摘要
背景与目的胆囊癌是胆道系统最常见的恶性肿瘤之一。胆囊癌的预后极差,手术治疗仍是目前治疗胆囊癌的主要方法。由于肿瘤的生物学异质性,仅依靠临床病理分期来评估预后有一定的局限性。细胞周期调控异常是细胞周期增殖失控而导致癌变的重要原因。参与细胞周期调控的分子有细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDK)和CDK抑制蛋白(CDKI)。细胞周期的不同时相由不同的调节因子控制,细胞周期调控异常,尤其是G1/S期调控点的异常,在肿瘤发生中起着很重要的作用。P27是近年来发现的一个重要的CDKI,主要通过抑制G1期的Cyclin-CDK的活性,使细胞停滞于G1期。CyclinE蛋白在细胞周期G1/S的转换中发挥重要的正性调控作用,与肿瘤的发生密切相关。P27作为一种抑癌基因,CyclinE作为一种癌基因,二者均作用于细胞周期G1/S期调控点而调节细胞的生长和分化。在肿瘤治疗的研究中癌基因、抑癌基因及其对细胞周期调控作用有着非常重要的意义。本课题主要通过研究P27和CyclinE在胆囊癌组织中的表达情况,探讨P27和CyclinE的表达与胆囊癌的发生、组织学分化程度、Nevin分期进展、肿瘤浸润深度、淋巴结转移和周围浸润等临床病理因素的关系,分析它们在胆囊癌发生、发展中的作用,进而揭示其在临床应用中的前景。
方法收集安徽医科大学第一附属医院及六安市人民医院2001年1月～2006年12月收治的经手术切除且临床资料完整的胆囊癌标本40例。收集慢性胆囊炎标本20例做为对照组。标本经福尔马林固定,石蜡包埋,切片后用免疫组织化学法(SP法)检测P27和CyclinE的表达状况。对结果进行统计学分析,揭示P27和CyclinE的表达与胆囊癌及其各临床因素的相互关系。
结果(1)慢性胆囊炎组织中P27的高表达阳性率为100%(20/20),明显高于胆囊癌的45%(18/40),(P<0.05)。CyclinE在胆囊癌组织中表达阳性率为67.5%(27/40),显著高于慢性胆囊炎的15.0%(3/20),(P<0.05)。(2)P27的表达与胆囊癌组织学分化程度、Nevin分期、肿瘤浸润深度、淋巴结转移、周围浸润有关(P<0.05),而与患者的性别、年龄、组织学类型、肿瘤大小无关(P>0.05)。CyclinE的表达与胆囊癌组织学分化程度、Nevin分期进展、肿瘤浸润深度有关(P<0.05),而与性别、年龄、组织学类型、肿瘤大小、淋巴结转移、周围浸润无关(P>0.05)。P27和CyclinE的表达与胆囊癌患者的预后明显相关(P<0.05)。P27可能成为胆囊癌的一个独立的预后指标物。(3)P27和CyclinE表达之间呈负相关关系(r=-0.375,P<0.05)。
结论P27表达下降和CyclinE阳性表达导致细胞周期的调控异常,可能参与了胆囊癌的发生、发展过程。
Background and objective Gallbladder carcinoma is one of the most popular malignant tumor in bile duct system. The prognosis of gallbladder carcinoma is very poor. Operation is the main treatment of gallbladder carcinoma. Histopathological stage is still the most popular indexes to predict patient' s prognosis. However, tumor stage may lead physician to a wrong way sometimes because of the biological heterogeneity of tumor. Recent studies have revealed that uncontrolled cell cycle is very important to uncontrolled cell proliferation and carcinogenesis. Cyclin-dependent kinase(CDK),together with cylin and CDK inhibitor, govern cell cycle progression in eukaryotic cell. In cell cycle, different phase is regulated by different factors. Abnormal control on cell cycle, especially the abnorm of the check point of G1/S stage, plays an important role in carcinogenesis.. P27 is an important CDK inhibitor, which binds to and inhibits preferentially G1-phase kinases thereby arresting the cell cycle at G1-phase. Proto-oncogene cyclinE, encodes cyclinE protein,which acts as an important positive regulator in G1/S transition,and appears to be closely linked with carcinogenesis. CyclinE,as an oncogene and p27, as an antioncogene modulate the growth and diferentiation of cells by working on the cell cycle check point of G1/S stage, role in carcinogenesis. The researches on oncogene, antioncogene and the role of cell cycle control have great significance to help us treat tumor. This research, by studying the expressions of cyclinE and p27 in the tissues of gallbladder carcinomas (GBC), was to explore the correlation of cyclinE and p27 and gallbladder carcinogenesis,the histological grade, nevin′s stage, depth of invasion, and hence evaluate their roles in carcinogenesis and their significance at clinic.
Methods The samples from 40 patients with gallbladder carcinoma, who underwent surgical resection at The First Afiliated Hosptial of Anhui Medical University and Lu’An Peaple’s Hospital from 2001 Jan to 2006 Dec, were used for this study. 20 chronic cholecystitis (CC) specimens were control group. The samples were routinely fixed in 10% formalin and embedded in paraffin. After sectioned into slices, we detected the expression of p27 and cyclinE protein by immunohistochemical staining technique(SP method). We analyzed the relationship between the expression of p27 and cyclinE protein with clinical parameters.
Results (1)The positive expression rate of p27 in gallbladder carcinoma was 45.0%(18/40), which was lower than that in chronic cholecystitis(100% ), P<0.05. The positive expression rate of cyclinE in gallbladder carcinoma was 67.5%(27/40), which was significantly higher than that in chronic cholecystitis(15.0%,3/20), P<0.05. (2)The expression level of p27 protein in gallbladder carcinomas showed an obvious difference in the histological grade, nevin′s stage, depth of invasion, lymph node metastasis, infiltration (P<0.05). But not markedly associated with patient′s age, , histological types, tumor size. The expression level of cyclinE protein in gallbladder carcinomas showed an obvious difference in the histological grade,nevin′s stage, depth of invasion (P<0.05). But not markedly associated with patient′s age, histological types, tumor size, lymph node metastasis, infiltration. Log rank test showed that the p27 and cyclinE expression were significantly related with prognosis of the patients (P<0.05).(3)There was negative correlation between p27 and cyclinE expression (r=-0.375,P<0.05).
Conclusion The decreased expression of p27 and the high expression of cyclin E result in abnomal regulation of cell cycle,which may be associated with gallbladder carcinogenesis and progression.
引文
[1] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature, 2001, 411(6835):342-348.
[2] 陈济,宋方洲. CyclinE-CDK2分子活性调节机制. 医学分子生物学杂志,2006, 3(3):220-222.
[3] Ravank K, Jarvinen K, Paasinen-Sohns A, et al. Loss of p27kip1 from cyclin E/CDKI but not from cyclin k1/CDK4, complexes in cells transformed by polyamine biosynthtetic enzymes. Cancer Res, 2000, 60(18):5244-5353.
[4] 郎庆华,吕建一,赵洪,等.原发性胆囊癌的诊断和治疗. 贵阳医学院学报, 2006, 31(5):476-477.
[5] Anthony Rhodes, Bharat Jasani, Andre J. Balaon, et al. Study of interlaboratory reliability and reproducibility of Estrogen and Progesterone receptor assays in Europe. American Jourmal of Clinical Pathology, 2001, 115(1):44-58.
[6] 陶绪雄, 王卫星,陈志丹,等.P27和PCNA在原发性胆囊癌中的表达和相关性.武汉大学学报(医学版), 2006, 27(3):361-362.
[7] Blagosklonny MV, Pardee AB. Exploiting cancer cell cycling for selective protection of normal cells. Cancer Res, 2001, 61(11):4301-4305.
[8] Muller-tidow C,Metzger R,Kugler K,et a1.CyclinE is the only cyclin-dependent kinase 2-associated cyclin that predictes metastasis and survival in early stage non-small cell lung cancer.Cancer Res,2001,61:647-652.
[9] Kamal T, Takagi K, Asami H, et al. Decreasing of p27kip1 and cyclinE prtein levels is associated with progression from superficial into invasive bladder cancer. Bri J Cancer, 2001, 84(9):1242-1251.
[10] Wu XS, Akiyama Y, Igari T, et a1.Expression of homeodomain proteinCDX2 in gallbladder carcinomas.J Cancer Res Clin 0ncoL, 2005, 131(5):271-178.
[11] Polyak K, Lee MH,Erdjument-Bromage H,et al.Cloning of p27Kip1,a cyclin -dependent kinase inhibitor and a potential mediator of extracellular antimitogentic signals.Cell. 1994, 78(1):59-66.
[12] Zhang DH, Li FR, Qi H,et a1. Effect of cyclin kinase inhibitor P27 on glomerular mesangial cel proliferation. China Journal of Modern Medicine. 2004, l4(8):64-66.
[13] Takano Y, Kato Y, Van-Diest PJ, et al. CyclinD2 overexpression and cyclinE inversely with a por prognosis in gastric cancer cases. Am J Path, 2000, 156(2):585-591.
[14] So JB, Samarasinge K, Raju GC, et al. Expression of cell-cycle regulators p27 and cyclinE correlates with survival in gastric carcinoma patients. J Surg Res. 2000, 94(1):56-60.
[15] Kim DH, Lee HI, Nam ES, et al. Reduced expression of the cell-cycle inhibitor p27Kip1 is associated with progression and lymph node metastasis of gastric carcinoma. Histopathology, 2000, 36(3):245-251.
[16] Watson NF, Durrant LG, Scholefield JH, et al. Cytoplasmic expression of p27(kip1) is associated with a favourable prognosis in colorectal cancer patients. World J Gastroenterol, 2006, 12(39):6299-6304.
[17] Tamiya T, Mizumatsu S, Ono Y, et al. High cyclinE / low p27kip1 expression is associated with poor prognosis in astrocytomas. Acta Neuropathol, 2001, 101(4):334-340.
[18] 戴艳枝,刘勇,袁晟,等. 采用组织芯片技术研究乳腺癌中p27和cyclinE的表达. 江西医药, 2003, 38(4):252-254.
[19] Kravtsov VG, Shakhmurov MG, Sukmanov OV, et al. Expression of cycline-dependent kinase p27 in the low differentiated gastricadenocarcinoma. Arkh Patol, 2006, 68(5):14-16.
[20] 董磊,宋张娟. 宫颈鳞癌中p16、p21~(WAF1)、Rb、cyclinE蛋白的表达. 临床与实验病理学杂志, 2004, 20(5):554-556.
[21] 牛坚,陈澍周,钱海鑫. CyclinE、P27在原发性胆囊癌中表达及临床意义. 肝胆胰外科杂志, 2004, 16(2):90-92.
[22] Sasaki T, Katayose Y, Suzuki M, et al. Adenovirus expressing mutant p27kip1 enhanced apoptosis against cholangiocarcinoma than adenovirus-p27kip1 wild type. Hepatogastroenterology, 2004, 51(55):68-75.
[23] Kim YT,Zhao M.Aberrant cell cycle regulation in cervical carcinoma.Yonsei Med J,2005, 46(5):597-613.
[24] Skp2与p27kip1在乳腺癌组织中的表达及临床意义.河北医科大学学报, 2006, 27(1):26-27.
[1] Ravank K, Jarvinen K, Paasinen-Sohns A, et al. Loss of p27kip1 from cyclin E/CDKI but not from cyclin k1/CDK4, complexes in cells transformed by polyamine biosynthtetic enzymes. Cancer Res, 2000,60(18):5244-5353.
[2] Polyak K, Lee MH, Erdjument-Bromage H,et al.Cloning of p27Kip1,a cyclin -dependent kinase inhibitor and a potential mediator of extracellular antimitogentic signals.Cell. 1994, 78(1):59-66.
[3] Pietenpol JA,Bohlander SK,Sato Y,etal.Assignment of the human P27kip1 gene to 12p13 and its analysis in Ieukemias.Cancer Res, 1995, 55(6):1206-1209.
[4] Verkhivker GM.Protein conformational transitions coupled to bind-ing in molecular recognition of unstructured proteins:hierarchy of structural loss from all-atom monte carlo simulations of P27Kip1 unfolding-unbinding. Biopolymers, 2004, 75:420-433.
[5] Verkhivker GM.Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins:deciphering the effect of intermolecular interactions on computational structure prediction of the P27Kip1 protein bound to the cyclin A-cyclin-dependent kinase 2 complex.Proteins, 2005, 58(3):706-716.
[6] Liu Z, Dong Z, Han B, et al. Regulation of expression by promoters versus internal ribosome entry site in the 5’-untranslated sequence of the humancyclin-dependent kinase inhibitor P27kip1. Nucleic Acids Res, 2005, 33(12):3763-3771.
[7] Sa G, Stacey DW. P27 expression is regulated by separate signaling pathways,downstream of Ras, in each cell cycle phase. Exp Cell Res, 2004 , 300(2):427-439.
[8] Sakakibara K, Kubota K, Worku B, et al. PDGF-BB regulates P27 expression through ERK-dependent RNA turn-over in vascular smooth muscle cells. J Biol Chem, 2005, 280(27):25470-25477.
[9] Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell, 2004, 5:417-421.
[10] 孙立春,隋广杰,于雁,等.Skp2与p27kip1在乳腺癌组织中的表达及临床意义.河北医科大学学报, 2006, 27(1):26-27.
[11] Hershko D, Bornstein G, Ben-Izhak O, et al. Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas. Cancer, 2001, 91(4):1745-1751.
[12] 郑祥毅,丁伟,谢立平,等.Skp2和p27kip1在前列腺癌组织中的表达与临床病理特征的相关性研究.癌症, 2004, 23(2):215-218.
[13] 何洪智,赵晓航,张立勇,等.蛋白质泛素化降解途径与肿瘤发生的关系.世界华人消化杂志, 2002, 10(12):1365-1368.
[14] Ungermannova D, Gao Y, Liu X. Ubiquitination of P27KIP1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2. J Biol Chem, 2005, 280(34):30301-30309.
[15] Kamura T, Hara T, Matsumoto M, et al. Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase. Nat Cell Biol, 2004, 6(12):1229-1235.
[16] Kotoshiba S, Kamura T, Hara T, et al. Molecular dissection of the interaction between P27 and Kip1 ubiquitylation-promoting complex, theubiquitin ligase that regulates proteolysis of P27 in G1 phase. J Biol Chem, 2005, 280(18):17694-17700.
[17] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature, 2001, 411(6835):342-348.
[18] Schmidt M, Lu Y,Liu BL,etal.Differential modulation of paclitaxel-mediated apoptosis by p21Waf1 and p27Kip1 Oncogen. 2000, 19(20):2423-2429.
[19] Zhang DH, Li FR, Qi H,et a1. Effect of cyclin kinase inhibitor P27 on glomerular mesangial cel proliferation. China Journal of Modern Medicine. 2004, l4(8):64-66.
[20] Muller-tidow C,Metzger R,Kugler K,et a1.CyclinE is the only cyclin-dependent kinase 2-associated cyclin that predictes metastasis and survival in early stage non-small cell lung cancer.Cancer Res,2001,61:647—652.
[21] Sasaki T, Katayose Y, Suzuki M, et al. Adenovirus expressing mutant p27kip1 enhanced apoptosis against cholangiocarcinoma than adenovirus-p27kip1 wild type. Hepatogastroenterology. 2004, 51(55): 68-75.
[22] Kamal T, Takagi K, Asami H, et al. Decreasing of p27kip1 and cyclinE prtein levels is associated with progression from superficial into invasive bladder cancer. Bri J Cancer. 2001, 84(9):1242-1251.
[23] Quaroni A, Tian JQ, Seth P, et al. P27(Kip1) is an inducer of intestinal epithelial cell differentiation. Ap Rhys Am J Physiol Cell Physiol. 2000 , 279(4):1045-1057.
[24] 夏可义,肖强.转录因子E2F与胃癌关系的研究进展.国外医学(外科学分册), 2005, 32(1):70-74.
[25] Myers TK, Andreuzza SE, Franklin DS. p18INK4c and P27KIP1 are requiredfor cell cycle arrest of differentiated myotubes. Exp Cell Res, 2004, 300:365-378.
[26] Groth A, Willumsen BM. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of P27(Kip) Cdk-complexes. Cell Signal, 2005, 17(9):1063-1073.
[27] Koh TY,Park SW,Park KH,et al. Inhibitory effect of P27(Kip1) gene transfer on head and neck squamous cell carcinoma cell lines. Head Neck , 2003, 25(1):44-49.
[28] Deng X, Mercer SE, Shah S, et al. The cyclin-dependent kinase inhibitor P27Kip1 is stabilized in G0 by Mirk/dyrk1B kinase, J Biol Chem, 2004, 279(21):22498-22504.
[29] Iancu Rubin C, Atweh GF. P27(Kip1) and stathmin share the stage for the first time. Trends Cell Biol, 2005, 15(7):346-348.
[30] Wu XS, Akiyama Y, Igari T, et a1.Expression of homeodomain protein CDX2 in gallbladder carcinomas.J Cancer Res Clin 0ncoL, 2005, 131(5): 271-278.
[31] 牛坚,陈澍周,钱海鑫. CyclinE、P27在原发性胆囊癌中表达及临床意义. 肝胆胰外科杂志, 2004, 16(2):90-92.
[32] Kim YT,Zhao M.Aberrant cell cycle regulation in cervical Carcinoma.Yonsei Med J,2005, 46(5):597-613.
[33] Blagosklonny MV, Pardee AB. Exploiting cancer cell cycling for selective protection of normal cells. Cancer Res, 2001 61(11):4301-4305.
[34] Teraishi F, Kagawa S, Watanabe T, et al. ZD1839(gefitinib, “Iressa”), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma. FEBS Lett. 2005, 579(19):4069-4075.
[35] Chen F, Kim E, Wang CC, et al. Ciglitazone2induced P27 genetranscriptional activity is mediated through Sp1 and is negatively regulated by the MAPK signaling pathway. Cell Signal, 2005, 17(12): 1572-1577.
[36] Chu I, Sun J, Arnaout A, et al. P27 phosphorylation by Src regulatates inhibition of cyclin E-Cdk2. Cell. 2007 Jan 26, 128(2):281-294.
[37] Fischer C, Sanchez-Ruderisch H, Welzel M, et al. Galectin-1 interacts with the alpha 5beta 1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and P27. J Biol Chem, 2005, 280(44):37266-37277.
[38] Zhang Q, Tian L, Mansouri A, et al. Inducible expression of a degradation-resistant form of P27(Kip1) causes growth arrest and apoptosis in breast cancer cells. FEBS Lett, 2005, 579(18):3932-3940.
[39] Harada K, Supriatno, Kawashina Y, et al. Down-regulation of S-phase kinase associated protein 2(Skp2) induces apotosis in oral cancer cell. Oral Oncol 2005, 41(6):623-630.
[40] Takano Y, Kato Y, Van-Diest PJ, et al. CyclinD2 overexpression and cyclinE inversely with a por prognosis in gastric cancer cases. Am J Path, 2000, 156(2):585-591.
[41] 陆云飞,曾健. p16和p27在乳腺癌中的表达及其预后意义. 华中医学杂志, 2001, 25(6):316-319.
[42] So JB, Samarasinge K, Raju GC, et al. Expression of cell-cycle regulators p27 and cyclinE correlates with survival in gastric carcinoma patients. J Surg Res. 2000, 94(1):56-60.
[43] Kim DH, Lee HI, Nam ES, et al. Reduced expression of the cell-cycle inhibitor p27Kip1 is associated with progression and lymph node metastasis of gastric carcinoma. Histopathology. 2000, 36(3):245-251.
[44] Tamiya T, Mizumatsu S, Ono Y, et al. High cyclinE / low p27kip1 expression is associated with poor prognosis in astrocytomas. Acta Neuropathol, 2001, 101(4):334-340.
[45] Watson NF, Durrant LG, Scholefield JH, et al. Cytoplasmic expression of p27(kip1) is associated with a favourable prognosis in colorectal cancer patients. World J Gastroenterol. 2006, 12(39):6299-6304.