Jian-Wei Zhu, Bao-Ming Yu, Yu-Bao Ji, Ming-Hua Zheng, Dong-Hua Li, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
Correspondence to: Dr. Jian-Wei Zhu, Shanghai Institute of Digestive Sugery, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China. tozhujw@263.net
Telephone: +86-21-64370045-662211
Received 2001-07-19 Accepted 2001-09-27

Abstract
AIM: To evaluate the effect of reactive oxygen species such as hydrogen peroxide on the progression of human colon cancer.

METHODS: Human colon carcinoma cell lines, LS174T and HCT8, were treated respectively with 10^-5,10^-7or 10^-9 mol·L^-1 hydrogen peroxide for 24h, and co-cultured with human endothelial cell line ECV-304. The migration of ECV-304 induced by cancer cells was calculated and the expression level of vascular endothelial growth factor in cancer cells was determined by RT-PCR analysis and ELISA. Dactinomycin of 1.5mg·L^-1 which could block transcription of cancer cells was applied to observing the effects of H₂O₂ on transcriptional activity and the relative half-life of VEGF mRNA. Finally, to evaluate the effect of H₂O₂ on NF-κB activity in colon cancer cells, NF-κB in cytoplasm and nucleus of the cells were detected with FITC-tagged antibody and its presence in the nucleus(Fn) vs cytoplasm(Fc) was monitored by measuring the green fluorescence integrated over the nucleus by laser scanning cytometry(LSC).

RESULTS: Exogenouse hydrogen peroxide of low concentration increased the migration of endothelial cell induced by colon cancer cells. When cancer cells were treated with 10^-5 mol·L^-1 H₂O₂, the migration number of endothelial cells induced by LS174T cells was 203±70, and the number induced by HCT8 cells was 145±65. The two values were significantly higher than those treated with other concentrations of H₂O₂ (P<0.01).The expression of vascular endothelial growth factor in cancer cells, which could be blocked by dactinomycin, were increased to a certain degree, while the relative half-life of VEGF mRNA was not prolonged after treatment with hydrogen peroxide. The activity of NF-κB in colon cells rose after the cells were exposed to hydrogen peroxide for 24h.The Fn values in HCT8 cells were 91±13 (0 mol·L^-1 H₂O₂) and 149±40(10^-5 mol·L^-1 H₂O₂) (P<0.05),in LS174T cells were 127±35(0 mol·L^-1 H₂O₂) and 192±11(10^-5 mol·L^-1 H₂O₂) (P<0.05). It is similar to the case of VEGF expression in cancer cells.

CONCLUSION: Hydrogen peroxide increases vascular endothelial growth factor expression in colon cancer cells, and it is likely that reactive oxygen species such as hydrogen peroxide facilitates the development of colon cancer.

Zhu JW, Yu BM, Ji YB, Zheng MH, Li DH. Upregulation of vascular endothelial growth factor by hydrogen peroxide in human colon cancer. World J Gastroenterol 2002;8(1):153-157

INTRODUCTION
Reactive oxygen species (ROS) can be easily produced in intracolonic cavity,due to large amounts of bacteria and dietary metabolites in it. Several reports on the relationship between ROS and cancer suggested that ROS such as oxygen radicals, hydroxyl radicals and hydrogen peroxide (H₂O₂), were involved in the pathogenesis of colon tumors^[1-14]. H₂O₂, a special intermediate in redox reaction, is able to cross cell membranes in a free manner and modify protein and nucleic acid after being changed into radicals, and now it is thought to be a kind of signal molecular which plays an important role in the growth of tumor cells. Evidences have been given in some reports that H₂O₂ could promote cell growth and related gene expression in human tumors such as prostate cancer and breast cancer. Considering the special environment in colon and rectum we think it is necessary to evaluate the effects of ROS, especially H₂O₂, on the progression of colorectal cancer^[15-27]. As angiogenesis, which induced by several factors such as vascular endothelial growth factor(VEGF), is often demonstrated in solid tumors, and thought to be an essential requirement for the development of malignant tumors ^[28-33], we investigated the effects of H₂O₂ on VEGF expression in colon cancers in this study to find evidences that ROS such as H₂O₂ plays a role in the progression of the tumor.

MATERIALS AND METHODS
Cell culture and culture conditions
Human colon cancer cell lines, LS174T and HCT8, and human umbilical vein endothelial cell line ECV-304 were purchased from American Type Culture Collection. These cell lines were cultured and maintained in RPMI1640 supplemented with 100 ml·L^-1 fetal bovine serum at 37℃ in 50 ml·L^-1 carbon dioxide and 950 ml·L^-1 air.

H₂O₂ treatment of cancer cells and MTT assay
To determinate the effects of H₂O₂ on the growth of cancer cells, LS174T and HCT8 cells were grown (10³ per well )on 96-well plates and treated with the culture media containing H₂O₂ (ten concentrations from 10^-10mol·L^-1 to 10^-1 mol·L^-1)(300 ml·L^-1 H₂O₂ solution was purchased from Sigma). After 48h, MTT assay showed that H₂O₂ inhibited the growth of cancer cells when its concentration was ＞10^-5 mol·L^-1, while had no effect on cell growth when its concentration was ≤10^-5 mol·L^-1. Therefore, those concentrations of H₂O₂, 10^-5,10^-7 and 10^-9 mol·L^-1, were used in subsequent studies.

Endothelial cell migration induced by cancer cells
To clarify the effects of H₂O₂ on the migration of vascular endothelial cells, which could be promoted by VEGF,!colon cancer cells were co-cultured with endothelial cells. LS174T and HCT8 cells were plated on 12-well plates (Falcon) at a density of 4×10⁵ per well. Four hours later, the cells were washed with serum-free medium, and exposed to 10^-5,10^-7 or 10^-9      mol·L^-1 H₂O₂ with the H₂O₂-containing complete media for 24h. Cell culture inserts with polyethylene terephthalate membranes(PET) and 8 μm pore size (Becton Dickinson, USA) were then placed into the 12-well plates, and endothelial cells ECV-304 were seeded into the inserts with a density of 1×10⁴. Six hours later, the cells on the upper surface of the PET membranes were wiped off completely, and the inserts were fixed and stained. The migration capacity of endothelial cells was estimated by counting the number of the cells beneath the PET membranes. The controls were the groups without cancer cells in the wells or without treating cancer cells with H₂O₂.

Expression of VEGF in colon cancer cell
To determine the effects of H₂O₂ on expression of VEGF, LS174T and HCT8 were grown to 90% confluence to avoid the effects of cell density and incubated in complete media in the presence of H₂O₂ (10^-5,10^-7 and 10^-9 mol·L^-1) for 24h. Total RNA was extracted and resuspended in sterile RNase-free water for storage at -70℃. Access RT-PCR system (Promega ) with the sensitive feature was used to determine the relative VEGF mRNA expression. All primers were synthesized by Life Technology, Hongkong.

VEGF sense: 5’-AAGCCATCCTGTGTGCCCCTG ATG-3’
antisense: 5’-GCGAATTCCTCCTGCCCGCGCTGAC-3’
β-actin sense: 5’-AACACCCAGCCATGTACGTTG-3’
antisense: 5’-CGGATGTCCACGTCACACTTCAT-3’     

      The 50μL mixture for reverse transcription and PCR amplification were added in one-tube including AMV reverse transcriptase 5U, Tf 1 DNA polymerase 5U, MgSO₄, 20μL dNTP mixture, reaction buffer and 20pmol of each primer and 0.1μg total RNA sample. The condition for RT-PCR included a 48℃ reverse transcription, a 94℃ AMV inactivation and denaturation, a 60℃ annealing and a 72℃ extension. PCR amplification was subjected to 40 cycles. A volume of 10μL RT-PCR products was added in 20g·L^-1 agarose gel containing 0.5mg·L^-1 EB. After electrophoresis, the density and area of each band were measured using Fluro-sTM image software (Bio-Rad, USA). The relative RNA level of VEGF in tumor cells was calculated using the house-keeping gene β-actin as an internal control. The experiments were repeated at least four times.

Analysis VEGF transcriptional activity and VEGF mRNA half-life
To confirm that the effect of H₂O₂ on expression of VEGF in colon cancer cells was due to an increase in transcription, transcription activity of cancer cells was blocked by Dactinomycin (ActD, purchased from Sigma ). LS174T cells were incubated in the presence of ActD(1.5mg·L^-1) for 4h before their exposure to 10^-5 mol/L H₂O₂ in serum-free medium. Total RNA was extracted from cells after 24h, and RT-PCR analysis was made. Control cells were treated with ActD without H₂O₂.
      To determine the effect of H₂O₂ on VEGF mRNA stability, LS174T cells were incubated in the presense or absence of 10^-5mol·L^-1 H₂O₂ for 24h. Further transcription in cells was then blocked by addition of 1.5mg·L^-1 ActD. Total RNA was extracted from cells at 0, 0.25, 0.5, 1, 2 and 4h. RT-PCR analysis was made and the relative level of VEGF mRNA expression at each point was compared with the control value (total RNA extracted from cells before ActD treatment was defined as 100% ). The relative half-life of VEGF mRNA was determined by plotting relative VEGF mRNA expression levels on a semilogarithmic axis versus time.

Determination of VEGF protein levels
The VEGF protein levels in the supernantant were determined with an enzyme-linked immunosorbent assay(ELISA) kit. Examinations were repeated three times.

Activity of transcriptional factor NF-κB in colon cancer cells
To evaluate the effects H₂O₂ on NF-κB activity in colon cancer cells, NF-κB in cytoplasm and nucleus of the cells was detected with FITC-tagged antibody and its presence in the nucleus vs cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus by laser scaning cytometry(LSC) according to the Deptala’s report. Briefly, the cells were first attached to the mocroscope slides, and exposed to 10^-5 mol·L^-1 H₂O₂ for 0.5, 1,3,6,12 and 24h. The cells on slides were fixed and incubated with NF-κB P65 antibody (Santa Cruz) and FITC-tagged goated-antirabbit Ig ( Santa Cruz )at room temperature. Cellular DNA was then counterstained by addition of a solution containing propidium iodide and RNase (Sigma). The cells were placed on microscope slides mounted under coverslips and analyzed by LSC. At least 10³ cells were analyzed by LSC per slide. Fluorescence intensity in nucleus (Fn) and in cytoplasm (Fc) were detected and the activity of NF-κB was detected by estimating the value of Fn or Fn/Fc.

Statistics
When appropriate, data were expressed as x±s . Analysis of variance and t test were applied to assess the significance of differences. Statistical significance was accepted atP< 0.05.

RESULTS
H₂O₂ promotes the migration of endothelial cells induced by colon cancer cells
The migration of endothelial cells induced by cancer cells was promoted to a certain degree when LS174T and HCT8 cells were exposed to 10^-5, 10^-7 or 10^-9 mol·L^-1 H₂O₂ for 24h. When cancer cells were treated with 10^-5 mol·L^-1 H₂O₂, the migration number of endothelial cells induced by LS174T cells was 203±70, and the number induced by HCT8 cells was 145±65. The two values were significantly higher than those treated with other concentrations of H₂O₂(Table 1,Figue 1). When there was no cancer cell in the co-culture system, the number of random motility of endothelial cells was about ten cells.

Upregulation of VEGF expression by H₂O₂
Electrophoresis of RT-PCR products showed the three positive bands, 243,375 and 509bp, representing VEGF121,VEGF165 andβ-actin respectively. The internal control demonstrated a stable expression in colon cells treated with each dose of H₂O₂ The analysis of electrophoresis showed that expression levels of VEGF were elevated in LS174T and HCT8 cells with H₂O₂ exposure, especially with 10^-5 mol·L^-1 H₂O₂ (Figure 2). After inhibition of transcriptional activity by ActD before addition of H₂O₂, induction of VEGF mRNA expression was completely inhibited in LS174T cells(Figure 3), indicating that H₂O₂ -induced VEGF expression possibly occurred at the transcriptional level. The relative half-life of VEGF mRNA in LS174T cells treated with H₂O₂ was similar to that of the cells without H₂O₂ exposure, demonstrating that the stability of VEGF mRNA was not affected by the treatment with H₂O₂(Figure 4). To determine whether secretion of VEGF is increased by H₂O₂ in colon cancer cells, the supernatant was assayed and the results showed that H₂O₂ also promoted the VEGF protein expression. The levels of VEGF protein peaked when LS174T and HCT8 cells were incubated in the media containing 10^-5 mol·L^-1 H₂O₂. This situation is similar to the increase of VEGF expression.

Table 1 The migration of endothelial cells induced by colon cancer cells (x±s)

^aP<0.01, t =3.4751 υs LS174T, without H₂O₂ treatment ^bP<0.01, t =3.4183 υs HCT8, without H₂O₂ treatment     

Figure 1 The migration of endothelial cells induced by LS174T cells was increased after the cancer cells were exposed to 10^-5mol·L^-1 H₂O₂. The regular circles is the 8μm pores located in PET membrane. A: induced by cancer cells without H₂O₂ treatment. B: induced by cancer cells with H₂O₂ treatment. ×200
Figure 2 Expression levels of VEGF were increased in LS174T and HCT8 after exposure to 10^-9,10^-7and 10^-5mol·L^-1 H₂O₂, demonstrating a dose-dependent feature.
Figure 3 LS174T cells were treated with 1.5mg·L^-1 Act D for 4h, followed by exposure to 10^-5 mol·L^-1 H₂O₂ for 24h. RT-PCR was done. The control was those without Act D treatment.
Figure 4(PDF) Effect of H₂O₂ on the half-life of VEGF mRNA in LS174T cells with or without H₂O₂ exposure following Act D treatment. No difference in half-life was showed between the two groups.

H₂O₂ increases the activity of NF-κB
LS174T and HCT8 cells were incubated in the presence of H₂O₂ (10^-5 mol·L^-1) for 0.5,1,3,6,12 and 24h in complete medium. NF-κB activity peaked after exposure to H₂O₂ for 24h. We investigated the changes of NF-κB activity in LS174T and HCT8 cells treated with 10^-5 , 10^-7 and 10^-9 mol·L^-1 H₂O₂ in media. Compared with the cells without H₂O₂ treatment, administration of 10^-5 mol·L^-1 H₂O₂ for 24h led to a more remarkable increase in green fluorescence intensity measured over nuclear area (Fn)(Table 2,Figure 5), indicating the increase of NF-κB activity in LS174T and HCT8 cells.

Table 2 Change of NF-κB activity in colon cancel cells (x±s)

^aP<0.05, t =3.4179 υs HCT8, no H₂O₂ treatment ^bP<0.05, t =3.0981 υs LS174T, no H₂O₂ treatment

Figure 5 Measurement of nuclear and cytoplasmic NF-κB associated fluorescence by LSC. The red fluorescence represents the nuclear area, and the intensity of green fluorescence over nucleus(Fn) reflects NF-κB activity. A, B: LS174T cells without H₂O₂ treatment. C, D: Increase in NF-κB activity of LS174T cells with H₂O₂ exposure. E, F: HCT8 cells without H₂O₂ treatment. G, H: Increase in NF-κB activity of LS174T cells with H₂O₂ exposure.

DISCUSSION
The reactive oxygen species(ROS), which are ubiquitous and occur naturally in all aerobic species, may be divided into two categories :free oxygen radicals (·OH,·NO and O₂^·－ ) and nonradical ROS such as H₂O₂. For decades, H₂O₂ has been one of the ROS that has been well investigated in flammatory response and oxidant-induced stress. Recently, numerous evidence has been presented to show that H₂O₂ can act as a signaling molecule involved in many cellular function such as apoptosis and proliferation^[1-10].And the regulation of series of genes involved in carcinogenesis and progression is associated with the function of H₂O₂^{[3,5,6,9,11-14]}.Several reports have suggested that ROS such as H₂O₂ plays a role in the pathogenesis of tumor in colon, where there are a great deal of bacteria and dietary metabolites^[15-27]. Diet rich in fat increased the formation of ROS in feces, which then possibly damaged the stem cells in the colon^[20,22,26]. However, up to date, little information has been available about the role of H₂O₂ the special reactive oxygen intermediate, in the biological behaviors of colon cancer cells.
      VEGF is a potent and unique angiogenic protein that stimulates capillary formation and has specific mitogenic and chemotactic activity for vascular endothelial cells^[28].In colon cancer, VEGF levels are elevated and correlated with a poor clinical outcome^[29-33]. VEGF expression is regulated by some pathological processes such as hypoxia^[34-36] and by numerous cytokines and growth factors including interleukin1β, interleukin 6, platelet-derived growth factor, transforming growth factorβ,epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, angiotensinⅡ, hypoxia -inducible factor Ⅰ and EIF4E etc.^[37-52] Recently,oncogene p53 is also found to be a regulator of VEGF gene in colon cancer cells^[53,54].In the present study, we found, to our knowledge, for the first time, that exogenous H₂O₂ could up-regulate the expression of VEGF in human colon cancer cells and the migration of endothelial cells induced by the cancer cells after we reviewed those results from RT-PCR assay, ELISA and migration experiment of endothelial cells. Considering the important role of VEGF in neovascurization in solid tumors, we believe that hydrogen peroxide may have the promoting effects on the progression of colon cancer. Related studies also found that hydrogen peroxide could not only increase the expression of VEGF in cultured human vascular smooth muscle cell, human retinal pigment epithelial, melanoma cells and glioblastoma cells, but also promote the growth of prostate and breast cancer cells^[55-58].
      NF-κB activation, as expressed by its translocation from the cytoplasm to nucleus, can be conveniently assayed by LSC by measuring the intensity of NF-κB-associated immunofluorescence over the area of cell nucleus and comparing it with the intensity over cytoplasm^[59]. NF-κB, as a transcriptional factor controlling a variety of target genes such as adhesion molecular and apoptosis, is closely related to the pathogenesis and progression of tumors. NF-κB activity in cells like leukocyte, could be increased by hydrogen peroxide and NF-κB activation was an essential step before VEGF expression level was increased by hydrogen peroxide in murine osteoblastic cells^[60,61].It is noteworthy in the present experiment that the increase of NF-κB activity was accompanied by the promotion of expression of VEGF in colon cancer cells exposed to hydrogen peroxide. Thereby, we estimate that the NF-κB activation may be the prerequisite of the effect of hydrogen peroxide on VEGF expression in colon cancer cells.
      In view that such reactive oxygen species as hydrogen peroxide are likely to promote the development of colon cancer, it would be helpful in releasing oxidative stress by antioxidants in the colon cancer therapy.

REFERENCES
1    Rhee SG. Redox signaling :hydrogen peroxide as intracellular messenger. Exper Mol Med 1999;35:53-59
2    Gata L,Paul J,Ba GN, Tew KD, Tapiero H. Oxidative stress induced in pathologies:the role of antioxdants. 
      Biomed Pharmaco Ther 1999;53:169-180
3    Schwieger A, Bauer L, Hanusch J, Sers C, Schafer R, Bauer G.Ras oncogene expression determines sensitivity for 
      intercellular induction of apoptosis. Carcinogenesis 2001;22:1385-1392
4    Chen Y, Kramer DL, Diegelman P, Vujcic S, Porter CW. Apoptotic signaling in polyamine analogue-treated SK-MEL-28 
      human melanoma cells. Cancer Res 2001;61:6437-6444
5    Del Bello B, Valentini MA, Zunino F, Comporti M, Maellaro E.Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis
      of human melanoma cells. Oncogene 2001;20:4591-4595
6    Chung YM, Yoo YD, Park JK, Kim YT, Kim HJ.Increased expression of peroxiredoxin II confers resistance to cisplatin.
      Anticancer Res 2001; 21:1129-1133
7    Dare E, Li W, Zhivotovsky B, Yuan X, Ceccatelli S.Methylmercury and H(2)O(2) provoke lysosomal damage in human 
      astrocytoma D384 cells followed by apoptosis. Free Radic Biol Med 2001;30:1347-1356
8    Moriya K, Nakagawa K, Santa T, Shintani Y, Fujie H, Miyoshi H, Tsutsumi T, Miyazawa T, Ishibashi K, Horie T, Imai K, 
      Todoroki T, Kimura S, Koike K. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C
      virus-associated hepatocarcinogenesis. Cancer Res 2001; 61:4365-4370
9    Joseph P, Muchnok TK, Klishis ML, Roberts JR, Antonini JM, Whong WZ, Ong T. Cadmium-induced cell transformation
      and tumorigenesis are associated withtranscriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of
      cellular calcium and reactive oxygen species. Toxicol Sci 2001;61:295-303
10  Huang RP, Peng A, Golard A, Hossain MZ, Huang R, Liu YG, Boynton AL. Hydrogen peroxide promotes transformation of 
      rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor. Mol Carcinog 
      2001;30:209-217
11  Chen YR, Shrivastava A, Tan TH.Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation 
      of JNK by hydrogen peroxide and pyrrolidine dithiocarbamate. Oncogene 2001;20:367-374
12  Zmijewski JW, Song L, Harkins L, Cobbs CS, Jope RS. Oxidative stress and heat shock stimulate RGS2 expression in 
      1321N1 astrocytoma cells. Arch Biochem Biophys 2001;392:192-196
13  Mendoza L, Carrascal T, De Luca M, Fuentes AM, Salado C, Blanco J,Vidal-Vanaclocha F.Hydrogen peroxide mediates 
      vascular cell adhesion molecule-1 expression from interleukin-18-activated hepatic sinusoidal endothelium: implications 
      for circulating cancer cell arrest in the murine liver. Hepatology 2001;34:298-310
14  Hardman RA, Afshari CA, Barrett JC. Involvement of mammalian MLH1 in the apoptotic response to peroxide-induced 
      oxidative stress. Cancer Res 2001;61:1392-1397
15  Risau W. Development and differentiation of endothelium. Kidney Int Suppl 1998; 67:s3-6
16  Landriscina M, Cassano A, Ratto C, Longo R, Ippoliti M, Palazzotti B, Crucitti F, Barone C. Quantitative analysis of basic
      fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer. Br J Cancer 
      1998;78:765-770
17  Ishigami SI, Arii S, Furutani M, Niwano M, Harada T, Mizumoto M, Mori A, Onodera H, Imamura M.Predictive value of
      vascular endothelial growth factor in metastasis and prognosis of human colorectal cancer. Br J Cancer 
      1998;78:1379-1384
18  Nanashima A, Ito M, Sekine I, Naito S,Yamaguchi H, Nakagoe T, Ayabe H. Significance of angiogenic factor in liver 
      metastatic tumors originating from colorectal cancers. Dig Dis Sci 1998;43:2634-2640
19  Wang MP, Cheung N, Yuen ST, Leung SY, Chung LP. Vascular endothelial growth factor is up-regulated in the early 
      pre-malignant stage of colorectal tumor progression. Int J Cancer 1999;81:845-850
20  Zebrowski BK, Liu W, Ramirez K, Akagi Y, Mills GB, Ellis LM. Markedly elevated level of vascular endothelial growth factor 
      in malignant ascites. Ann Surg Oncol 1999;6:373-378
21  Dachs GU, Chaplin DJ. Microenviromental control of gene expressions:implications for tumor angiogenesis, progression 
      and metastasis. Semi Radiat Oncol 1998; 8:208-216
22  Ortega N, L,Fagihi FE, Plouet J. Control of vascular endothelial growth factor angiogenic activity by the extracellular 
      matrix. Biol Cell 1998; 90:381-390
23  Neufeld G, Cohen T,Gengrinovitch S,Poltorak Z.Vascular endothelial growth factor and its receptors. FASEB J
      1998;90:381-390
24  Lufc FC, Mervaala E, Muller DN, Gross V, Schmidt F,Park JK, Schmitz C, Lippoldt A, Breu V, Dechend R, Dragun D, 
      Schneider W, Ganten D, Haller H. Hypertension-induced end organ damage:A new thansgenic approach to an old 
      problem. Hypertention 1999;33:212-218
25  Semenza GL, Agani F, Iyer N, Kotch L, Laughner E, Leung S, Yu A. Regulation of cardiovascular development and 
      physiology by hypoxia-inducible factor 1. Ann N Y Acad Sci 1999;874:262-268
26  Nanthan CA, Flanklin S, Abreo KW, Nassar R, Debenedatti A, Williams J,Stucker FJ.Expression of eIF4E during head and
      neck tumorigenesis :possible role in angiogenesis. Laryngoscope 1999;109:1253-1258
27  Arri S,Mori A, Uchida S, Fujimoto K,Shimada Y,Imamura M. Implication of vascular endothelial growth factor in the 
      development and metastasis of human cancers. Hum Cell 1999;12:25-30
28  Holash J, Wiegand SJ, Yancoponlos GD. New model of tumor angiogenesis :dynamic balance between vessel regression 
      and growth mediated by angiopoietins and VEGF. Oncogene 1999;18:5356-5362
29  Lamszus K, Laterra J, Westphal m, rosen EM. Scatter factor/hepatocyte growth factor content and function in
      human gliomas. Int J Dev Neurosci 1999;17:517-530
30  Saaristo A, Karpanen T, Alitalo K. Mechanism of angiogenesis and their use in the inhibition of tumor growth and
      mestastasis. Oncogene 2000;19:6122-6129
31  Akagi Y, Liu W, Zebrowski B, Xie K and Ellis LM. Regulation of vascular endothelial growth factor expression in human 
      colon cancer by insulin-like growth factor 1. Cancer Res 1998;58:4008-4014
32  Akagi Y, liu W, Xie K, Zebrowski B Shaheen RM and Ellis LM. Regulation of vascular endothelial growth factor expression 
      in human colon cancer by Interleukin-1β. Br J Cancer 1999;80:1506-1511
33  Bouvet M, Ellis LM, Nishizaki M, Fujiwara T, Liu W, Bucana CD, Fang B, Lee JJ and Roth JA. Adenovirus-mediated wild-
      type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in 
      human colon cancer. Cancer Res 1998;58:2288-2292
34  KondoY, Arii S, Furutani M, Isigami S, Mori A, Onodera H, Chiba T, Imamura M. Implication of vascular endothelial 
      growth factor and p53 status for angiogenesis in noninvasive colorectal carcinoma. Cancer 2000;88:1820-1827
35  Pool-Zobel BL, Abrahamese SL, Collins AR, Kark W, Gugler R,Oberreuther D,Siegel Treptow-van Lishaut S, 
      Rechkemmer G. Analysis of DNA strand breaks, oxidized bases,and glutathione S-transferases in human colon cells 
      from biopsies. Cancer Epidemiol biomarkers prev 1999;8:609-614
36  Kondo S, Toyokuni S, Iwasa Y, tanaka T, Onodera H, Imamura M. Persistent oxidative stress in human colorectal 
      carcinoma, but not in adenoma. Free Radic Biol Med 1999;27:401-410
37  Bras A, Sanches R, Cristovao L,Fidalgo P,Mexia J, Leitao N, Rueff J. Oxidative stress in familial adenomatous polyposis.
      Eur J Cancer 1999;8:305-310
38  wen RW, Giacosa A,Hull WE, Haubner R, Spiegelhalder B, Bartsch H. The antioxidant/anticancer potential of phenolic 
      compounds isolated from olive oil. Eur J Cancer 2000;36:1235-1247
39  Van Rossen ME, Sluiter W, Bonthuis F, Jeekel H, Marquet RL, Van Eijck CH. Scanvenging of reactive oxygen species 
      leads to diminished peritoneal tumor recurrence. Cancer Res 2000;60:5625-5629
40  Owen RW, Spiegelhalder B, Bartsch H. Generation of reactive oxygen species by the faecal matrix. Gut 
      2000;46:225-232
41  Edmiston KH, Shoji Y, Mizoi T,Ford R, Nachman A, Jessup JM. Role of nitric oxide and superoxide anion in elimination 
      of low metastatic colorectal carcinomas by unstimulated hepatic sinusoidal endothelial cell. Cancer Res 
      1998;58:1524-1531
42  Giardina C, Inan MS. Nonsteroidal anti-inflammatory drugs,short-chain fatty acids, and reactive oxygen metabolism in 
      human colorectal cancer cells. Biochim Biophys Acta 1998;1401:277-288
43  Polyak K, Li Y, Zhu H, Lengauer C, Willson JK, Markowitz SD, Trush MA, Kinzler KW, Vogelsrein B. Somatic mutations 
      of the mitochondrial genome in human colorectal tumor. Nat Genet 1998;20:291-293
44  Jessup JM, Battle P, Waller H, Edmiston KH, Stolz DB, Watkins SC, Locker J, Skena. Reactive nitrogen and oxygen 
      radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells. Cancer Res 
      1999;59:1825-1829
45  Liegibel UM, Abrahamse SL, Pool-Zobel BL, Rechkemmer G. Application of confocal laser scanning microscopy to detect
      oxidative stress in human colon cells. Free Radic Res 2000;32:535-547
46  Zheng ZH, Zhang H, Pan YX, Gao Y, Yang JZ. Prevention of postoperative abdominal adhesions by an antibody to VEGF in
      mice. Shijie Huaren Xiaohua Zazhi 1999;7:227-229
47  Pan X, Ke CW, Pan W，He X, Cao GW, Qi ZT. Killing effect of DT/VEGF system on gastric carcinoma cell. Shijie Huaren
      Xiaohua Zazhi 2000;8:393-396
48  Mao H, Yuan AL, Zhao MF, Lai ZS, Zhang YL, Zhou DY. Effect of p38MAPK signal pathway on ultrastructural change of 
      liver cancer cells induced by VEGF. Shijie Huaren Xiaohua Zazhi 2000;8:536-538
49  Pan X, Pan W, Ni CR, Ke CW, Cao GW, Qi ZT. Killing effect of tetracycline controlled expression of DT/VEGF system on 
      liver cell cancer. Shijie Huaren Xiaohua Zazhi 2000;8:867-873
50  Pan X, Pan W, Ke CW, Zhang B, Cao GW, Qi ZT. Tetracycline controlled DT/VEGF system gene therapy mediated by
      adenovirus vector. Shijie Huaren Xiaohua Zazhi 2000;8:1121-1126
51  Assy N, Paizi M, Gaitini D, Baruch Y, Spira G. Clinical implication of VEGF serum levels in cirrhotic patients with or without
      portal hypertension. World J Gastroenterol 1999;5:296-300
52  Tian XJ, Wu J, Meng L, Dong ZW, Shou CC. Expression of VEGF121 in gastric carcinoma MGC803 cell line.
      World J Gastroenterol 2000;6:281-283
53  Bruce WR, Giacca A, Medline A. Possible mechanisms relating diet and risk of colon cancer.
      Cancer Epidemiol Biomarkers Prev 2000;9:1271-1279
54  Bianchi NO, Bianchi MS, Richard SM. Mitochondrial genome instability in human cancers. Mutat Res 2001;488:9-23
55  Ellis EA, Guberski DL, Somogyi-Mann M, Grant MB. Increased H₂O₂,vascular endothelial growth factor and receptors 
      in the retina of the BBZ/Wor diabetic rat. Free Radic Biol Med 2000;28:91-101
56  Castilla MA, Carmelo C, Gazapo RM, Martin O, Gonzalez-Pacheco FR, Tejedor A, Bragado R, Arroyo MV. Role of vascular
      endothelial growth factor(VEGF) in endothelial cell protection against cytotoxic agents. Life Sci 2000; 67:1003-1013
57  Haklar G, Sayin-Ozveri E, Yuksel M, Aktan AO, Yalcin AS. Different kinds of reactive oxygen and nitrogen species were 
      detected in colon and breast tumors. Cancer Lett 2001;165:219-224
58  Wartenberg M, Diedershagen H, Hescheler J, Sauer H. Growth stimulation versus induction of cell quiescence by 
      hydrogen peroxide in prostate tumor spheroids is encoded by the duration of the Ca2+ response. J Biol Chem 
      1999; 274:27759-27767
59  Deptala A, Bedner E, Gorczyca W, Darzynkiewicz Z. Activation of nuclear factor KappaB (NF-kB) assayed by laser scaning
      cytometry. Cytometry 1998; 33: 376-382
60  Kaul N, Choi J, Forman HJ. Transmembrane redox signaling activates NF-KappaB in macrophages. Free Radical Biol Med
      1998; 24: 202-207
61  hua CC, Hamdy RC, Chua BH. Mechanism of transforming growth factor -beta 1 induced expression of vascular 
      endothelial growth factor in murine osteoblastic MC3T3-E1 cells. Biochim Biophys Acta 2000; 1497: 69-76 

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