisplatin induced c-fos gene expression in ovarian carcinoma cells. J Biol Chem 1990;265:11285 – 8. [22] Endo Y, Kanai K, Oda T, et al. Clinical significance of alpha-fetoprotein in hepatitis and liver cirrhosis. Ann NY Acad Sci 1975;259:234 – 8. [23] Sugimoto M, Itsukaichi T, Ishii K, et al. Marked increase in serum a-fetoprotein level in cirrhosis: a case report. Am J Gastroenterol 1987;82:69 – 72. [24] Nakazaki H. Preoperative and postoperative cytokines in patients with cancer. Cancer 1992;70:709 – 13. [25] Leist M, Gantner F, Naumann H, et al. Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 1997;112:923 – 34. [26] Wang C-Y, Cusack JC Jr, Liu R, Baldwin AS Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kB. Nat Med 1999;5:412 – 7.
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Expression of P-glycoprotein in rat hepatocarcinogenesis by diethylnitrosamine and the modulation by anticancer drugs Yasuo Takeuchi a, Motonobu Sugimoto a,*, Kaori Ochiai a, Kinji Ito b a
Second Department of Internal Medicine, Toho Uni6ersity School of Medicine, 6 -11 -1 Omori-nishi, Ota-ku, Tokyo 143 -8541, Japan b Second Department of Pathology, Toho Uni6ersity School of Medicine, 5 -21 -16 Omori-nishi, Ota-ku, Tokyo 143 -8541, Japan Received 31 October 2000; received in revised form 15 May 2001; accepted 15 June 2001
Abstract P-glycoprotein (P-GP) is known to be a multidrug resistant 1 gene product and to exhibit resistance to a broad range of drugs including anticancer drugs such as epirubicin. Its overexpression is reported in human hepatocellular carcinoma and in adenomatous hyperplasia of the liver as well. In order to clarify the evolution of P-GP expression during hepatocarcinogenesis and its modulation by anticancer drugs, we performed an immunohistochemical study in male Wistar rat livers exposed to diethylnitrosamine (DEN) for 12 weeks. Some rats were pretreated with cisplatin or epirubicin 1 week prior to the exposure, and some rats were treated with them at the 10th week after the exposure. While there was no P-GP expression in the liver of the control, cisplatin, and epirubicin (DEN-free) rats, expression was confirmed in the hepatocytes of DEN-treated rats. The immunostaining of hyperplastic nodules was significantly more intense than in well-differentiated hepatocellular carcinomas, and no staining was observed in poorly-differentiated carcinomas. Markedly intense staining was observed in the early hyperplastic nodules of cisplatin-pretreated rats, as well as in epirubicin-pretreated rats. Plasma a-fetoprotein levels were markedly elevated in DEN-treated rats, while tumor necrosis factor-a levels were not. In conclusion, the results suggest that P-GP confers a protective effect against anticancer drugs and provides a great advantage to the initiated cells. Furthermore, in addition to epirubicin, cisplatin also promotes the induction of P-GP in the initiated cell. © 2002 Elsevier Science B.V. All rights reserved. Keywords: P-glycoprotein; Hepatocarcinogenesis; Hepatocellular carcinoma; Hyperplastic nodule; Diethylnitrosamine; Cisplatin; Epirubicin
1. Introduction P-glycoprotein (P-GP) is known to be a multidrug resistant 1 (MDR1) gene product and to * Corresponding author. Tel.: + 81-3-3762-4151; fax: + 813-3763-8542. E-mail address: [email protected] (M. Sugimoto).
exhibit resistance to a broad range of drugs including anticancer drugs [1–3]. Although it is also known to normally express in the surface of epithelial cells of various organs such as liver, kidney, adrenal gland, jejunum and colon [4–6], its overexpression is obviously seen in human hepatocellular carcinoma (HCC) [7,8]. Recently it was reported that P-GP expression inversely correlate
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with chemotherapeutic response in human HCC treated with VP-16 or doxorubicine [9]. Since epirubicine (EPIR) is a derivative of doxorubicine, EPIR may be effective in tumors without P-GP expression. However, there have been no reports regarding cisplatin (CDDP) despite its worldwide utilization. On the other hand, P-GP was found to overexpress in adenomatous hyperplasia of the liver and its immunostaining grade to be stronger than HCC [10]. Some human HCCs are etiologically linked to adenomatous hyperplasia [11,12], which mimics hyperplastic nodules (HPN) in rat and mouse hepatocarcinogenesis [13]. Several workers found increased MDR1 mRNA levels or P-GP expression in HPN induced by chemical hepatocarcinogenesis [14–17]. However, few studies have been performed regarding basic experiments using anticancer drugs. In the present study, in order to clarify the evolution of P-GP expression during hepatocarcinogenesis and the modulation by anticancer drugs, we performed immunohistochemical study in rat livers treated with diethylnitrosamine (DEN), with or without CDDP or EPIR pre- and post-treatment. The experimental schedule was made under the hypothesis that EPIR pretreatment may induce P-GP expression in the DENtreated rat liver, whereas CDDP pretreatment may not.
2. Materials and methods Male Wistar rats weighing approximately 200 g were employed. Eighty rats were once treated intraperitoneally with DEN (Wako Pure Chemical Industries, Osaka, Japan) at a dose of 200 mg/kg, and subsequently fed water containing 0.05% DEN for 12 weeks (DEN-treated group). Within this group, 1 week prior to DEN treatment, 24 rats were pretreated intraperitoneally once with CDDP (Nippon Kayaku Co, Tokyo, Japan) at a dose of 2 mg/kg (CDDP +DEN group), and another 24 rats were pretreated intraperitoneally once with EPIR (Pharmacia Upjohn Co, Tokyo, Japan) at a dose of 1.5 mg/kg (EPIR +DEN group). Eight rats of DEN group
were prepared for post-treatment with CDDP (1 mg/kg) (DEN+ CDDP group), and another eight rats were prepared for post-treatment with EPIR (0.75 mg/kg) (DEN + EPIR group) at the 10th week after DEN treatment. Remaining 16 rats were fed with water containing DEN, without any anticancer drugs (DEN group). Forty-eight rats were simultaneously fed without DEN treatment (DEN-free group). Within this group, 16 rats were treated with single injection of CDDP (CDDP group), and another 16 rats were treated with EPIR (EPIR group). Remaining 16 rats were normally fed without any drugs (control group). Every 4 weeks after DEN treatment, three rats were randomly chosen from each group, and under the ether anesthesia livers were removed and blood samples were drown from cardiac cavity. Intact rats and rats with single injection of CDDP or EPIR were also sacrificed as controls. Rats of DEN + CDDP and DEN +EPIR groups were sacrificed only at the 12th week (Fig. 1). Two parts of each liver seemed to be representative lesions were utilized for the immunohistochemical study. The liver tissue was fixed in 4% paraformaldehyde, dehydrated with ethanol and embedded in paraffin, then cut into 5 mm thick section. The sections were stained with hematoxylin and eosin (HE). For immunohistochemistry, the sections were deparaffinized, blocked with 10% anti-goat serum (Wako Pure Chemical Industries, Osaka, Japan), and then reacted with primary antibody, anti-P-GP (Oncogene Research Products, Cambridge, MA, USA) diluted with phosphatebuffered saline (PBS) (1:400) overnight. After being rinsed in PBS, sections were then reacted with secondary antibody, biotin-conjugated antirabbit goat IgG (Wako Pure Chemical Industries, Osaka, Japan) for 3 h. Subsequently they were stained with streptavidin– biotin–peroxidase complex (Wako Pure Chemical Industries, Osaka, Japan) and 3,3%-diamino-benzidine tetrachloride (Wako Pure Chemical Industries, Osaka, Japan). Histological evaluation was performed for two liver tissues from each liver with HE staining. Immunohistochemical evaluation was performed for same two liver tissues from each liver with
Y. Takeuchi et al. / Hepatology Research 22 (2002) 107–118
immunostaining. The negative immunostaining (no P-GP expression) was rated as − ; positive staining (P-GP expression) was rated as + ; intense staining (P-GP overexpression) was rated as + + + ; and intermediate staining between + and + + + was rated as + +. For the objective evaluation, 1000 hepatocytes were examined and the number of positively stained hepatocytes (PGP positive cell count) was used to grade P-GP expression. The blood samples were immediately centrifuged and plasma fractions were obtained. They were stored at − 80 °C until use. Alpha-fetoprotein (AFP) and tumor necrosis factor (TNF)-a were assayed with enzyme linked immuno-solvent assay (ELISA). Statistical analysis was performed using Student’s t-test.
Fig. 1. Experimental schedule. DEN, diethylnitrosamine; CDDP, cisplatin; EPIR: epirubicin. Male Wistar rats were once treated intraperitoneally with DEN at a dose of 200 mg/kg, and subsequently fed water containing 0.05% DEN for 12 weeks (DEN-treated group). One week prior to the DEN treatment, some rats were pretreated intraperitoneally once with CDDP at a dose of 2 mg/kg (CDDP+ DEN group), and some were pretreated intraperitoneally once with EPIR at a dose of 1.5 mg/kg (EPIR+DEN group). Ten weeks after the DEN treatment, some rats were treated with CDDP (1 mg/kg) (DEN+ CDDP group), and some were treated with EPIR (0.75 mg/kg) (DEN +EPIR group). Remaining rats were fed with water containing DEN, without any anticancer drugs (DEN group). Rats without DEN treatment were simultaneously fed as controls (DEN-free group). Some rats were once treated with CDDP (CDDP group), and some were once treated with EPIR (EPIR group). Remaining rats were normally fed without any drugs (control group). Every 4 weeks after the experiment, three rats were sacrificed for the evaluation.
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3. Results Out of 16 rats of DEN group, one rat died by the 4th week, two by the 8th week, and three by the 12th week. Out of 24 rats of CDDP+ DEN group, nine rats died by the 4th week, five by the 8th week, and four by the 12th week. Out of 24 rats of EPIR+ DEN group, four rats died by the 4th week, two by the 8th week, and three by the 12th week. Out of eight rats of DEN+ CDDP group, two rats died by the 8th week, and one by the 12th week. Out of eight rats of DEN+ EPIR group, two rats died by the 8th week, and three by the 12th week. Out of 16 rats each of CDDP group, EPIR group and control group, none died through experimental periods. Since every 4 weeks after DEN treatment, three rats were sacrificed for experiments, none of CDDP+ DEN group rats were left at the 12th week. Therefore, no evaluation was performed on rats at the 12th week of this group. Macroscopically, most rats at the 8th week of DEN-treated groups showed hepatomegaly with uneven liver surface. Most rats at the 12th week of DEN-treated groups revealed jaundice, ascites, and liver tumor. On the other hand, no rats of control, CDDP and EPIR (DEN-free) groups showed remarkable changes through experimental periods. Microscopically, while DEN-free group rats showed no pathological changes until 12th week (Fig. 2A), rats at the 4th week of the DEN group showed degeneration, spotty necrosis and regenerative foci in centrilobular areas (Fig. 3A). DENtreated group rats revealed scattered hyperplastic nodules by 8 weeks after DEN treatment (Fig. 4A). Twelve weeks after DEN treatment, hyperplastic nodules were evident and well-differentiated, and partly poorly-differentiated HCCs were also found (Fig. 5A). Rats of DEN+ CDDP and DEN + EPIR group showed no tumor regression. Immunohistochemically, there was no P-GP expression in the livers of DEN-free groups through 12 weeks (Fig. 2B). In the liver of DEN-treated group rats, the expression of P-GP was confirmed correspondingly with the hepatocellular membrane of the bile canalicular side at the 4th week (Fig. 3B). The overexpression of P-GP was ob-
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Fig. 2. Microscopic finding of a control group rat liver at the 12th week of the experiment. (A) Hematoxylin and eosin (HE) staining shows no pathological changes ( × 20). (B) Immunostaining shows no P-GP expression in the liver ( × 20).
served in the hepatocytes of hyperplastic nodules at the 8th week after DEN treatment (Fig. 4B). However, there was sparse expression in the well-
differentiated HCCs, and no expression in the poorly-differentiated HCCs (Fig. 5B). In the CDDP + DEN group (Fig. 6A), as well as
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EPIR + DEN group (Fig. 6B), immunostaining was rather stronger than DEN group at the 4th week after DEN treatment. In the DEN+ CDDP group, as well as DEN+EPIR group, immunostaining was negative for HCCs (Table 1).
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P-GP positive cell counts in DEN group rats at the 4th, 8th, and 12th week were 2329 123, 6759 128, and 2099 58, respectively. The 8th week value was significantly higher than the 4th and 12th week values. At the 4th week, the values
Fig. 3. Microscopic finding of a DEN group rat liver at the 4th week of the experiment. (A) HE staining shows a degeneration, spotty necrosis and regenerative foci in centrilobular areas ( × 20). (B) Immunostaining shows the expression of P-GP correspondingly with the hepatocellular membrane of the bile canalicular side ( ×20).
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Fig. 4. Microscopic finding of a DEN group rat liver at the 8th week of the experiment. (A) HE staining shows a hyperplastic nodule ( × 20). (B) Immunohistostaining shows overexpression of P-GP in hepatocytes of the hyperplastic nodule ( × 20).
were high in CDDP+ DEN group (6429 118), EPIR +DEN group (4419219), and DEN group (2329 123) in that order. Differences between
them were statistically significant. At the 8th week, the values were high in DEN group (6759 128), EPIR+ DEN group (4369 160), and
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CDDP + DEN group (4369150), in that order. There were statistically significant differences between the DEN and CDDP +DEN groups, and
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the DEN and EPIR + DEN groups. P-GP positive cells were mainly in hyperplastic nodules. At the 12th week, the values were high in the
Fig. 5. Microscopic finding of a DEN group rat liver at the 12th week of the experiment. (A) HE staining shows a well-differentiated, and partly poorly-differentiated HCC ( × 20). (B) Immunostaining shows sparse expression of P-GP in the well-differentiated HCC, and no expression in the poorly-differentiated HCC ( × 20).
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Fig. 6. Immunohistochemical finding of a CDDP+ DEN and EPIR+ DEN group rat liver at the 4th week of the experiment. (A) In a CDDP+ DEN group rat liver, the expression of P-GP is much stronger than in DEN group rat liver ( × 20). (B) In a EPIR+ DEN group rat liver, the expression of P-GP is rather stronger than DEN group ( ×20).
EPIR + DEN group (4179 190) and DEN group (209958), in that order. There was a statistically significant difference between the DEN and
EPIR + DEN groups. P-GP positive cells were mainly from well-differentiated HCCs. The values of DEN-free group rats, as well as those in the
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DEN +CDDP and DEN + EPIR groups were too low to be calculated (Fig. 7). Plasma AFP and TNF-a values were not always assayed for three rats each time, because some rats died or some blood samples were inappropriate for the assays. Plasma AFP levels of DEN-free group rats showed no elevation until 12th week. Mean value of control rats was 2995 ng/ml. On the other hand, those of DEN-treated group rats showed marked elevation. Mean values of DEN group rats at the 4th, 8th, and 12th week were 3430, 11 300,
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and 290 000 ng/ml, respectively. The values of DEN + CDDP group and DEN + EPIR group rats were apparently lower than those of DEN group rats (Fig. 8). Statistical analyses among them were not performed because of their small samples. Plasma TNF-a values of control group and CDDP group rats were less than 16 ng/ml until the 12th week. The value of one rat of ERIR group at the 4th week showed 22 ng/ml. Those of DEN group rats were less than 16 ng/ml until the 8th week, though one rat of this group at the 12th week showed 29 ng/ml. The value of one rat each
Table 1 Common histological findings observed in the present study 4W
8W
12W
HE
P-GP
HE
P-GP
HE
P-GP
DEN-free Control CDDP EPIR
Normal Normal Normal
− − −
Normal Normal Normal
− − −
Normal Normal Normal
− − −
DEN-treated DEN CDDP+DEN EPIR+DEN DEN+CDDP DEN+EPIR
Degeneration HPN HPN – –
+ +++ ++
HPN HPN HPN – –
+++ ++ ++
HCC (Dead) HCC HCC HCC
well +
poor −
well ++ poor − well − poor − well − poor −
HE, hematoxylin and eosin staining; P-GP, P-glycoprotein immunostaining; HPN, hyperplastic nodule; HCC, hepatocellular carcinoma; well, well-differentiated; poor, poorly-differentiated.
Fig. 7. P-GP positive cell count in each rat group. The value presents the number of P-GP positive cells among 1000 hepatocytes. *, Under estimation.
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Fig. 8. Plasma a-fetoprotein (AFP) level in each rat group.
Fig. 9. Plasma tumor necrosis factor (TNF)-a level in each rat group.
of CDDP +DEN group and EPIR + DEN group at the 8th week showed 32 and 26 ng/ml, respectively. The value of two rats of DEN+ CDDP group were 21 and 26 ng/ml, and the value of one rat DEN +EPIR group was 18 ng/ml (Fig. 9).
4. Discussion P-GP is normally expressed in the bile canalicular membrane of the hepatocyte [4–6], though the present immunohistochemical study showed no P-GP expression in the liver of the control, CDDP, or EPIR groups, probably due to the poor sensitivity of the primary antibody utilized in our study. However, overexpression of P-GP
was observed in the hyperplastic nodules in the liver at the 8th week after DEN treatment. There was sparse expression in the well-differentiated HCCs and no expression in the poorly-differentiated HCCs. These findings are consistent with earlier human studies showing expression of P-GP in adenomatous hyperplasias of the liver more extensive than HCC [10]. Recent pathological study of 101 cases of HCC revealed that P-GP positive immunostaining was seen in 92% of the tumors, but the expression was less extensive in the tumors than in adjacent nontumorous liver tissue [18]. The results of the present study suggest that P-GP confers a protective effect against multiple substances, including anticancer drugs, and provides a great advantage to the initiated cells.
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Moreover, in the present study, the CDDP+ DEN group, as well as the EPIR+ DEN group, showed more intense immunostaining than the DEN group at the 4th week after DEN treatment. This suggests that not only EPIR, which is a known substrate of P-GP [19,20], but also CDDP, is a substrate-like substance of P-GP. CDDP and EPIR may be effective in tumors without P-GP expression. Furthermore, inhibition of P-GP should be considered to enhance the effectiveness of anticancer treatment. Although varapamil [20] and cyclosporin-A [21] have been used as P-GP inhibitors in overcoming anticancer drug-resistant neoplasms, newer compounds with fewer side effects should be utilized. AFP is a well known representative tumor marker of human HCC. The profile of rat plasma AFP in the current study revealed a prominent increase, which correlated with DEN-induced tumor progression. However, plasma AFP elevation was seen even at the 4th week after DEN treatment. While high levels of plasma AFP are believed to be strongly suggestive of HCC [22], some cases of chronic hepatitis and liver cirrhosis in which AFP exceeds 400 ng/ml, without co-existing of HCC, have been reported. Earlier, we encountered a case of cirrhosis with transitory AFP elevation as high as 7190 ng/ml [23]. It is possible that regeneration after acute exacerbation of chronic liver disease might have caused the increase in AFP production. In the present experimental study, the same mechanism might function during the DEN-induced liver injury, adding to the chemical hepatocarcinogenesis. However, although high levels of plasma TNFa have been reported in human HCC patients [24], it remains controversial as a tumor marker of human HCC. TNF-a is thought to be an indicator of apoptosis of hepatocytes [25]. Recently, it was reported that inhibition of transcription factor NF-kB through the adenoviral delivery of a modified form of IkBa-an inhibitor of NF-kBsensitizes chemoresistant tumors to the apoptotic potential of TNF-a, resulting in tumor regression [26]. However, the profile of rat plasma TNF-a in the present study showed no correlation with DEN-induced tumor progression.
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In conclusion, our results show that P-GP may confer a protective effect against anticancer drugs, and that both EPIR and CDDP increase P-GP expression when given before DEN exposure. However, the mechanism responsible and the significance of the findings will require further investigation.
Acknowledgements The present study was supported in part by a grant from the Japanese Ministry of Education, Science and Culture (1996–1998). The authors are grateful to Professor Neil Kaplowitz (University of Southern California School of Medicine, Los Angeles, USA) for his critical comments and advice in preparing the manuscript.
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