线粒体琥珀酸脱氢酶复合物在结直肠癌中的表达及临床意义
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摘要
研究背景与目的：结直肠癌是消化道常见的恶性肿瘤。迄今为止,结直肠癌的发病机制仍不十分清楚。早在1931年,Otto Warburg就发现肿瘤细胞中糖酵解增强,有氧氧化减弱。近年来,能量代谢紊乱在肿瘤发生与演进中的作用及机制逐渐受到人们的重视。近几年研究发现琥珀酸脱氢酶(succinate dehydrogenase,SDH)是核基因编码的线粒体抑瘤基因,对研究糖酵解增强的触发因素提供了重要线索,并首次将线粒体酶功能紊乱同肿瘤形成联系起来。研究表明,由于SDH的失活,线粒体内积聚的琥珀酸渗漏到胞浆中,进而抑制了脯氨酰羟化酶。脯氨酰羟化酶的抑制导致了肿瘤细胞对某些凋亡信号产生抵抗和／或激活缺氧诱导因子(hypoxia-inducible factor,HIF)从而促进了肿瘤生成与发展。国内外至今尚未见SDH在结直肠癌中的发生和演进中所起作用的相关文献。本课题旨在通过研究SDH复合物在结直肠癌组织中的表达情况,并分析SDH的表达与HIF-1α以及与肿瘤临床病理特征、病人预后的关系,为下一步研究SDH在结直肠癌中的作用机制奠定基础。
方法：本研究采用336个点阵的结直肠组织芯片,利用免疫组化技术检测SDH四个亚基(SDHA、SDHB、SDHC、SDHD)蛋白表达,并进一步分析蛋白表达与结直肠组织临床病理特征及病人预后的关系。同时抽提了三株结肠癌细胞以及不同分化阶段结直肠癌组织的蛋白通过Western blot检测SDHB与HIF-1α的表达情况。
结果：(1)SDHB蛋白在结直肠癌组织较正常结直肠粘膜的表达明显下调,而SDHA、SDHC、SDHD在结直肠癌组织和正常粘稠的表达无明显差异。SDHB在结直肠癌组织的阳性表达与分化程度相关,在低分化结直肠癌组织中表达较中、高分化明显减弱。Western blot检测亦发现SDHB在低分化结直肠癌组织中表达明显减弱。(2)Western blot检测发现不同分化程度的结直肠癌组织和结直肠正常粘膜不表达活化的HIF-1α;此外在常氧状态下培养的SW480、SW620及HT-29三株结肠癌细胞中,只有HT-29细胞株表达SDHB蛋白,而三株细胞均未表达有活性的HIF-1α;通过氯化钴诱导HT-29细胞化学缺氧后,Western blot检测到有活性的HIF-1α蛋白表达。(3)年龄、Dukes分期、有无淋巴结转移及有无远处转移这四项临床病理参数与结直肠癌患者预后相关,而现有数据尚不能证明SDHB的表达与结直肠癌患者的预后存在相关性。
结论：(1)本实验首次研究了SDH四个亚基在结直肠组织中的表达水平：SDHA、SDHC与SDHD在结直肠正常粘膜和结直肠癌组织中高表达,正常粘膜与癌组织中的表达无显著性差异；SDHB在癌组织中表达下调,且SDHB蛋白表达与结直肠癌组织分化程度相关,在低分化癌组织中表达较中、高分化癌组织明显减弱。现有数据尚不能证明SDHB的表达与结直肠癌患者的预后存在相关性。(2)影响结直肠癌患者预后的因素有年龄、Dukes分期、有无淋巴结转移及有无远处转移。
Background and objective:Colorectal cancer is a common malignant tumor in digestive tractor, the exact mechanisms of which still remain unknown. As early as in 1931,Otto Warburg found the phenomenon of enhanced glycolysis and decreased aerobic oxidation of glucose in tumours, but increasingly importance was attached to the role and mechanism of abnormal energy metabolism in tumorigenesis and tumor progression until recent years.Recent studies have found that succinate dehydrogenase is a tumor suppressor encoded by nuclear gene, which provided an important clue to the study of the triggers of enhanced glycolysis.And it was the first time to associate mitochondrial enzymes and their dysfunction with tumorigenesis.Further research forward showed that succinate, which accumulate in mitochondria owing to the inactivation of SDH, leak out to the cytosol,where they inhibit a family of prolyl hydroxylase enzymes (PHDs).Depending on the PHD inhibited, affected tumor cells become resistant to certain apoptotic signals and/or activate hypoxia-inducible factor (HIF),which result in the tumor formation and maintenance. There is no related literature regarding the role of SDH in tumor occurrence and maintenance in colorectal cancer domestic and abroad. This research was to study the expression of SDH in colorectal cancer and analyze the relationship among its expression, clinicopathological characteristics and prognosis, and analyze the relationship between SDH and HIF-1α, which lay a foundation for further study of the mechanism of SDH in colorectal cancer.
Methods:In the present study, the expression of the protein level of the four subunits of SDH(SDHA、SDHB、SDHC and SDHD)were examined in 336 colorectal tissue cores using tissue microarray analysis by immunohistochemical analysis.Further analysis was performed to clarify the relationship between the protein expression, clinicopathological parameters and prognosis. Meanwhile, the expression of SDHB and HIF-la were analyzed by western blot in three colon cancer cell lines and colon cancer tissues with different differentiation status.
Results:(1)Immunohistochemistry assay revealed decreased expression levels of SDHB in colorectal cancer compared with the adjacent mucosa and adjacent normal mucosa of primary tumor, while no significant difference was found in the expression level of SDHA, SDHC and SDHD in colorectal cancer and normal mucosa. Moreover, a close association was observed between SDHB expression and the tumor differentiation grade. The expression level of SDHB significantly reduced in poorly differentiated colorectal cancer than moderately differentiated and well-differentiated colorectal cancer. Western blot showed similar result.(2)There was no activated HIF-1αexpression in colorectal cancer tissues with differencial SDHB expression level.SDHB protein is high expressed in HT-29 cell line but not in SW480, SW620 cell lines.No activated HIF-1αprotein expressed could be detected in all these three cell lines in normoxia. However, western blot assay showed cobalt chloride could induce the expression of activated HIF-la protein. (3) Survival analysis revealed that "age", "Duke's stage", "distant metastasis" and "lymph node metastasis" were closely related with the prognosis of patients with colorectal cancer. While no correlation between the expression of SDHB and the prognosis was found at present.
Conclusion:(1)In this research, the expression level of four subunits of SDH was firstly studied in colorectal cancer. Colorectal cancer and normal mucosa showed high expression levels of SDHA, SDHC and SDHD, and there was no significant difference in the level of protein expression among colorectal carcinoma, adjacent mucosa and adjacent normal mucosa of primary tumor. The expression level of SDHB protein decreased in colorectal cancer as compared with normal colorectal mucosa. The expression of SDHB decreased more severely in poorly differentiated colorectal cancer than in moderately differentiated and well-differentiated colorectal cancer. No correlation between the expression of SDHB and the prognosis was found at present. (2) Survival analysis revealed that "age","Duke's stage","distant metastasis" and "lymph node metastasis" are closely related with the prognosis of patients with colorectal cancer.
方法：本研究采用336个点阵的结直肠组织芯片,利用免疫组化技术检测SDH四个亚基(SDHA、SDHB、SDHC、SDHD)蛋白表达,并进一步分析蛋白表达与结直肠组织临床病理特征及病人预后的关系。同时抽提了三株结肠癌细胞以及不同分化阶段结直肠癌组织的蛋白通过Western blot检测SDHB与HIF-1α的表达情况。
结果：(1)SDHB蛋白在结直肠癌组织较正常结直肠粘膜的表达明显下调,而SDHA、SDHC、SDHD在结直肠癌组织和正常粘稠的表达无明显差异。SDHB在结直肠癌组织的阳性表达与分化程度相关,在低分化结直肠癌组织中表达较中、高分化明显减弱。Western blot检测亦发现SDHB在低分化结直肠癌组织中表达明显减弱。(2)Western blot检测发现不同分化程度的结直肠癌组织和结直肠正常粘膜不表达活化的HIF-1α;此外在常氧状态下培养的SW480、SW620及HT-29三株结肠癌细胞中,只有HT-29细胞株表达SDHB蛋白,而三株细胞均未表达有活性的HIF-1α;通过氯化钴诱导HT-29细胞化学缺氧后,Western blot检测到有活性的HIF-1α蛋白表达。(3)年龄、Dukes分期、有无淋巴结转移及有无远处转移这四项临床病理参数与结直肠癌患者预后相关,而现有数据尚不能证明SDHB的表达与结直肠癌患者的预后存在相关性。
结论：(1)本实验首次研究了SDH四个亚基在结直肠组织中的表达水平：SDHA、SDHC与SDHD在结直肠正常粘膜和结直肠癌组织中高表达,正常粘膜与癌组织中的表达无显著性差异；SDHB在癌组织中表达下调,且SDHB蛋白表达与结直肠癌组织分化程度相关,在低分化癌组织中表达较中、高分化癌组织明显减弱。现有数据尚不能证明SDHB的表达与结直肠癌患者的预后存在相关性。(2)影响结直肠癌患者预后的因素有年龄、Dukes分期、有无淋巴结转移及有无远处转移。
Background and objective:Colorectal cancer is a common malignant tumor in digestive tractor, the exact mechanisms of which still remain unknown. As early as in 1931,Otto Warburg found the phenomenon of enhanced glycolysis and decreased aerobic oxidation of glucose in tumours, but increasingly importance was attached to the role and mechanism of abnormal energy metabolism in tumorigenesis and tumor progression until recent years.Recent studies have found that succinate dehydrogenase is a tumor suppressor encoded by nuclear gene, which provided an important clue to the study of the triggers of enhanced glycolysis.And it was the first time to associate mitochondrial enzymes and their dysfunction with tumorigenesis.Further research forward showed that succinate, which accumulate in mitochondria owing to the inactivation of SDH, leak out to the cytosol,where they inhibit a family of prolyl hydroxylase enzymes (PHDs).Depending on the PHD inhibited, affected tumor cells become resistant to certain apoptotic signals and/or activate hypoxia-inducible factor (HIF),which result in the tumor formation and maintenance. There is no related literature regarding the role of SDH in tumor occurrence and maintenance in colorectal cancer domestic and abroad. This research was to study the expression of SDH in colorectal cancer and analyze the relationship among its expression, clinicopathological characteristics and prognosis, and analyze the relationship between SDH and HIF-1α, which lay a foundation for further study of the mechanism of SDH in colorectal cancer.
Methods:In the present study, the expression of the protein level of the four subunits of SDH(SDHA、SDHB、SDHC and SDHD)were examined in 336 colorectal tissue cores using tissue microarray analysis by immunohistochemical analysis.Further analysis was performed to clarify the relationship between the protein expression, clinicopathological parameters and prognosis. Meanwhile, the expression of SDHB and HIF-la were analyzed by western blot in three colon cancer cell lines and colon cancer tissues with different differentiation status.
Results:(1)Immunohistochemistry assay revealed decreased expression levels of SDHB in colorectal cancer compared with the adjacent mucosa and adjacent normal mucosa of primary tumor, while no significant difference was found in the expression level of SDHA, SDHC and SDHD in colorectal cancer and normal mucosa. Moreover, a close association was observed between SDHB expression and the tumor differentiation grade. The expression level of SDHB significantly reduced in poorly differentiated colorectal cancer than moderately differentiated and well-differentiated colorectal cancer. Western blot showed similar result.(2)There was no activated HIF-1αexpression in colorectal cancer tissues with differencial SDHB expression level.SDHB protein is high expressed in HT-29 cell line but not in SW480, SW620 cell lines.No activated HIF-1αprotein expressed could be detected in all these three cell lines in normoxia. However, western blot assay showed cobalt chloride could induce the expression of activated HIF-la protein. (3) Survival analysis revealed that "age", "Duke's stage", "distant metastasis" and "lymph node metastasis" were closely related with the prognosis of patients with colorectal cancer. While no correlation between the expression of SDHB and the prognosis was found at present.
Conclusion:(1)In this research, the expression level of four subunits of SDH was firstly studied in colorectal cancer. Colorectal cancer and normal mucosa showed high expression levels of SDHA, SDHC and SDHD, and there was no significant difference in the level of protein expression among colorectal carcinoma, adjacent mucosa and adjacent normal mucosa of primary tumor. The expression level of SDHB protein decreased in colorectal cancer as compared with normal colorectal mucosa. The expression of SDHB decreased more severely in poorly differentiated colorectal cancer than in moderately differentiated and well-differentiated colorectal cancer. No correlation between the expression of SDHB and the prognosis was found at present. (2) Survival analysis revealed that "age","Duke's stage","distant metastasis" and "lymph node metastasis" are closely related with the prognosis of patients with colorectal cancer.
引文
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[2]Scheffler IE. Molecular genetics of succinate:quinone oxidoreductase in eukaryotes[J].Prog Nucleic Acid Res Mol Biol,1998,60:267-315.
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[9]Reisch N, Peczkowska M, Januszewicz A, et al. Pheochromocytoma:presentation, diagnosis and treatment[J].J Hypertens,2006,24(12):2331-2339.
[10]Bender BU, Gutsche M, Glasker S,et al.Differential genetic alterations in von Hippel-Lindau syndrome-associated and sporadic pheochromocytomas[J].J Clin Endocrinol Metab,2000,85(12):4568-4574.
[11]Baysal BE, Ferrell RE, Willett-Brozick JE, et al. Mutations in SDHD, a mitochondrial complex Ⅱ gene, in hereditary paraganglioma[J].Science,2000, 287(5454):848-851.
[12]Niemann S,Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3[J].Nat Genet,2000,26(3):268-270.
[13]Astuti D, Latif F, Dallol A, et al.Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma[J].Am J Hum Genet,2001,69(1):49-54.
[14]Knudson AG Jr. Genetics of human cancer[J].Annu Rev Genet,1986, 20:231-251.
[15]Gimenez-Roqueplo AP, Favier J, Rustin P, et al. The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex Ⅱ in the mitochondrial respiratory chain and activates the hypoxia pathway[J].Am J Hum Genet,2001,69(6):1186-1197.
[16]Gimenez-Roqueplo AP, Favier J, Rustin P, et al. Functional consequences of a SDHB gene mutation in an apparently sporadic pheochromocytoma[J].J Clin Endocrinol Metab,2002,87(10):4771-4774.
[17]Douwes Dekker PB,Hogendoorn PC, Kuipers-Dijkshoorn N, et al. SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology[J].J Pathol,2003,201(3):480-486.
[18]Baysal BE, Willett-Brozick JE, Filho PA, et al.An Alu-mediated partial SDHC deletion causes familial and sporadic paraganglioma[J].J Med Genet,2004, 41(9):703-709.
[19]Schiavi F, Boedeker CC, Bausch B, et al.Predictors and prevalence of paraganglioma syndrome associated with mutations of the SDHC gene[J].JAMA, 2005,294(16):2057-2063.
[20]Neumann HP, Pawlu C, Peczkowska M, et al.Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations[J]. JAMA,2004,292(8):943-951.
[21]Benn DE, Gimenez-Roqueplo AP, Reilly JR, et al.Clinical presentation and penetrance of pheochromocytoma/paraganglioma syndromes[J].J Clin Endocrinol Metab,2006,91(3):827-836.
[22]Vanharanta S,Buchta M, McWhinney SR, et al. Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma[J].Am J Hum Genet,2004,74(1):153-159.
[23]Gimenez-Roqueplo AP, Favier J, Rustin P, et al.The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex Ⅱ in the mitochondrial respiratory chain and activates the hypoxia pathway[J].Am J Hum Genet,2001,69(6):1186-1197.
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