乙酰肝素酶HPA与S100A4在结肠癌中的表达及与肿瘤侵袭和转移的关系
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摘要
目的
结肠癌是消化道肿瘤中最常见的恶性肿瘤。近几年来的死亡率逐步攀升,引起死亡的主要原因是肿瘤的浸润和转移。近年来的试验和临床数据都表明,乙酰肝素酶HPA在结肠癌、胃癌、肝癌、食管癌等消化道肿瘤中高表达,在一些侵袭转移性强的恶性肿瘤中表达更高。其表达量也与病人的预后有紧密联系。
S100蛋白家族是一类EF-手型钙结合蛋白,通过对钙离子的调节及与靶蛋白的相互作用,发挥重要的生物学作用,参与细胞周期活动、细胞分化、肿瘤生长以及细胞外基质分泌活动等过程,且其多个成员在多种肿瘤中表达异常,特别是S100A4的异常表达,与肿瘤的侵袭与转移相关。因此S100家族与肿瘤的发生发展关系密切。
本研究通过检测结肠癌组织中和正常结肠中HPA和S100A4的表达,探讨两者在结肠癌表达中的相关性,以便为结肠癌患者的诊断和预后提供科学有效的评价指标。
方法
1、材料
70例结肠癌标本来自2007年至2009年在中国医科大学附属第一医院行外科手术切除的结肠癌标本。在这70例标本中均取相应断端未见癌组织作为正常组织进行对照。20例结肠癌新鲜标本来自2009年3月至2009年12月在中国医科大学附属第一医院行外科手术切除的新鲜标本。所有标本均取癌、癌旁(癌组织周围3CM以内)、正常组织(癌组织周围3CM以外)做对照。
2、免疫组织化学染色
兔抗人乙酰肝素酶多克隆抗体购自Santa公司,兔抗人S100A4多克隆抗体购自中杉金桥公司,SP免疫组化试剂盒、DAB酶底物显色试剂盒购白福州迈新生物技术开发公司。染色方法根据试剂盒说明进行。
3、Western Blot
将含80μg总蛋白的组织裂解产物进行10% SDS-PAGE电泳1.5h后,转印至PVDF膜(65V,2h),脱脂奶粉室温封闭2h,经TTBS冲洗3次,抗HPA多克隆抗体(1：300)4℃孵育过夜后,辣根过氧化物酶标记二抗37℃孵育2h,TTBS洗三次后ECL发光。以β-actin为内对照,以β-actin为内对照,比较各条带灰度值。
4、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。p<0.05有统计学意义。
结果
乙酰肝素酶HPA与S100A4在结肠癌组织中的阳性表达率明显高于正常结肠组织。并且两者与患者的性别、年龄、病理组织学类型、分化程度之间没有明显的差异,而与肿瘤的浸润深度和有无淋巴结转移则密切相关,两者的关系为正相关。
结论
在结肠癌组织中,乙酰肝素酶HPA与S100A4蛋白的表达变化与肿瘤的进展密切相关,是判断其生物学行为,预测转移趋势极具价值的指标。联合检测乙酰肝素酶HPA与S100A4蛋白的表达可用于评定结肠癌的转移潜能。
Introduction
Colon cancer is the most common gastrointestinal tumor malignancy. In recent years the death rate gradually increased, leading cause of death is caused by tumor invasion and metastasis. In recent years, experimental and clinical data indicate that heparanase HPA in the colon, stomach, liver, esophageal and other gastrointestinal tumors with high expression of invasion and metastasis in some malignant tumors expressed a higher intensity. The expression level and patient prognosis are closely associated. The expression level and patient prognosis are closely associated.
S100 protein family is a class of EF-hand type calcium binding protein, through the regulation of calcium and interaction with the target protein, play an important biological role in cell cycle activity, cell differentiation, tumor growth and secretion of extracellular matrix activities, processes, and many members of its abnormal expression in tumors, especially in the abnormal expression of S100A4 with tumor invasion and metastasis. S100 family, and therefore the development of tumors close.
This study is to determine the normal colon cancer tissues and the expression of S100A4 in the HPA and discussed both in the strap of the relevance of colon cancer, colon cancer patients for the diagnosis and prognosis of scientific and effective evaluation.
Materials and Methods
1. Patients and specimens
70 cases of colon cancer specimens from 2007 to 2009 at the First Affiliated Hospital of China Medical University, underwent surgical resection of colon cancer specimens. In these 70 specimens were taken in the ends corresponding normal tissue, as no cancer were compared. Fresh specimens from 20 patients with colon cancer from March 2009 to December 2009 at the First Hospital of China Medical University, surgical removal of the fresh specimens. All specimens were obtained cancer, adjacent (within cancer tissues around the 3CM), normal tissue (tumor tissue around the outside 3CM) as control.
2. Immunofluence
Rabbit polyclonal anti-human heparanase antibodies were purchased from Santa Inc., rabbit polyclonal anti-human S100A4 antibody was purchased from the Shan Golden Bridge Corporation, SP immunohistochemistry kit, DAB chromogenic enzyme substrate kit was purchased from Fuzhou, the taking of new bio-technology development company. Staining according to kit instructions.
3. Western Blot
The organization with 80μg total protein cleavage products were 10% SDS-PAGE electrophoresis 1.5h, the transfer to PVDF membrane (65V,2h), nonfat dry milk closed at room temperature 2h, washed three times by TTBS, anti-HPA polyclonal antibody (1 300) 4℃overnight incubation, the horseradish peroxidase labeled secondary antibody at 37℃for 2h, TTBS washing three times ECL light. Toβ-actin as internal control, to P-actin as internal control, compared with a gray value of each.
4. Statistical analysis
All statistical calculations were performed by SPSS version 13.0 for Windows software. p values less than 0.01 were considered statistically significant.
Results
Heparanase HPA and S100A4 in human colon cancer positive rate was significantly higher than normal colon tissue. Both the patients and the sex, age, histological type, differentiation, there is no significant difference between. With tumor invasion and lymph node metastasis are closely related. And positively correlated between the two.
Conclusions
In colon adenocarcinoma, heparanase HPA and S100A4 protein expression is closely related with tumor progression, is predicting the biological behavior of the transfer trend forecasts valuable indicators. Combined detection of heparanase HPA and S100A4 protein expression can be used to assess colon cancer metastasis.
结肠癌是消化道肿瘤中最常见的恶性肿瘤。近几年来的死亡率逐步攀升,引起死亡的主要原因是肿瘤的浸润和转移。近年来的试验和临床数据都表明,乙酰肝素酶HPA在结肠癌、胃癌、肝癌、食管癌等消化道肿瘤中高表达,在一些侵袭转移性强的恶性肿瘤中表达更高。其表达量也与病人的预后有紧密联系。
S100蛋白家族是一类EF-手型钙结合蛋白,通过对钙离子的调节及与靶蛋白的相互作用,发挥重要的生物学作用,参与细胞周期活动、细胞分化、肿瘤生长以及细胞外基质分泌活动等过程,且其多个成员在多种肿瘤中表达异常,特别是S100A4的异常表达,与肿瘤的侵袭与转移相关。因此S100家族与肿瘤的发生发展关系密切。
本研究通过检测结肠癌组织中和正常结肠中HPA和S100A4的表达,探讨两者在结肠癌表达中的相关性,以便为结肠癌患者的诊断和预后提供科学有效的评价指标。
方法
1、材料
70例结肠癌标本来自2007年至2009年在中国医科大学附属第一医院行外科手术切除的结肠癌标本。在这70例标本中均取相应断端未见癌组织作为正常组织进行对照。20例结肠癌新鲜标本来自2009年3月至2009年12月在中国医科大学附属第一医院行外科手术切除的新鲜标本。所有标本均取癌、癌旁(癌组织周围3CM以内)、正常组织(癌组织周围3CM以外)做对照。
2、免疫组织化学染色
兔抗人乙酰肝素酶多克隆抗体购自Santa公司,兔抗人S100A4多克隆抗体购自中杉金桥公司,SP免疫组化试剂盒、DAB酶底物显色试剂盒购白福州迈新生物技术开发公司。染色方法根据试剂盒说明进行。
3、Western Blot
将含80μg总蛋白的组织裂解产物进行10% SDS-PAGE电泳1.5h后,转印至PVDF膜(65V,2h),脱脂奶粉室温封闭2h,经TTBS冲洗3次,抗HPA多克隆抗体(1：300)4℃孵育过夜后,辣根过氧化物酶标记二抗37℃孵育2h,TTBS洗三次后ECL发光。以β-actin为内对照,以β-actin为内对照,比较各条带灰度值。
4、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。p<0.05有统计学意义。
结果
乙酰肝素酶HPA与S100A4在结肠癌组织中的阳性表达率明显高于正常结肠组织。并且两者与患者的性别、年龄、病理组织学类型、分化程度之间没有明显的差异,而与肿瘤的浸润深度和有无淋巴结转移则密切相关,两者的关系为正相关。
结论
在结肠癌组织中,乙酰肝素酶HPA与S100A4蛋白的表达变化与肿瘤的进展密切相关,是判断其生物学行为,预测转移趋势极具价值的指标。联合检测乙酰肝素酶HPA与S100A4蛋白的表达可用于评定结肠癌的转移潜能。
Introduction
Colon cancer is the most common gastrointestinal tumor malignancy. In recent years the death rate gradually increased, leading cause of death is caused by tumor invasion and metastasis. In recent years, experimental and clinical data indicate that heparanase HPA in the colon, stomach, liver, esophageal and other gastrointestinal tumors with high expression of invasion and metastasis in some malignant tumors expressed a higher intensity. The expression level and patient prognosis are closely associated. The expression level and patient prognosis are closely associated.
S100 protein family is a class of EF-hand type calcium binding protein, through the regulation of calcium and interaction with the target protein, play an important biological role in cell cycle activity, cell differentiation, tumor growth and secretion of extracellular matrix activities, processes, and many members of its abnormal expression in tumors, especially in the abnormal expression of S100A4 with tumor invasion and metastasis. S100 family, and therefore the development of tumors close.
This study is to determine the normal colon cancer tissues and the expression of S100A4 in the HPA and discussed both in the strap of the relevance of colon cancer, colon cancer patients for the diagnosis and prognosis of scientific and effective evaluation.
Materials and Methods
1. Patients and specimens
70 cases of colon cancer specimens from 2007 to 2009 at the First Affiliated Hospital of China Medical University, underwent surgical resection of colon cancer specimens. In these 70 specimens were taken in the ends corresponding normal tissue, as no cancer were compared. Fresh specimens from 20 patients with colon cancer from March 2009 to December 2009 at the First Hospital of China Medical University, surgical removal of the fresh specimens. All specimens were obtained cancer, adjacent (within cancer tissues around the 3CM), normal tissue (tumor tissue around the outside 3CM) as control.
2. Immunofluence
Rabbit polyclonal anti-human heparanase antibodies were purchased from Santa Inc., rabbit polyclonal anti-human S100A4 antibody was purchased from the Shan Golden Bridge Corporation, SP immunohistochemistry kit, DAB chromogenic enzyme substrate kit was purchased from Fuzhou, the taking of new bio-technology development company. Staining according to kit instructions.
3. Western Blot
The organization with 80μg total protein cleavage products were 10% SDS-PAGE electrophoresis 1.5h, the transfer to PVDF membrane (65V,2h), nonfat dry milk closed at room temperature 2h, washed three times by TTBS, anti-HPA polyclonal antibody (1 300) 4℃overnight incubation, the horseradish peroxidase labeled secondary antibody at 37℃for 2h, TTBS washing three times ECL light. Toβ-actin as internal control, to P-actin as internal control, compared with a gray value of each.
4. Statistical analysis
All statistical calculations were performed by SPSS version 13.0 for Windows software. p values less than 0.01 were considered statistically significant.
Results
Heparanase HPA and S100A4 in human colon cancer positive rate was significantly higher than normal colon tissue. Both the patients and the sex, age, histological type, differentiation, there is no significant difference between. With tumor invasion and lymph node metastasis are closely related. And positively correlated between the two.
Conclusions
In colon adenocarcinoma, heparanase HPA and S100A4 protein expression is closely related with tumor progression, is predicting the biological behavior of the transfer trend forecasts valuable indicators. Combined detection of heparanase HPA and S100A4 protein expression can be used to assess colon cancer metastasis.
引文
1 Boyd DD, Nakajima M. Involvement of heparanase in tumor metastases:a new target in cancer therapy. J Natl Cancer Inst.2004; 96(16):1194-1195.
2 Ohkawa T, Naonoto Y, Nobuhisa T, et al. Heparanase expression at the invasion front of human head and neck cancers and correlation with poor prognosis. Clin Cancer Res,2005; 11(8):2899-2906.
3 Nasser NJ. Heparanase involvement in physiology and disease. Cell Mol Life Sci.2008; 65(11):1706-1715.
4 Zheng LD, Jiang GS, Pu JR, et al. Stable knockdown of heparanase expression in gastric cancer cells in vitro.2009; 15(43):442-448.
5 Gupta S, Hussain T, Maclennan GT, et al. Differential expression of S100A2 and S100A4 during progression of human prostare adenocarcinoma. J Clin Oncol.2003; 21(1):106-112.
6 Cho YG, Nam SW, Kim TY, et al. Overexpression of S100A4 is closely related to the aggressiveness of gastric cancer. APMIS.2003; 111(5):539-545.
7 Pathuri P, Vogeley L, Luecke H. Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form. J Mol Biol.2008; 383(1):62-77.
8 Ismail NI, Kaur G, Hashim H, et al. S100A4 overexpression proves to be independent marker for breast cancer progression. Cancer Cell Int.2008; 5(8):12.
9 Gongoll S, Peters G, M engel M, et al. Prognostic significance of calcium-binding protein S100A4 in colorectal cancer. Gastroenterology.2002; 123(5):1478-1484.
10 Cho YG, Kim CJ, Nam SW, et al.Overexdression of S100A4 is closely associated with progression of colorectal cancer. World J Gastroenterol.2005; 11(31):4852-4856.
11 Flatmark K, Pedersen KB, Nesland JM, et al. Nuclear localization of the etastasis-related protein S100A4 correlates with tumour stage in colorectal cancer. J Pathol.2003 200(5):589-595.
12 Taylor S, Herrington S, Prime W,et al. S100A4 (p9Ka) protein in colon carcinoma and liver metastases:association with carcinoma cells and T-lymphocytes. Br J Cancer.2002 86(3):409-416.
13 Komatsu K, M urata K, Kameyama M, et al. Expression of S100A6 and S100A4 in matched samples of human colorectal mucosa, primary colorectal adenocarcinomas and liver metastases. Oncology.2002; 63(2):192-200.
14 Bronckart Y, Decaestecker C, Nagy N, et al. Developm ent and progression of malignancy in human colon tissues are correlated with expression of specific Ca2+-binding S100 proteins. Histol H istopathol.2001; 16(3):707-712.
15 Curran S, Dundas SR, Buxton J, et al. Matrix metallopeoteinase/tissue inhibitors of matrix metalloproteinase phenotype identifies poor prognosis eolorectal cancers. Clin Cancer Res. 2004; 10(24):8229-8234.
16 Gongoll S, Peters G, Mengel M, et al. Prognostic significance of calcium-binding protein S100A4 in coloreetal cancer. Gastroenterology.2002; 123(5):1478-1484.
17 Hemandas AK, Salto-Tellez M, Maricar SH, et al. Metastasis-associated protein S100A4-a potential prognostic marker for colorectal cancer. J Surg Oncol.2006; 93(6):498-503.
18 Mazzuccheli L. Protein S100A4:too long overlooked by pathologists. Am J Pathol 2002;160(1):7-13.
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20 Tarabykina S, Griffiths TR, Tulchinsky E, et al. Metastasis-associated protein S100A4: spotlight on its role in cell migration. Curr Cancer Drug Targets.2007; 7(3):217-228.
21 Gongoll S, Peters G, M engel M, et al. Progostic significace of calcium binding protein S100A4 in colrectal cancer. Gastroco terolgy.2002;123(5):1478-1484.
22 Cho YG, Kim CJ, Nam SW, et al. Overexpression of S100A4 is closely Associated with progression of colorectal cancer. World J Gastroenterol.2005;11(31):4852-4856.
23 Flatmark K, Pedersen KB, Nesland JM, et al. Nuclear localization of the metastasisrelated protein S100A4 correlates with tumour stage in colorectal cancer. J Pathol, 2003;200(5):589-595.
24 Takaoka M, Naomoto Y, Ohkawa T,et al. Heparanase expression corelates with invasion and poor prognisis in gastric cancer. Lab Invest.2003;83(5):613-622.
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2 Ohkawa T, Naonoto Y, Nobuhisa T, et al. Heparanase expression at the invasion front of human head and neck cancers and correlation with poor prognosis. Clin Cancer Res,2005; 11(8):2899-2906.
3 Nasser NJ. Heparanase involvement in physiology and disease. Cell Mol Life Sci.2008; 65(11):1706-1715.
4 Zheng LD, Jiang GS, Pu JR, et al. Stable knockdown of heparanase expression in gastric cancer cells in vitro.2009; 15(43):442-448.
5 Gupta S, Hussain T, Maclennan GT, et al. Differential expression of S100A2 and S100A4 during progression of human prostare adenocarcinoma. J Clin Oncol.2003; 21(1):106-112.
6 Cho YG, Nam SW, Kim TY, et al. Overexpression of S100A4 is closely related to the aggressiveness of gastric cancer. APMIS.2003; 111(5):539-545.
7 Pathuri P, Vogeley L, Luecke H. Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form. J Mol Biol.2008; 383(1):62-77.
8 Ismail NI, Kaur G, Hashim H, et al. S100A4 overexpression proves to be independent marker for breast cancer progression. Cancer Cell Int.2008; 5(8):12.
9 Gongoll S, Peters G, M engel M, et al. Prognostic significance of calcium-binding protein S100A4 in colorectal cancer. Gastroenterology.2002; 123(5):1478-1484.
10 Cho YG, Kim CJ, Nam SW, et al.Overexdression of S100A4 is closely associated with progression of colorectal cancer. World J Gastroenterol.2005; 11(31):4852-4856.
11 Flatmark K, Pedersen KB, Nesland JM, et al. Nuclear localization of the etastasis-related protein S100A4 correlates with tumour stage in colorectal cancer. J Pathol.2003 200(5):589-595.
12 Taylor S, Herrington S, Prime W,et al. S100A4 (p9Ka) protein in colon carcinoma and liver metastases:association with carcinoma cells and T-lymphocytes. Br J Cancer.2002 86(3):409-416.
13 Komatsu K, M urata K, Kameyama M, et al. Expression of S100A6 and S100A4 in matched samples of human colorectal mucosa, primary colorectal adenocarcinomas and liver metastases. Oncology.2002; 63(2):192-200.
14 Bronckart Y, Decaestecker C, Nagy N, et al. Developm ent and progression of malignancy in human colon tissues are correlated with expression of specific Ca2+-binding S100 proteins. Histol H istopathol.2001; 16(3):707-712.
15 Curran S, Dundas SR, Buxton J, et al. Matrix metallopeoteinase/tissue inhibitors of matrix metalloproteinase phenotype identifies poor prognosis eolorectal cancers. Clin Cancer Res. 2004; 10(24):8229-8234.
16 Gongoll S, Peters G, Mengel M, et al. Prognostic significance of calcium-binding protein S100A4 in coloreetal cancer. Gastroenterology.2002; 123(5):1478-1484.
17 Hemandas AK, Salto-Tellez M, Maricar SH, et al. Metastasis-associated protein S100A4-a potential prognostic marker for colorectal cancer. J Surg Oncol.2006; 93(6):498-503.
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