Introduction

The development of polymerase chain reaction (PCR) technique enables us to investigate the relationship between bacteria, especially the anaerobic, and tumor occurrence.[1-5] Since the relationship between bacterial infection and gallbladder carcinoma is obscure, we amplified the bacterial gene fragments in the gallbladder tissues from 46 patients with gallbladder carcinoma, and in 18 of them bile was also detected by PCR. The aim of this study was to assess the role of bacteria in the development of gallbladder carcinoma.[6]

Methods

Patients

Seven male and 11 female patients, aged 18-69 years (average 41.1 years) were subjected to operations, including U-tube drainage (1), right or left biliary tube drainage (4), radical cholecystectomy (9), and cholecystorrhaphy (4). Their bile underwent PCR amplification. The tissue fragments of gallbladder carcinoma from the remaining 28 patients were taken from formalin-fixed, paraffin-embedded tissues. All samples obtained from the Affiliated Hospital of Medical College, Qingdao University, Qingdao and the First Affiliated Hospital of Xi?/SPAN>an Jiaotong University, Xi?/SPAN>an, China were pathologically confirmed.

Methods

According to the literature,[3] the sequences of bacterial primers were designed for amplifying bacterial 16S rRNA: P1 5?/SPAN>-TGCGGTTGGATCACCTCCT-3?/SPAN>, P2 5?/SPAN>-TCCCCACCTTCCTCCAGTT-3?/SPAN> (Institute of Cellular Biology, Shanghai, China). Specimens of fresh gallbladder carcinoma tissues of 18 patients were obtained at operation, and stored immediately at -72 ℃ after washing with brine. At the beginning of the test, 0.2 g iced tissue was added with 10 mmol/L Tris (trishydroxymethylaminomethane)-EDTA (ethylendiamine tetraacetic acid) at pH8.0 and 1% sodium dodecyl sulfate (SDS), digested by 100 μg/μl proteinase K. DNA was precipitated for one night at 37 ℃. The total incubation volume was 50 μl, containing 29 μl sterilized water, 5 μl primers (P1, P2), 4 μl 4?/SPAN>dNTP (200 mmol/L), 2 units Taq DNA, 5 μl amplified DNA, resuspended in 50 μl TE buffer (Tris/EDTA). DNA was extracted by the traditional purification method. The PCR cycle took place at 95 ℃ for 45 s, 58 ℃ for 45 s, and 72 ℃ for 1 min. Thirty-four cycles were carried out for each reaction. PCR-amplified products (10 μl) were applied to agarose gel containing ethidum bromide, and after electrophoresis (60 V) for 3 hours, DNA was visualized under ultraviolet light, recorded and photographed. Blank and standard contrast was provided in each group.

Results

The positive rate of bacterial DNA in the gallbladder carcinoma tissue was 78.3% (36/46). The sequence of 16S ribosomal RNA gene fragments amplified by PCR were approximately 371 base pairs (bp). Multiple kinds of standard bacterial gene fragments obtained from 36 patients included Colibacillus, B.fragilis, Klebsiella, C.perfringens and Clostridium with a positive rate of 78.3% (36/46). Among these patients, 14 patients with gallbladder carcinoma received operation. Their relative bile at operation was detected bacterial gene fragments, with a positive rate of 77.8% (14/18). This result was quite close to that in gallbladder carcinoma tissues.

Discussion

Bacterial rRNA is divided into 5S, 16S and 23S rRNA according to their descending coefficients. The 16S rRNA bacterial gene is the comparative DNA sequence in serial number of bacterial chromosome, which is known to exist in all kinds of bacterial chromosomes.[7-11] Its internal structure can be divided into changeable and unchangeable areas; but no significant difference exists in the unchangeable area. The changeable area is formed in the process of bacterial evolution, which makes the difference of bacteria. Theoritically, different primers can be designed to detect all kinds of bacteria by PCR amplification.[12-18] In this experiment a pair of common primers was designed in the unchangeable area of bacteria; five kinds of different bacteria standard strains were amplified by PCR, and all produced 371bp of DNA fragments.[19-27] The result was similar to that of gallbladder carcinoma tissues. We conclude that bacterial fragments exist in the core center of gallbladder carcinoma tissues.[128-34]

The major causative factors of gallbladder carcinoma are very complex. Cholecystitis with gallstone was reported one of the most important factors. Many research revealed that cholecystitis or gallstone can give rise to epithelial hyperplasia of gallbladder mucusa or canceration secondarily. In this study, 78.3% patients (36/46) were subjected to amplification of bacteria gene fragments, most of which was C.perfringens. This indicates that relationship exists between gallbladder carcinoma and infection of anaerobic bacteria, especially C.perfringers. The gallbladder mucosa stimulated by anaerobic and aerobic bacteria might be the major cause for the development of carcinoma.[35-43]

Competing interest

The author or authors do not choose to response to the statements listed in Instructions for Authors.

References

1 Shi JS, Wang JS, Liu G, Yu YL, Lu Y, Jiao XY, et al. Early diagnosis of primary gallbladder carcinoma. Hepatobiliary Pancreat Dis Int 2002;1:273-275.

2 Tsuchiya T, Shimokawa I, Higami Y, Ohtani H, Shigeoka Y, Ohshima K, et al. Primary low-grade MALT lymphoma of the gallbladder. Pathol Int 2001;51:965-969.

3 Su WC, Chan KK, Lin XZ, Lin PW, Chow NH, Shin JS, et al. A clinical study of 130 patients with biliary tract cancers and periampullary tumors. Oncology 1996;53:488-493.

4 Kato H, Koyabu S, Aoki S, Tamai T, Sugawa M, Watanabe M, et al. An autopsy case of gallbladder cancer developing in a Japanese man with cerebrotendinous xanthomatosis: genetic analysis of the sterol 27-hydroxylase and p53 genes. Pathology 2003;35:141-144.

5 Parwani AV, Geradts J, Caspers E, Offerhaus GJ, Yeo CJ, Cameron JL, et al. Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions. Mod Pathol 2003;16:299-308.

6 Lu Y, Xiang TH, Shi JS, Sun Z. Bile anaerobic bacteria detection and antibiotic susceptibility in patients with gallstone. Hepatobiliary Pancreat Dis Int 2003;2:431-434.

7 Kohya N, Kitajima Y, Jiao W, Miyazaki K. Effects of E-cadherin transfection on gene expression of a gallbladder carcinoma cell line: repression of MTS1/S100A4 gene expression. Int J Cancer 2003;104:44-53.

8 Wadler S, Yu B, Tan JY, Rozenblit A, Kaufiman H, Edelman M, et al. Persistent replication of the modified chimeric adenovirus ONYX-015 in both tumor and stromal cells from a patient with gall bladder carcinoma implants. Clin Cancer Res 2003;9:33-43.

9 Kim YT, Kim J, Jang YH. Genetic alterations in gallbladder adenoma, dysplasia and carcinoma. Cancer Lett 2001;169:59-68.

10 Tazuma S, Kajiyama G. Carcinogenesis of malignant lesions of the gall bladder. The impact of chronic inflammation and gallstones. Langenbecks Arch Surg 2001;386:224-229.

11 Kohya N, Kitajima Y, Kitahara K, Miyazaki K. Mutation analysis of K-ras and beta-catenin genes related to O6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma. Int J Mol Med 2003;11:65-69.

12 Rashid A, Ueki T, Gao YT, Houhhan PS, Wallace C, Wang BS, et al. K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China. Clin Cancer Res 2002;8:3156-3163.

13 Zhang W, Yue B, Wang GQ. Serum and ascites levels of macrophage migration inhibitory factor, TNF-α and IL-6 in patients with chronic virus hepatitis B and hepatitis cirrhosis. Hepatobiliary Pancreat Dis Int 2002;1:577-580.

14 Feng HY, Shi YJ, WU CX. Mononuclear macrophages in pathogenesis of acute lung injury during acute obstructive cholangitis. Hepatobiliary Pancreat Dis Int 2002;1:587-591.

15 Takai S, Shiratori Y, Kanematsu M, Yamzaki K, Baiki T, Yasuka I, et al. Usefulness of MR imaging in the postsurgical monitoring of gallbladder cancer in a patient with bile duct cancer that developed 7 years after resection of mucinous adenocarcinoma of the gallbladder. J Gastroenterol 2001;36:787-789.

16 Tsuchida A, Itoi T, Aoki T, Kayanage K. Carcinogenetic process in gallbladder mucosa with pancreaticobiliary maljunction (Review). Oncol Rep 2003;10:1693-1699.

17 Koda M, Yashima K, Kawaguchi K. Expression of Fhit, Mlh1, and P53 protein in human gallbladder carcinoma. Cancer Lett 2003;25:199:131-138.

18 Caca K, Feisthammel Jl. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines. Int J Cancer 2002;97:481-488.

19 Kimura Y, Furuhata T, Mukaiya M, Kihara C, Kawakami M, Okita K, et al. Frequent beta-catenin alteration in gallbladder carcinomas. J Exp Clin Cancer Res 2003;22:321-328.

20 Nakayama K, Konno M, Kanzaki A. Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct. Cancer Lett 2001;166:135-141.

21 Wistuba II, Ashfaq R. Fragile histidine triad gene abnormalities in the pathogenesis of gallbladder carcinoma. Am J Pathol 2002;160:2073-2079.

22 Matsumoto Y, Fujii H, Itakura J. Recent advances in pancreaticobiliary maljunction. J Hepatobiliary Pancreat Surg 2002;9:45-54.

23 Tsuchiya T, Shimokawa I, Higami Y, Otani H, Komastsu T, Chiba T, et al. Primary low-grade MALT lymphoma of the gallbladder. Pathol Int 2001;51:965-969.

24 Rashid A, Gao YT, Bhakta S. Beta-catenin mutations in biliary tract cancers: a population-based study in China. Cancer Res 2001;61:3406-3409.

25 Ahrendt SA, Rashid A, Chow JT. p53 overexpression and K-ras gene mutations in primary sclerosing cholangitis-associated biliary tract cancer. J Hepatobiliary Pancreat Surg 2000;7:426-431.

26 Otani K, Shimizu S, Chijiiwa K, Yamaguchi K, Noshiro H, Tanaka M. Immunohistochemical detection of 8-hydroxy-2?/SPAN>-deoxyguanosine in gallbladder epithelium of patients with pancreaticobiliary maljunction. Eur J Gastroenterol Hepatol 2001;13:1363-1369.

27 Saetta A, Lazaris AC, Michalopoulos NV. Genetic alterations involved in the development of gallbladder carcinomas from Greek patients. Hepatogastroenterology 2001;48:1284-1288.

28 Kiguchi K, Carbajal S, Chan K, Beitran L, Ruffino L, Shen J, et al. Constitutive expression of ErbB-2 in gallbladder epithelium results in development of adenocarcinoma. Cancer Res 2001;61:6971-6976.

29 Kashiwagi K, Ikeda H, Hirohashi Y. Analysis of a shared pancreatic cancer antigen recognized by an HLA-A*2601-restricted cytotoxic T-lymphocyte clone. Pancreas 2003;26:E81-88.

30 Quan ZW, Wu K, Wang J. Association of p53, p16, and vascular endothelial growth factor protein expressions with the prognosis and metastasis of gallbladder cancer. J Am Coll Surg 2001;193:380-383.

31 Mollenhauer J, Herbertz S, Helmke B, Kollender G, Krabes I, Madsen K, et al. Deleted in Malignant Brain Tumors 1 is a versatile mucin-like molecule likely to play a differential role in digestive tract cancer. Cancer Res 2001;61:8880-8886.

32 Su WC, Shiesh SC, Liu HS. Expression of oncogene products HER2/Neu and Ras and fibrosis-related growth factors bFGF, TGF-beta, and PDGF in bile from biliary malignancies and inflammatory disorders. Dig Dis Sci 2001;46:1387-1392.

33 Wistuba II, Tang M, Maitra A. Genome-wide allelotyping analysis reveals multiple sites of allelic loss in gallbladder carcinoma. Cancer Res 2001;61:3795-3800.

34 Nakano K, Todo T, Chijiiwa K. Therapeutic efficacy of G207, a conditionally replicating herpes simplex virus type 1 mutant, for gallbladder carcinoma in immunocompetent hamsters. Mol Ther 2001;3:431-437.

35 Boudny V, Nakano S. Src tyrosine kinase but not activated Ras augments sensitivity to taxanes through apoptosis in human adenocarcinoma cells. Anticancer Res 2003;23:7-12.

36 Tao KS, Lu YG, Wang T, Dou KF. Procedures for congenital choledochal cysts and curative effect analysis in adults. Hepatobiliary Pancreat Dis Int 2002;1:442-445.

37 Miyasaka K, Takata Y, Funakoshi A. Association of cholecystokinin A receptor gene polymorphism with cholelithiasis and the molecular mechanisms of this polymorphism. J Gastroenterol 2002;37(Suppl 14):102-106.

38 Matsubara T, Sakurai Y, Zhi LZ. K-ras and p53 gene mutations in noncancerous biliary lesions of patients with pancreaticobiliary maljunction. J Hepatobiliary Pancreat Surg 2002;9:312-321.

39 Kim YW, Park YK, Yoon TY. Expression of the GLUT1 glucose transporter in gallbladder carcinomas. Hepatogastroenterology 2002;49:907-911.

40 Ku JL, Yoon KA, Kim IJ, Kim WH, Jang JY, Suh KS, et al. Establishment and characterisation of six human biliary tract cancer cell lines. Br J Cancer 2002;87:187-193.

41 Zhou YM, Li YM, Cao N, Feng Y, Zeng F. Significance of expression of epidermal growth factor (EGF) and its receptor (EGFR) in chronic cholecystitis and gallbladder carcinoma . Ai Zheng 2003;22:262-265.

42 Rashid A. Cellular and molecular biology of biliary tract cancers. Surg Oncol Clin N Am 2002;11:995-1009.

43 Matsuo K, Kuroki T, Kitaoka F, Tajima Y, Kanamatsu T. Loss of heterozygosity of chromosome 16q in gallbladder carcinoma. J Surg Res 2002;102:133-136.

Received August 29, 2002

Accepted after revision November 6, 2002