Transforming growth factor-b1 in invasion and metastasis in colorectal cancer

Bin Xiong, Hong-Yin Yuan, Ming-Bo Hu, Feng Zhang, Zheng-Zhuan Wei, Ling-Ling Gong, Guo-Liang Yang

Bin Xiong ,Hong-Yin Yuan, Ming-Bo Hu, Feng Zhang, Zheng-Zhuan Wei, Ling-Ling Gong, Guo-Liang Yang, Department of Oncology, Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071,Hubei Province, China
Supported by Hubei Province Natural Science Foundation, No.2000J054
Correspondence to: Dr. Bin Xiong, Department of Oncology, Affiliated Zhongnan Hospital of Wuhan University,Wuhan 430071, Hubei Province, China. xbxh@public.wh.hb.cn
Telephone: +86-27-87325716
Received 2001-08-08 Accepted 2001-09-20


Abstract
AIM: To investigate the role of TGF b1 in invasion and metastasis in colorectal cancer by analysing TGFb1 correlated with depth of tumor invasion, stage and metastasis.

METHODS: Serum TGFb1 levels were determined in 50 patients with colorectal cancer and 30 healthy volunteers using a TGFb1 enzyme-linked immunosorbent assay. TGFb1 expression in primary and lymph node metastatic lesions were detected in 98 cases of colorectal cancer by immunohistochemical staining and in situ hybridization.

RESULTS: Serum levels of TGFb1 in patients with colorectal cancer(40±18 ug·L^-1) were significantly higher than those in the healthy control group(19±8 ug·L^-1),P<0.05. Elevated levels of serum TGFb1 were found in 60 % of patients with colorectal cancer when the mean +2 s was used as the upper limit of the normal range (35.1 ug·L^-1). Increases in serum TGFb1 levels were significantly associated with Duke's stage (P<0.05), but there was no significant difference between Duke's stage B patients and Duke's stage C patients. In the cytoplasm of cancer cells, TGFb1 was immunostained in 37.8 % (37/98) of colorectal cancer, and this expression was confirmed by in situ hybridization. Among 35 cases of colorectal cancer with lymph node metastatic lesions, TGFb1 positive staining was found in 18 (51.4 %) cases of primary tumor, and 25 (71.4 %) cases with lymph node metastatic lesions, respectively. Of 17 cases w ith no staining in the primary lesion, 7 (41.2%) casesshowed TGFb1 staining in the metastatic lesion. Serum TGFb1 levels and TGFb1 expression in colorectal cancer tissues were correlated significantly with depth of tumor invasion, stage and metastasis. Patients in stage C-D,T3-T4 and with metastasis had significantly higher TGFb1 levels than patients in stage A-B,T1-T2 and without metastasis (P<0.05).

CONCLUSION: These results suggest that transforming growth factor-b1 is closely related to the invasion and metastasis of colorectal cancer. It increased the invasive and metastatic potential of tumor by altering a tumor microenvironment. TGFb1 may be used as a possible biomarker.

Xiong B, Yuan HY , Hu MB, Zhang F, Wei ZZ, Gong LL, Yang GL. Transforming growth factor-b1 in invasion and metastasis in colorectal cancer.World J Gastroenterol 2002; 8(4):674-678



INTRODUCTION
The incience of colorectal cancer has become high in China and its biology is a hot topic of research^[1-44].Tumor invasion and metastasis are complex processes in which cancer cells detach from the original tumor mass to establish metastatic foci at distant sites. Metastatic cells characteristically lose growth inhibitory responses, undergo alteration in adhesiveness and demonstrate enhanced production of enzymes that can degrade extracellular matrix components. Since it is the development of metastatic disease that is primarily responsible for cancer mortality, an understanding of the mechanisms that facilitate metastatic tumor progression is of great importance^[1]. Transforming growth factor(TGF)-b1 is a 25-kd polypeptides. This growth factor regulates cell growth and differentiation in both normal and transformed cells. TGF-b1 was found to inhibit the growth of normal and neoplastic cells. Resistance to the negative growth-regulating properties of TGF-b1 has been observed in epithelial and mesenchymal tumors. Tumor cell lines that lack TGF-b receptors lose responsiveness to TGF-b1, and the escape of cells from TGF-b1 mediated negative regulation is linked to tumor progression^[45]. Colorectal cancer is one of the most malignant neoplasms. TGF-b1 plays a crucial role in tumor extension. We examined the expression of TGF-b1 in primary and lymph node metastatic lesions in colorectal cancer, as well as serum TGF-b1 levels in the peripheral veins. Our objective is to determine the clinical significance of TGF-b1 in advance of colorectal cancer.

MATERIALS AND METHODS
Patients
Serum TGF-b1 assays were performed in 50 patients treated from July 1999 to June 2000. There were 32 men and 18 women, and their age ranged from 23 to 74 years (means, 53±11 years). According to Duke's staging criteria, 9 cases were stageⅠ, 18 stageⅡ,18 stage Ⅲ and 5 stage Ⅳ. Thirty healthy volunteers were selected as control group among whom there were 17 men and 13 women. Their age ranged from 20 to 56 years (means, 45±8 years).
      A total of 98 colorectal adenocarcinoma patients (including the 50 patients above)who had undergone surgical resection in the Affiliated Zhongnan Hospital of Wuhan University (Wuhan, China) from July 1998 to December 2000, TGFb1 and TGFbRⅡ immunohistochemical staining and in situ hybridization were performed. There were 53 men and 45 women, and their age ranged from 23 to 74 years (means, 56±11 years). Among 98 patients, 17 were well differentiated adenocarcinoma, 47 moderately differentiated adenocarcinoma and 34 poorly differentiated adenocarcinoma. According to Duke's staging criteria, 34 cases were stageⅠ, 29 stageⅡ, 30 stage Ⅲ and 5 stage Ⅳ.

Methods
Preparation of serum sample and TGFβ1 assay  Two mL of blood sample, collected from the peripheral vein before surgery, were stored for approximately 3 h at 4 ℃ until the samples were centrifuged. Blood samples were centrifuged at 3 000 g for 20 min. The serum was separated and stored frozen at -70 ℃ until the time of analysis. TGFb1 was assayed using human TGFb1 enzyme-linked immunoabsorbent assay kits. The ELISA kits were obtained from Quantikine Co. of USA. The TGFb1 assay was performed according to the methods outlined in the package insert. Standard samples of 200 mg were added to each well, and incubated for 3 h at room temperature. After complete wash of each well, 200 ul TGFb1 conjugate was added to each well and these were incubated for 1.5 h at room temperature. We repeated the aspiration/wash and added 200 ul of substrate solution to each well and incubated for 20 min. Finally, we added 50 ml of stop solution and absorbances in each well were measured using a spectrophotometric plate reader at a wavelength of 490 nm. To determine the TGF-b1 concentration in each sample, we first calculated the average absorbance value in each set of duplicates. Serum levels of TGF-b1 were calculated from linear regression equation.
Immunohistochemistry All the tissue specimens were fixed in 100 mL.L^-1 neutral formalin and embedded in paraffin. Five-um thick sections were xylene dehydrated in ethanol. Tissue sections were washed three times in 0.05 moL.L^-1 PBS,and incubated in endogenous peroxidase blocking solution. Non-specific antibody binding was blocked by pretreatment with PBS containing 5 g.L^-1 bovin serum albumin. Sections were then rinsed in PBS and incubated overnight at 4 ℃ with diluted anti-TGFb1 and anti-TGFbRⅡ protein polyclonal antibody. The steps were performed using Immunostain kit according to the manufacturer抯 instructions. PBS was used as substitutes of protein antibody for negative control groups. The sections were examined under light microscopy. Anti-TGFb1 and anti-TGFbRⅡprotein polyclonal antibody were purchased from Bosden Comp. (Wuhan, China). S-P detection kit was purchased from Fuzhou Maixin Comp. (Fuzhou, China). Anti-TGFb1 and anti-TGFbRⅡ protein polyclonal antibody were diluted to 1:100.
In situ hybridization All the tissue specimens were fixed in 100 mL.L^-1 neutral formalin and embedded in paraffin. Six-um thick sections were xylene dehydrated in ethanol, and digested with 10 mg.L^-1 proteinase for 10 min. Sections were washed in 0.5 moL.L^-1 PBS for 15 min. They were incubated overnight at 37 ℃ with the 500 ug.L^-1 digoxigenin-labeled RNA probe in hybridization buffer. After hybridization, sections were washed in 2× SSC for 10 min at 37 ℃ and finally in 0.2×SSC for 15 min at 37 ℃. Sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody for 60 min at 37 ℃. The steps were performed using in situ hybridization kit according to the manufacturer's instructions. The kits were purchased from Bosden Comp. (Wuhan, China).
TGFb1 in situ hybridization probe sequences were:
(1)5-CGTTTCACCAGCTCCATGTCGATGGTCTTGCAAT-3'
(2)5_CTTGATTTTAATCTCTGCAAGCGCAGCTCTGCACG-3'
(3)5_TTGGTATCCAGGGCTCTCCGGTGCCGTGAGCTGTG-3'

Statistical analysis
The difference between each group was analyzed by Chi-square test and correlativity.
The limit of significant difference was P<0.05.

RESULTS
Serum TGFb1 levels in patients with colorectal cancer
Serum TGFb1 levels in patients with colorectal cancer (40.4±17.6 ug.L^-1)were significantly higher than in normal controls(19.2±8.0 ug.L^-1),P<0.01. Elevated levels of serum TGFb1 were found in 60 % of patients with colorectal cancer when the mean +2 s was used as the upper limit of the normal range (35.1 ug.L^-1,Figure 1). Increased in serum TGFb1 levels were significantly associated with Duke's stage (P<0.05), but there was no significant difference between Duke's stage B patients and Duke's stage C patients. Serum levels of TGFb1 were 29.2±7.3 ug.L^-1 in Duke's stage A patients, 39.5±11.9 ug.L^-1 in Duke's stage B patients, 43.1±15.8ug.L^-1 in Duke's stage C patients, and 57.8±16.2 ug.L^-1 in Duke's tage D patients. Serum levels of TGFb1 in each stage were significantly higher than those in the control group(Figure 2). Serum levels of TGFb1 were not correlated with age, gender, tumor size and differentiation degree of tumor.

Figure 1 Serum TGFb1 in patients with colorectal cancer. Bars represent mean ± standard deviation.

Figure 2 Serum TGFb1 in patients with colorectal cancer according to Duke's stage. Bars represent mean ± standard deviation.

      TGFb1 and TGFbRⅡ Expression in colorectal cancer tissue The TGFb1 and TGFbRⅡ protein were stained mainly in the cytoplasm and cell membrane of cancer cells, as shown in Figures 3 and 4. Staining was classified as negative if less than 10 % of the cells were positive and as positive if more than 10 % were positive^[2].Thirty-seven(37.8 %) of 98 tissues from colorectal cancer patients were positive for TGFb1 staining and forty-six(46.9 %) were positive for TGFbRⅡ staining. The expression of TGFb1 and TGFbRⅡ was correlated significantly with the depth of invasion, stage of disease and metastasis (lymph node and distant metastasis). Patients in T₃-T₄, stage C-D and with metastasis had significantly higher expression of TGFb1 than patients in T₁-T₂, stage A-B and without metastasis (P<0.05). Patients in T3-T4, stage C-D and with metastasis had significantly lower expression of TGFbRⅡ than patients in T₁-T₂, stage A-B and without metastasis (P<0.05). The expression of TGFb1 and TGbRⅡ was not correlated with age, gender, tumor size and differentiation degree of tumor (Table1).

Table 1  Clinicopathologic characteristics of colorectal cancer with expression of TGFb1 and TGFbRⅡ

^aP<0.05, T₁-T₂ vs T₃-T₄, Present vs Absent, A, B vs C+D

TGFb mRNA expression in colorectal cancer tissue
The expression of TGFb1mRNA in 50 colorectal cancer tissues was examined. The degree of staining was prominent in cases of TGFb1 positive immunohistochemical staining, but it was rare for negative cases of immunohistochemical staining. The pattern of distribution of TGFb1mRNA is the same as immunohistochemical staining (Figure 5).
Relationship of TGFb1 expression between primary and lymph node metastic lesions Among 35 cases of colorectal cancer with lymph node metastatic lesions, the TGFb1 positive rate was 51.4 %（18/35) for primary lesions and 71.4 % (25/35)for metastatic lesions in the lymph nodes. Of 17 cases with no staining in the primary lesion, 7 cases (41.2 %) showed TGFb1 staining in the metastatic lesion (Figure 6).

Figure 3 TGFb1 staining in cytoplasm of cancer cells. ×400

Figure 4 TGFbRⅡstaining in cytoplasm of cancer cells. ×400

Figure 5 TGFb1mRNA expression in colorectal cancer in situ hybridization. ×400

Figure 6 TGFb1 Expression in lymph node metastatic lesions. ×400


DISCUSSION
TGFb1 has been found to be overexpressed locally in many tumors, and is believed to play a role in tumor transformation and progression, as well as in tumor regression^[46-48]. Although TGFb1 acts as a potent inhibitor of cell growth and tumor progression, loss of this negative regulation can lead to tumor development. TGFb1 switches to a growth stimulator with tumor progression. Growth inhibition is replaced in many tumors by the opposite response, growth stimulation. This paradoxical switch in the responsiveness of tumor cell to TGFb1 during neoplastic progression may be due to the inactivation of the TGFb1 signaling pathway such as mutations in the type 2 TGFb receptor gene or through reduced expression and increase of blood supply to a tumor mass and inhibition of immunologic mechanisms involved in tumor identification and cytolysis. In cases of breast cancer, expression of TGFb1 was positively associated with invasion and matastasis. Maehara et al^[45] reported that the expression of TGFb1 in gastric cancer cells was closely related to infiltrative growth of the cancer and to the higher rate of lymph node metastasis.
      We found that TGFb1 levels in the serum of patients with colorectal cancer were significantly elevated compared with normal controls. TGFb1 was overexpressed in colorectal cancer tissue compared with normal colorectal mucosa. Elevated serum levels of TGFb1 and over-expression of TGFb1 in colorectal cancer tissue were correlated significantly with invasion and metastasis of colorectal cancer. Patients in T₃-T₄, stage C-D and with metastasis had significantly higher expression of TGFb1 in tumor tissue and TGFb1 levels in the serum than patients in T₁-T₂, stage A-B and without metastasis (P<0.05). The expression of TGFb1 in tumor tissue and TGFb1 levels in the serum were not correlated with age, gender and differentiation degree of tumor. Shim et al^[49] reported that patients of colorectal cancer in stage C-D had significantly higher expression of TGFb1 in tumor tissues and TGFb1 levels in the serum than patients in stage A-B (P<0.05). TGFb1 in colorectal cancer patients may be associated with disease progression. Among 35 cases of colorectal cancer with lymph node metastasis lesions, the TGFb1 positive rate was 51.4 %(18/35) for primary lesions and 71.4 %(25/35) for metastatic lesions in the lymph nodes. Of 17 cases with no staining in the primary lesion, 7 cases(41.2 %)showed TGFb1 staining in the metastatic lesion. The preferential expression of TGFb1 in lymph node metastases suggests a clonal selection of tumor cells with TGFb1 expression, specific for the higher potential of lymph node metastasis in tumor advance, and TGFb1 plays a role related to the malignant progression of colorectal cancer.
      We also found that TGFbRⅡ expression was significantly lower in colorectal cancer tissues than in normal mucosa. The expression of TGFTGFbRⅡ in tumor tissues of Patients in T₃-T₄, stage C-D and with metastasis was significantly lower than that in patients in T₁-T₂, stage A-B and without metastasis (P<0.05). The expression of TGFbRⅡin tumor tissues was not correlated with age, gender and differentiation degree of tumor. The lower expression of TGFbRⅡ in colorectal cancer may be associated with disease progression. In our previous study^[50,51], we found that TGFb1 expression was correlated significantly with angiogenesis in colorectal cancer tissues and inhibition of immunologic mechanisms involved in tumor identification and cytolysis.TGFb1 is closely related to the invasion and matastasis of colorectal cancer, and it may be used as a possible biomarker.

REFERENCES
1    Mcearchern JA, Kobie JJ, Mack V, Arteaga CL, Dumout N, Mary JC ,Akporiaye ET. Invasion and metastasis of a mammary
      tumor involve TGFb signaling. Int J Cancer 2001;91:76-82
2    Xiao B, Jing B, Zhang YL, Zhou DY, Zhang WD. Tumor growth inhibition effect of hIL-6 on colon cancer cells transfected with
      the target gene by retroviral vector. World J Gastroenterol 2000;6:89-92
3    Huang PL, Zhu SN, Lu SL, Dai SZ, Jin YZ. Inhibitor of fatty acid synthase induced apoptosis in human colonic cancer cells.
      World J Gastroenterol 2000;6:295-297
4    Xie B, He SW, Wang XD. Effect of gastrin on protein kinasec and it抯 subtype in human colon cancer cell line SW 480.
      World J Gastroenterol 2000;6:304-306
5    Guo WJ, Zhon ZD, Wu HJ, Liu YQ, WuRG, Zhang WD. Ultrastructural localization of glutathione s-transferase-pi in human
      colorectal cancer cells. World J Gastroenterol 2000;6:454-455
6    Yuan P, Sun MH, Zhang JS, Zhu XZ, Shi DR. APC and K-ras gene mutation in aberrant crypt foci of human colon.
      World J Gastroenterol 2001;7:352-356
7    Zheng CX, Zhan WH, Zhao JZ, Zheng D, Wang DP, He YL, Zheng ZQ. The prognostic value of preoperative serum levels of CEA
      CA19-9 and CA72-4 in patients with colorectal cancer. World J Gastroenterol 2001;7:431-434
8    Peng ZH, Xing TH, Qiu GQ, Tang HM. Relationship between Fas/Fasl expression and apoptosis of colon adenocarcinoma cell
      lines. World J Gastroenterol 2001;7:88-92
9    Li XG, Song JD, Wang YQ. Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer.
      World J Gastroenterol 2001;7:551-554
10  Wang W, Luo HS,Yu BP. Telomerase and colorectal cancer. Shijie Huaren Xiaohua Zazhi 2000;8:800-802
11  Ying YQ, Zhou G, Wu P. Huang WB. Significance of TGF-a and TGF-β1 expressions in the tissue of colorectal cancer.
      Shijie Huaren Xiaohua Zazhi 2001;9:223-225
12  Xie B, He SW, Wang DK. Inhibition of neomycin on gastrin-stimulating cell proliferation of human colon cell line SW480.
      Shijie Huaren Xiaohua Zazhi 1999;7:249-251
13  Qiao Q, Wu JS, Zhang J, Ma QJ, Lai DN. Expression and significance of apoptosis related gene bcl-2, bax in human large
      intestine adenocarcinoma. Shijie Huaren Xiaohua Zazhi 1999;7:936-938
14  Jiang HY, Qing SH. Research on related factors with lymph node metastasis of colorectal cancer.
      Shijie Huaren Xiaohua Zazhi 1999;7:982-984
15  Wang Q, Wu JS, Lai DN, Ma QJ, Pan BR. Expression and significance of p16 protein in colorectal carcinoma.
      Shijie Huaren Xiaohua Zazhi 1999; 7:1047-1048
16  Cao JB, Li SR. Relation between human papilloma virus and colorectal cancer. Shijie Huaren Xiaohua Zazhi 1999;7:1070-1071
17  Zhang LL, Zheng ZS, Zhang YL, Wu BP, Guo W, Liu XX, Zhou DY. Microsatellite instability in multiple primary colorectal cancers.
      Shijie Huaren Xiaohua Zazhi 1999;7:397-399
18  Li M, Wang H, Yu BM, Zheng MH. Effect of p53 gene mutation and tumor markers on the prognosis of patients with colorectal
      cancer. Shijie Huaren Xiaohua Zazhi 1999;7:425-426
19  Wang SM, Wu JS, Yao X, He ZS, Pan BR. Effect of TGFa, EGFR anti-sense oligodeoxynucleotides on colon cancer cell line.
      Shijie Huaren Xiaohua Zazhi 1999;7:522-524
20  Li WS, Li JS. Surgical treatment of left colon carcinoma obstruction. Shijie Huaren Xiaohua Zazhi 1999;7:533-534
21  Yao XQ, Qing SH. Detection of multidrug resistance gene in progressive colon cancer and its significance.
      Shijie Huaren Xiaohua Zazhi 1999;7:535-536
22  Fan YF, Huang ZH. Progress in the studies of gene therapy for colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:427-430
23  Wang CH, Zhang XM, Zhan M, Tang FX, Li L. TGF-βand its receptor expression in human colorectal cancer.
      Shijie Huaren Xiaohua Zazhi 2001;9:462-3
24  Fan RY, Li SR, Wu ZT, Wu X. Detection of p53 protein, k-ras and APC gene mutation in sporadic colorectal cancer tissue and
      exfoliative epithelial cell in stool. Shijie Huaren Xiaohua Zazhi 2001;9:771-775
25  Sheng ZX, Chao G, Sun J. Clinical significance of Cox-2 mRNA expression in colorectal cancer tissue.
      Shijie Huaren Xiaohua Zazhi 2001;9:1082-1083
26  Yu BM, Zhao R. Molecular biology of colorectal carcinoma. Shijie Huaren Xiaohua Zazhi 1999;7:173-175
27  Deng YC, Zhen YS, Zheng S, Xue YC. Activity of boanmycin against colorectal cancer. World J Gastroenterol 2001;7:93-97
28  Bai DJ, Yang GL,Yuan HY, Li Y, Wang K. Effect of cimetidine on T-lymplocyte subsets in periopenative gastrointestinal cancer
      patients, Shijie Huaren Xiaohua Zazhi 2000;8:147-149
29  Yang JH, Rao BQ, Wang Y, Tu XH, Zhang LY, Chen SH, Ou-Yong XN, Dai XH.Clinical significance of defecting the circulating
      cancer cells in peripheral blood from colorectal cancer. Shijie Huaren Xiaohua Zazhi 2000;8:187-189
30  Li SR. Early diagnosis of colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:780-782
31  Sheng JQ, Chen ZM. Colorectal cancer related gene in the screen of colorectal cancer.
      Shijie Huaren Xiaohua Zazhi 2001;9:783-785
32  Gu F, Lu YM. Treatment of colorectal adenoma. Shijie Huaren Xiaohua Zazhi 2001;9:785-786
33  Han RY. Early endoscopic diagnosis of colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001; 9:789-790
34  Han YY. Endoscopic treatment of colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:790-792
35  Wang YB, Yang ZX. Routine diagnosis of colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:792-793
36  Ma Q, Zhang ZS, Wang QY. Reverse of multidrug resistance to colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:822-825
37  Gu HP, Ni CR, Zhan RZ. Relationship of expression of CD15, CD44V6 and nm23H1 mRNA with metastasis and prognosis of colon
      carcinoma. Shijie Huaren Xiaohua Zazhi 2000;8:887-891
38  Zha R, Yu BM, Zhang GC, Zheng MH, Li DH, Huang L, Zhou HZ. Effect of selenium on immunity and anti-oxidative functions in
      patients with colorectal carcinoma. Shijie Huaren Xiaohua Zazhi 2000;8:1013-1016
39  Jia L, Chen TX, Sun JW, Na ZM, Zhang HH. Relationship between microvessel density and proliferating cell nuclear antigen and
      prognosis in colorectal cancer. Shijie Huaren Xiaohua Zazhi 2000;8:74-76
40  Ji DJ, Cao Y, Zhang YL, Jiang B, Yu N, Feng FC, Zhou DY. Synchronous studies on variation of p53 gene transcriptions and
      expression in HT-29, Lovo cells. Shijie Huaren Xiaohua Zazhi 2000;8:77-79
41  Zhao R, Zhang GC, Yu BM, Zheng MH, Li DH, Zhu YM, Hu BY. Effect of selemicm on T lymphocytc against colonic cancer cells.
      Shijie Huaren Xiaohua Zazhi 2000;8:80-83
42  Cao JB,Li SR, Zhu QP, Gu SY, Li YJ, Chen ZM, Zhang HG. The study of relationship between human papillomaviras and
      colorectal cancer. Shijie Huaren Xiaohua Zazhi 2000;8:111-112
43  Chen J, Gu GG, Ling WH, Luo YH. Microsatellite instability in 46 cases with non-hereditary colorectal cancer. Shijie Huaren
      Xiaohua Zazhi 2000;8:350-352
44  Wang ZW, Cheng RX, Zhou GJ, Shen HD, Chon YQ. CK20 mRNA expression in the peripheral blood of colorectal cancer
      patients. Shijie Huaren Xiaohua Zazhi 2000;8:818-820
45  Maehara Y, Kakeji Y, Kabashima A. Role of transforming growth factor-β1 in invasion and metastasis in gastric carcinoma.
      J Clin Onclo 1999;17:607-614
46  Rodeck V, Nishiyma J, Mauriel A. Independent regulation of growth and Smad-mediated transcription by TGF-b in human
      melanoma cells. Cancer Res 1999;53:547-550
47  Shyr M, Sheen C, Han SC, Chin WS, Hock CE, Wei JC. Serum levels of TGF-b1 in patients with breast cancer. Arch Surg
      2001;136:937-940
48  Farina AR, Coppa A, Tiberio A, Turco A, Colletta G, Mackay AR. TGF-b1, enhances the invasiveness of human MDA-MB-231
      breast cancer cells by up-regulating urokinase activity. In J Cancer 1998;75:721-730
49  Shim K S, Kim K H, Han WS, Park E B. Elevated serum levels of transforming growth factor-b1 in patients with colorectal
      carcinoma. Cancer 1999;85:554-561
50  Xiong B, Yuan HY, Zhang F, Hu MB, Yang GL. The expression of transforming growth factorb1 in colorectal cancer and its
      relation to angiogenesis. World J Gastroenterol 2002;8:496-498
51  Xiong B, Yuan HY, Hu WB, Ydan T, Yang GD. Serum levels of transforming growth factor-b1 correlating with T cell subsets
      and natural killer cell activity in colorectal cancer. Shijie Huaren Xiaohua Zazhi 2001;9:1194-1195

Edited by Ma JY