Introduction
Hepatitis C virus (HCV) is recognized as a kind of serious infectious disease detrimental to the health of the human being.[1] It could ultimately lead to cancerization. Recent studies, however, have suggested that HCV may be related to the pathogenesis of cholangiocarcinoma.[2-4] Successful use of antisense oligodeoxynucleotide (asODN) to inhibit the replication and expression of HCV in vitro has been reported recently.[5-11] These effects are probably due to an unspecific binding of ODNs to intracellular and extracellular proteins, resulting in the alteration of cellular processes.
To study the inhibitory effect of asODN on hepatitis C virus in cholangiocarcinoma and find out a new therapy for HCV infected cholangiocarcinoma, we transfected QBC939 cells by a recombinant HCV containing HCV core gene cloned in vector of PBK-CMV (PBK-HCVC),a recombinant HCV containing HCV core gene. They were treated by 14-mers phosphorothioate ODN complementary to the HCV core genomic region. The variation of HCVmRNA level was detected by RT-PCR. Moreover, the inhibitory effect of asODN was also observed in nude mice.
Methods
Materials
PBK-HCVC transfected cholangiocarcinoma cells
PBK-HCV encompassing the core and envelope genomic regions of HCV, containing from 330nt to 2020nt of HCV II strain, was provided as a gift by Professor Zhi Chen, Institute of Infectious Disease, Zhejiang University, with restriction sites of Pst I and EcoR I. The prokaryotic expressing vector of PBK-CMV containing multiple cloning site (MCS), the neomycin and kanamycin-resistance gene, SV40 poly (A), was purchased from Stratagene Co., USA. In our study, recombinant plasmid of the HCV-core gene was constructed by molecular cloning. It was identified by restriction enzymes, and then transfected into QBC939 cells with lipofection. After it was selected with G418 made by the presence of neomycin and kanamycin resistance gene, resistant colonies were obtained. The QBC939 cells (a cholangiocarcinoma cell line) were a generous gift from Professor Shu-Guang Wang, Third Military Medical University, China. The QBC939 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640, supplemented with 100 ml/L FBS and incubated at 37 ℃ in an atmosphere of 50 ml/L CO2.
Nude mice
Twenty-five male nude mice were purchased from the Experimental Anamial Center of Hubei province, Wuhan, China.
Reagents
Lipofection was provided by Boehringer Mannheim Co., Germany, and Trizol reagent was purchased from Gibco BRL Co., USA.
Antisense oligodeoxynucleotide
14-mers phosphorothioate ODN complementary to the HCV core genomic region was synthesized by Shanghai GeneCore Bio-Technologies Co., Shanghai, China. Complementary sites varied from 341nt to 354nt of HCV II strain: 5?/SPAN>-TTTGAGGTTTAGGA-3?/SPAN>.
PCR Primers
Two primers were designed according to the sequence of HCV core genomic regions and synthesized by Shanghai GeneCore Bio-Technologies Co., Shanghai, China: primer 1: 5?/SPAN>-TGAGCACGAATTTCTAAAC-3?/SPAN>, primer 2: 5?/SPAN>-GCGCCGACAAGCGGAATGT-3?/SPAN>; and ?/SPAN>-actin mRNA primer 1: 5?/SPAN>-CAGAGCAAGAGAGGCATCCT-3?/SPAN>, primer 2: 5?/SPAN>-GGATAGCACAGCCTGGATAG-3?/SPAN>. The products of HCV and ?/SPAN>-actin RT-PCR were 403bp and 250bp.
Methods
RT-PCR for HCVmRNA
Total RNA was prepared from the QBC 939 cell line with the Trizol reagent (Gibco Brl, USA) according to the manufacturer?/SPAN>s instructions. RT-PCR was performed using the RT-PCR kit (Promega, Madison, Wisconsin, USA) according to the manufacturer?/SPAN>s protocol. cDNA synthesis was carried out with 2 ?/SPAN>g of total RNA. PCR reactions of 20 ?/SPAN>l were processed in a MJ. A peltier thermal cycler-100 (PTC-100) Thermocycler was used under the following conditions: 60 ℃ for 30 minutes, 94 ℃ for 1 minute, 55 ℃ for 30 seconds, 72 ℃ for 1 minute for a total of 35 cycles, and 72 ℃ at 10 minutes. The RT-PCR products were analyzed on 2% agarose gel. Amplified human ?/SPAN>-actin served as a control for sample loading and integrity.
RT-PCR for HCVmRNA after adding asODN
After adding different concentration of ODNs (0, 1.0, 2.0, 4.0, 5.0, 6.0 ?/SPAN>mol/L), RT-PCR amplification was done formerly.
Test of proliferation effect on tumor
The 25 male nude mice (Balb/c) of 6-8 weeks were divided into 3 groups: 10 mice for injection of QBC939 cells transfected of PBK-HCVC vector, 5 mice for inoculation of QBC939 cells, and 10 mice for injection of PBK-HCVC+asODN. Each mouse was injected subcutaneously at two sites (neck and back), each of which was inoculated 5×107 cells/0.1 ml. Six weeks after inoculation, the mean tumor size and number were measured. The differences between the 3 groups were analyzed by the t-test.
Results
Expression of HCVmRNA core protein in PBK-HCVC transfected QBC939 cells
The cell sections transfected with PBK-HCVC and control plasmid (PBK-CMV) were amplified by RT-PCR. The QBC939 cells of PBK-HCVC transfected were seen approximately 400bp (HCVmRNA) and 250bp (?/SPAN>-actin mRNA). The cells transfected with control plasmid (PBK-CMV) were only seen approximately 250bp (?/SPAN>-actin mRNA) (Fig. 1).
Fig. 1. Electrophoretic analysis of RT-PCR product. M: marker DL2000; Lane 1: PBK-CMV transfected QBC939 cells; Lane 2: PBK-HCVC transfected QBC939 cells; Lane 3: negative control.
Fig. 2. Different concentration of asODN on HCVmRNA expression.
Fig. 3. RT-PCR products of HCVmRNA after action of asODN. M: marker DL2000; Lane 1: control; Lane 2-6: after action of asODN; Lane 3: negative control.
Expression of HCVmRNA after use of ODN
After adding ODNs (0-6 ?/SPAN>mol/L), the ratio of HCVmRNA to ?/SPAN>-actin mRNA was gradually decreasing. At 6 ?/SPAN>mol/L ODNs, the ratio was zero (Figs. 2 and 3).
ODN restraining effect of cholangiocarcinoma cells on nude mice
Tumors were found in the groups of PBK-HCVC transfection, control and PBK-HCVC+asODN (P<0.05) at rates of 60% (6/10), 65% (13/20) and 25% (5/20). The time of tumor appearance was 10.1 days in the PBK-HCVC transfection group, 12.0 days in the control group and 15.3 days in the PBK-HCVC+asODN group. The time in the PBK-HCVC transfection group was earlier than that in the control group and PBK-HCVC+asODN group (P<0.01, P<0.01). The mean tumor size in the PBK-HCVC transfection group was larger than that in the control group (P<0.05) and PBK-HCVC+asODN group (P<0.01, Table). It was suggested that the tumorigenicity in nude mice was greatly depressed after action of ODN.
Discussion
Hepatitis C virus infection, an important cause of morbidity and mortality worldwide, produces a spectrum of liver disease ranging from an asymptomatic carrier state to end-stage liver disease. The HCV core protein could act as a transcriptional regulator of various viral and cellular promoters to potentially disrupt normal cellular functions.[12-17] The core protein may cooperate with ras oncogene and transform primary rat embryo fibroblasts to a tumorigenic phenotype.[18-21] It may also cause anti-apoptosis by ression of the tumor suppressor gene-p53 and activation of NF-?/SPAN>B and implicate a mechanism by which HCV may evade the host?/SPAN>s immune surveillance, leading to viral persistence and possibly to carcinogenesis.[22-29] Thus, the HCV core protein plays a major role in the malignant transformation of cells.
Cholangiocarcinoma is the second cancer of the hepatobiliary system with an increasing incidence and mortality. Approximately 3000 new patients are diagnosed in the USA each year, but the pathogenesis of cholangiocarcinoma is not clear enough. Previously HCV was considered hepatotropic virus, and viral replication and cellular injury are largely confined to the liver. Recent studies, however, have suggested that HCV may replicate in tissues other than hepatocytes. Researchers reported the presence of HCV RNA and HCV-antigens in the epithelial cells of lymph nodes, pancreas, ovary, kidney, heart and bile duct.[30-34] The infection of HCV could lead to damage and loss of the bile duct, which are defined as epithelial, lymphocytic infiltration[35-38] and steatosis. The higher rate of HCV infection in the bile duct provides an evidence that epithelial cells of the bile duct could be an important reservoir of HCV and coutribute to the pathogenesis of cholangiocarcinoma. Recent laboratory and epidemic studies found that the infection of HCV is also related to the development of cholangiocarcinoma.[39-45]
In our study we observed the inhibitory effect of asODN on HCV in cholangiocarcinoma. The QBC939 cells transfected by PBK-HCVC, a recombinant HCV containing the HCV core gene, were treated by 14-mers phosphorothioate ODN complementary to the HCV core genomic region. The variation of HCVmRNA level was detected by RT-PCR. Moreover, the inhibitory effect of asODN was observed in nude mice. HCVmRNA was detected in the transfected-QBC939 cells.The 14-mers complementary asODN showed effective inhibition on HCVmRNA and unexpression of HCVmRNA at 6 ?/SPAN>mol/L. The tumorigenicity of transfected-QBC939 cells incubated with asODN in nude mice was greatly inhibited. These data have shown that ODNs directed against the HCV RNA specifically and significantly inhibit vial translation in vitro. Completely phosphorothioate-modified ODNs are currently undergoing phase I, II and III clinical trials for the translation inhibition of various RNAs such as cytomegalo virus (CMV) in HIV patients. Our results suggest the feasibility of asODN in the treatment of HCV-infected cholangiocarcinoma.
Competing interest
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
References
1 Chen YD, Liu MY, Yu WL, Li JQ, Peng M, Dai Q, et al. Hepatitis C virus infections and genotypes in China. Hepatobiliary Pancreat Dis Int 2002;1:194-201.
2 Kimura Y, Leung PS, Kenny TP, van de Water J, Nishioka M, Giraud AS, et al. Differential expression of intestinal trefoil factor in biliary epithelial cells of primary biliary cirrhosis. Hepatology 2002;36:1227-1235.
3 Liu XF, Zou SQ, Qiu FZ. Construction of HCV-core gene vector and its expression in cholangiocarcinoma. World J Gastroenterol 2002;8:135-138.
4 Okuda K, Nakanuma Y, Miyazaki M. Cholangiocarcinoma: recent progress. Part 1: epidemiology and etiology. J Gastroenterol Hepatol 2002;17:1049-1055.
5 Mizutani T, Kato N, Hirota M, Sugiyama K, Murakami A, Shimotohno K. Inhibition of hepatitis C virus replication by antisense oligonucleotide in culture cells. Biochem Biophys Res Commun 1995;212:906-911.
6 Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotide. In vitro model for selection of target sequence. J Biol Chem 1994;269:14205-14210.
7 Fukuma T, Walton CM, Wu CH, Wu GY. Conjugation of an antisense oligodeoxynucleotide to ribonuclease h results in sequence-specific cleavage and intracellular inhibition of HCV gene expression. Bioconjug Chem 2003;14:295-301.
8 Ideo G, Bellobuono A. New therapies for the treatment of chronic hepatitis C. Curr Pharm Des 2002;8:959-966.
9 Caselmam WH, Eisehardt S, Alt M. Synthetic antisense oligodeoxynucleotides as potential drugs against hepatitis C. Intervirology 1997;40:394-399.
10 Alt M, Renz R, Hofschneider PH, Paumgartner G, Caselmann WH. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology 1995;22:707-717.
11 Alt M, Renz R, Hofschneider PH, Caselmann WH. Core specific antisense phosphorothioate oligodeoxynucleotides as potent and specific inhibitors of hepatitis C viral translation. Arch Virol 1997;142:589-599.
12 Ray RB, Lagging LM, Meyer K, Ray R. Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 1995;37:209-220.
13 Tsuchihara K, Hijikata M, Fukuda K, Kuroki T, Yamamoto N, Shimotohno K. Hepatitis C virus core protein regulates cell growth and signal transduction pathway transmitting growth stimuli. Virology 1999;258:100-107.
14 Yao ZQ, Ray S, Eisen-Vandervelde A, Waggoner S, Hahn YS. Hepatitis C virus: immunosuppression by complement regulatory pathway. Viral Immunol 2001;14:277-295.
15 Basu A, Meyer K, Ray RB, Ray R. Hepatitis C virus core protein is necessary for the maintenance of immortalized human hepatocytes. Virology 2002;298:53-62.
16 Watashi K, Shimotohno K. The roles of hepatitis C virus proteins in modulation of cellular functions: a novel action mechanism of the HCV core protein on gene regulation by nuclear hormone receptors. Cancer Sci 2003;94:937-943.
17 Ishikawa T, Shibuga K, Vasui K, Mitamura K, Veda S. Expression of hepatitis C virus core protein associated with malignant lymphoma in transgenic mice. Comp Immunol Microbiol Infeet Dis 2003;26:115-124.
18 Ray RB, Steele R, Meyer K, Ray R. Transcriptional repression of p53 promoter by hepatitis C virus core protein. J Biol Chem 1997;272:10983-10986.
19 Reeves ME, Dematteo RP. Genes and viruses in hepatobiliary neoplasia. Semin Surg Oncol 2000;19:84-93.
20 Moradpour D, Englert C, Wakita T, Wends JR. Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 1996;222:51-63.
21 Zhang H. Lethality in mice infected with recombinant vaccinia virus expressing hepatitis C virus core protein. Hepatobiliary pancreat Dis Int 2003;2:374-382.
22 Ray RB, Meyer K, Ray R. Suppression of apoptotic cell death by hepatitis C virus core protein. Virology 1996;226:176-182.
23 Marusawa H, Hijikata M, Chiba T, Shimotohno K. Hepatitis C virus core protein inhibits Fas and tumor necrosis factor alpha-mediated apoptosis via NF-?/SPAN>B activation. J Virol 1999;73:4713-4720.
24 Ray RB, Meyer K, Ray R. Hepatitis C virus core protein promotes immortalization of primary human hepatocytes. Virology 2000;271:197-204.
25 Ray RB, Ray R. Hepatitis C virus core protein: intriguing properties and functional relevance. Micrbiol Lett 2001;202:149-156.
26 Hahn CS, Cho YG, Kang BS, Lester IM, Hahn YS. The HCV core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology 2000;276:127-137.
27 Sacco R, Tsutsumi T, Suzuki R, Otsuka M, Aizaki H, Sakamoto S, et al. Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of capase-activated DNase. Virology 2003;17:24-35.
28 Kountouras J, Zavos C, Chatzopoulos D. Apoptosis in hepatitis C. J Viral Hepat 2003;10:335-342.
29 Ruggieri A, Murdolo M, Rapicetta M. Induction of Fas ligand expression in a human hepatoblastoma cell line by HCV core protein. Virus Res 2003;97:103-110.
30 Moldvay J, Deny D, Pol S, Brechot C, Lamas E. Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization. Blood 1994;38:269.
31 Shimizu YK, Iwamoto A, Hijikata M, Purcell RH, Yoshikura H. Evidence of in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci USA 1993;89:5477-5481.
32 Kato N, Nakazawa T, Mizutani T, Shimotohno K. Susceptibility of human T-lymphotropic virus type I infected cell line MT-2 to hepatitis C virus infection. Biochem Bio Res Commun 1995;26:863-869.
33 Dominguez MH, Gaytan GS. Hepatocellular carcinoma: an update. Ultrastruct Pathol 2001;25:497-516.
34 Zhao W, Wan JM, Liu W, Liu QJ, Zhang L, Zhou ZX, et al. Hepatitis gene chip in detecting HBV DNA, HCV RNA in serum and liver tissue samples of hepatitis patients. Hepatobiliary Pancreat Dis Int 2003;2:234-241.
35 Goldin RD, Patel NK, Thomas HE. Hepatitis C and bile duct loss. J Clin Pathol 1996;49:836.
36 Rozario R, Ramakrishna B. Histopathological study of chronic hepatitis B and C: a comparison of two scoring systems. J Hepatol 2003;38:223-229.
37 Uenishi T, Kubo S, Hirohashi K, Yamamoto T, Kinoshita H. Expression of bile duct-type cytokeratin in hepatocellular carcinoma in patients with hepatitis C virus and prior hepatitis B virus infection. Cancer Lett 2002;178:107-112.
38 Chu C, Hwang S, Luo J, Wang Y, Lu R, Lai C, et al. Clinical, virological, immunological, and pathological significance of GB virus C/hepatitis G infection in patients with chronic hepatitis C. Hepatol Res 2001;19:225-236.
39 Herrmann G. Immunohistochemical study of HBV antigen in 338 liver cell carcinomas. Z Gastroenterol 1999;37:329-342.
40 Lu H, Ye MQ, Thung SN, Dash S, Gerber MA. Detection of hepatitis C virus RNA sequences in cholangiocarcinomas in Chinese and American patients. Chin Med J 2000;113:1138-1141.
41 Kobayashi M, Ikeda K, Saitoh S, Suzuki F, Tsubota A, Suzuki Y, et al. Incidence of primary cholangiocellular carcinoma of the liver in Japanese patients with hepatitis C virus-related cirrhosis. Cancer 2000;88:2471-2477.
42 Yamamoto M, Takasaki K, Nakano M, Saito A. Minute nodular intrahepatic cholangiocarcinoma. Cancer 1998;82:2145-2149.
43 Guo ZJ, Li Q, He K. Diagnosis and treatment of hepatic cholangiocarcinoma: report of 52 cases. Hepatobiliary Pancreat Dis Int 2003;2:62-65.
44 Suriawinata A, Ivanov K, BenHaim M, Schwartz ME. A 67-year-old man with hepatitis C virus infection and a liver tumor. Semin Liver Dis 2000;20:227-231.
45 Liu XF, Zou SQ, Qiu FZ. Pathogenesis of cholangiocarcinoma in the porta hepatis and infection of hepatitis virus. Hepatobiliary Pancreat Dis Int 2003;2:285-289.
Received July 17, 2003
Accepted after revision December 2, 2003
