整合素連接激酶在胰腺癌中的表達(dá)及意義

劉偉 宋少偉 馬剛 石蕊 程穎 劉寧 郭克建

·論著·

整合素連接激酶在胰腺癌中的表達(dá)及意義

劉偉 宋少偉 馬剛 石蕊 程穎 劉寧 郭克建

目的探討整合素連接激酶(intergrin linked kinase, ILK)在胰腺癌組織中的表達(dá)及其臨床意義。方法采用免疫組織化學(xué)法和Western blotting法檢測(cè)ILK在60例胰腺癌和32例癌旁正常胰腺組織中的表達(dá)，并分析其與臨床病理參數(shù)的關(guān)系。結(jié)果免疫組化結(jié)果顯示，ILK蛋白在胰腺癌細(xì)胞胞質(zhì)內(nèi)和胞膜上以及間質(zhì)中均有表達(dá)，其陽性表達(dá)率為65.0%(39/60)，顯著高于正常胰腺組織的18.8%(6/32，P<0.05)。Western blotting結(jié)果顯示，ILK在胰腺癌組織中的相對(duì)表達(dá)量為303933±195116，顯著高于正常胰腺組織的144613±30074(P<0.05)。在胰腺癌組織中，ILK表達(dá)與腫瘤的臨床分期和淋巴結(jié)轉(zhuǎn)移有關(guān)(P<0.05)，而與腫瘤分化程度無關(guān)。結(jié)論ILK在胰腺癌組織高表達(dá)，并與腫瘤的惡性程度相關(guān)。

胰腺腫瘤; 整合素連接激酶; 免疫組織化學(xué); 蛋白質(zhì)印跡法

材料與方法

一、臨床資料

60例胰腺癌和32例癌旁正常胰腺組織標(biāo)本均來自中國醫(yī)科大學(xué)附屬第一醫(yī)院1990年至2008年手術(shù)切除的存檔蠟塊。2007年5月至2008年9月手術(shù)切除的10例胰腺癌患者另有術(shù)中從原發(fā)腫瘤及周圍胰腺組織中獲取并置-70℃冰箱保存的直徑約0.5～1 cm新鮮組織塊。

60例患者中男38例，女22例。年齡40～73歲，中位年齡57歲。高分化13例，中分化25例，低分化22例；按日本JPS臨床分期，Ⅰ期9例，Ⅱ期17例，Ⅲ期24例，Ⅳ期10例。有淋巴結(jié)轉(zhuǎn)移34例，無淋巴結(jié)轉(zhuǎn)移26例。

二、免疫組化染色法檢測(cè)ILK蛋白表達(dá)

60例胰腺癌及32例胰腺正常組織均行抗整合素連接激酶(ILK)的免疫組化染色。每一存檔蠟塊標(biāo)本連續(xù)切片2張，經(jīng)脫蠟、水化后嚴(yán)格按照鏈霉素抗生物素蛋白-過氧化物酶(S-P)法試劑盒(北京中衫金橋生物技術(shù)開發(fā)公司)說明書步驟進(jìn)行。兔抗人ILK多克隆抗體(美國ABGENT公司)工作濃度為1∶50。以PBS代替一抗作為陰性對(duì)照。ILK蛋白表達(dá)于細(xì)胞質(zhì)，黃色或淡黃染色。每張切片由3名病理科醫(yī)師選擇3個(gè)高倍視野，每視野計(jì)數(shù)100個(gè)細(xì)胞，染色細(xì)胞占總細(xì)胞數(shù)30%以上為陽性。

三、Western blotting檢測(cè)ILK蛋白表達(dá)

100 mg新鮮胰腺組織加入1 ml細(xì)胞裂解緩沖液，提取組織總蛋白。經(jīng)Larry法定量后，取50 μg蛋白行SDS-聚丙烯酰胺凝膠電泳，然后將蛋白質(zhì)印跡到硝酸纖維素膜上，用抗ILK抗體和抗β-actin抗體孵育，最后加入用堿性磷酸酶標(biāo)記的山羊抗兔二抗，用o-DIANISIDINE,TETRAZOTIZED和β-NAPHTHYL ACID PHOSPHATE顯色。應(yīng)用Fluor Chemv2.0 Stand Alone電泳凝膠分析軟件掃描測(cè)定各條帶灰度值，以目的條帶與β-actin條帶灰度值之比作為ILK蛋白相對(duì)表達(dá)量。

四、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、胰腺組織ILK蛋白表達(dá)

免疫組化結(jié)果顯示，ILK蛋白在胰腺癌細(xì)胞胞質(zhì)內(nèi)及胞膜上呈陽性表達(dá)，在胰腺癌組織的間質(zhì)中也有陽性表達(dá)(圖1)。胰腺癌組織ILK蛋白陽性表達(dá)率為65.0%(39/60)；癌旁正常胰腺組織的陽性表達(dá)率為18.8%(6/32)，兩者差異有顯著的統(tǒng)計(jì)學(xué)意義(P<0.05)。Western blotting結(jié)果顯示，ILK在胰腺癌中及癌旁正常胰腺組織中的相對(duì)表達(dá)量分別為303 933±195 116和144 613± 30 074(圖2)，癌組織ILK蛋白表達(dá)明顯高于癌旁正常胰腺組織(t=2.55，P<0.05)。

二、胰腺癌組織ILK蛋白表達(dá)與腫瘤臨床病理參數(shù)的關(guān)系

ILK蛋白表達(dá)與淋巴結(jié)轉(zhuǎn)移、JPS臨床分期相關(guān)(P<0.05)，與腫瘤分化程度無關(guān)(表1)。

表1 胰腺癌組織ILK蛋白表達(dá)與臨床病理參數(shù)的關(guān)系

討 論

整合素連接激酶是一種相對(duì)分子質(zhì)量為59 000的絲/蘇氨酸蛋白激酶，有4個(gè)錨蛋白重復(fù)序列，能與粘著斑復(fù)合物結(jié)合，并與生長因子受體酪氨酸激酶信號(hào)連接。研究表明，整合素介導(dǎo)細(xì)胞與細(xì)胞外基質(zhì)相互作用，從而調(diào)節(jié)細(xì)胞的生存、增殖、分化和遷移[1]。ILK與整合素在胞質(zhì)內(nèi)相互作用[2]，作為一個(gè)支架連接整合素到肌動(dòng)蛋白和信號(hào)轉(zhuǎn)導(dǎo)通路形成多蛋白復(fù)合體。通過黏附到細(xì)胞外基質(zhì)和磷脂酰肌醇-3激酶(PI3K)依賴途徑的生長因子激活I(lǐng)LK。在上皮細(xì)胞中，ILK的過表達(dá)導(dǎo)致停泊非依賴性細(xì)胞的增長[3]，細(xì)胞周期的延長，并且上調(diào)細(xì)胞調(diào)節(jié)蛋白D和細(xì)胞周期蛋白A的表達(dá)[4]以及在裸鼠中的致瘤性[5]，ILK能激活E-caherin的抑制物S-nail，可使E-cadherin表達(dá)下調(diào)、細(xì)胞間連接消失，導(dǎo)致腫瘤侵襲性增加[6]。在體外，ILK能磷酸化PKB/Akt，從而激活PKB轉(zhuǎn)導(dǎo)通路，抑制上皮細(xì)胞的凋亡。ILK同時(shí)能使其下游的關(guān)鍵靶點(diǎn)GSK-3磷酸化，促使細(xì)胞由G1期向S期過渡[7]。ILK能上調(diào)MMP-9表達(dá)，并參與生長因子信號(hào)通路，從而在降解細(xì)胞外基質(zhì)、促進(jìn)腫瘤血管生成并活躍細(xì)胞運(yùn)動(dòng)能力等方面促進(jìn)治療細(xì)胞向淋巴結(jié)轉(zhuǎn)移[8]。應(yīng)用ILK抑制劑(QLT-0254)可明顯抑制腫瘤的生長[9]。最近的研究表明，應(yīng)用siRNA條件性地沉默ILK表達(dá)，結(jié)果幾乎完全地抑制蛋白激酶B或者AKT激酶的活性，抑制絲氨酸GSK3β的磷酸化和cyclinD1表達(dá)[10]。另外，ILK在腫瘤血管的發(fā)生中起重要作用[11]。目前已證實(shí)，黑素瘤、卵巢癌、前列腺癌、非小細(xì)胞肺癌、胃癌、乳腺癌和結(jié)腸癌[12-17]均高表達(dá)ILK。ILK的表達(dá)與腫瘤的浸潤、轉(zhuǎn)移和血管發(fā)生關(guān)系密切。

圖1ILK在胰腺癌中呈陽性表達(dá)(1a.細(xì)胞質(zhì)中；1b.間質(zhì)中；1c.細(xì)胞膜上； ×200)圖2胰腺癌組織(1、3、5、7、9和癌旁正常胰腺組織(2、4、6、8、10)ILK蛋白的表達(dá)

本實(shí)驗(yàn)結(jié)果顯示，ILK在胰腺癌細(xì)胞中的陽性表達(dá)率為65%，明顯高于在胰腺正常組織中的陽性表達(dá)率，ILK表達(dá)與腫瘤淋巴結(jié)轉(zhuǎn)移和臨床分期相關(guān)，這與ILK在胃癌和黑色素瘤中的研究結(jié)果一致，與Sawai等[18]在胰腺癌中的研究結(jié)果相同。但I(xiàn)LK在惡性腫瘤中的表達(dá)與腫瘤的分化是否有關(guān)結(jié)果不一。ILK在前列腺癌和卵巢癌中與腫瘤細(xì)胞分化相關(guān)，在胃癌中的表達(dá)與胃癌分化程度無關(guān)，Sawai等和本實(shí)驗(yàn)結(jié)果均顯示ILK在胰腺癌的表達(dá)與胰腺癌的分化程度無關(guān)。因此，在這一方面還有待于進(jìn)一步研究。

[1] Hehlgans S,Haase M,Cordes N.Signalling via integrins:implications for cell survival and anticancer strategies.Biochim Biophys Acta,2007,1775:163-180.

[2] Hannigan GE,Leung-Hagesteijn C,Fitz-Gibbon L,et al.Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.Nature,1996,379:91-96.

[3] Radeva G,Petrocelli T,Behrend E,et al.Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.J Biol Chem,1997,272:13937-13944.

[4] D′Amico M,Hulit J,Amanatullah DF,et al.The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element binding protein-dependent pathways.J Biol Chem,2000,275:32649-32657.

[5] Tan C,Costello P,Sanghera J,et al.Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor,snail,in APC-/- human colon carcinoma cells.Oncogene,2001,20:133-140.

[6] Persad S,Dedhar S.The role of integrin-linked kinase (ILK) in cancer progression.Cancer Metastasis Rev,2003,22:375-384.

[7] Troussard AA,Costello P,Yoganathan TN,et al.The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9).Oncogene,2000,19:5444-5452.

[8] Yau CY,Wheeler JJ,Sutton KL,et al.Inhibition of integrin-linked kinase by a selective small molecule inhibitor,QLT0254,inhibits the PI3K/PKB/mTOR,Stat3,and FKHR pathways and tumor growth, and enhances gemcitabine-induced apoptosis in human orthotopic primary pancreatic cancer xenografts.Cancer Res,2005,65:1497-1504.

[9] Troussard AA,Mawji NM,Ong C,et al.Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation.J Biol Chem,2003,278:22374-22378.

[10] Tan C,Cruet-Hennequart S,Troussard A,et al.Regulation of tumor angiogenesis by integrin-linked kinase (ILK).Cancer Cell,2004,5:79-90.

[11] Dai DL,Makretsov N,Campos EI,et al.Increased expression of integrin-linked kinase is correlated with melanoma progression and poor patient survival.Clin Cancer Res,2003,9:4409-4414.

[12] Ahmed N,Riley C,Oliva K,et al.Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid.J Pathol,2003,201:229-237.

[13] Graff JR,Deddens JA,Konicek BW,et al.Integrinlinked kinase expression increases with prostate tumor grade.Clin Cancer Res,2001,7:1987-1991.

[14] Takanami I.Increased expression of integrin-linked kinase is associated with shorter survival in non-small cell lung cancer.BMC Cancer,2005,5:1.

[15] Ito R,Oue N,Zhu X,et al.Expression of integrin-linked kinase is closely correlated with invasion and metastasis of gastric carcinoma.Virchows Arch,2003,442:118-123.

[16] Chen P,Shen WZ,Karnik P.Suppression of malignant growth of human breast cancer cells by ectopic expression of integrin-linked kinase.Int J Cancer,2004,111:881-891.

[17] Bravou V,Klironomos G,Papadaki E,et al.ILK over-expression in human colon cancer progression correlates with activation of beta-catenin,down-regulation of E-cadherin and activation of the Akt-FKHR pathway.J Pathol,2006,208:91-99.

[18] Sawai H,Okada Y,Funahashi H,et al.Integrin-linked kinase activity is associated with interleukin-1a-induced progressive behavior of pancreatic cancer and poor patient survival.Oncogene,2006,25:3237-3246.

2009-04-07)

(本文編輯：屠振興)

Expressionandsignificanceofintegrinlinkedkinaseinpancreaticcarcinoma

LIUWei,SONGShao-wei,MAGang,SHIRui,CHENGYing,LIUNing,GUOKe-jian.

DepartmentofGastrointestrinalandPancreaticSurgery,FirstAffiliatedHospital,ChinaMedicalUniversity,Shenyang110001,China

SONGShao-wei,Email：songsw10@hotmail.com

ObjectiveTo investigate the expression and clinical significance of intergrin linked kinase (ILK) in pancreatic carcinoma.MethodsILK protein was detected by immunohistochemistry and Western blotting in 60 cases of pancreatic carcinoma and 32 cases of normal pancreatic tissue, and the relationship with the clinicopathological characteristics were analyzed.ResultsImmunohistochemistry showed ILK was expressed in cytoplasm and membrane of pancreatic carcinoma cells and the positive rate was 65% (39/60), which was significantly higher than 18.75%(6/32) of normal pancreatic tissue(P<0.05). Western blotting showed the expression of ILK in pancreatic carcinoma tissue was 303933±195116, which was significantly higher than 144613±30074 of normal pancreatic tissue (P<0.05). In pancreatic carcinoma, the expression of ILK was correlated with clinical stage and lymph metastasis (P<0.05), but not correlated with tumor cell differentiation (P>0.05).ConclusionsILK protein was highly expressed in pancreatic carcinoma tissue and it was correlated with the degree of malignancy.

Pancreatic neoplasms; Intergrin linked kinase; Immunohistochemistry; Western blotting

整合素連接激酶(integrin linked kinase，ILK)作為一種絲/蘇氨酸蛋白激酶是多種致瘤相關(guān)因素的上游交叉點(diǎn)，在細(xì)胞與細(xì)胞外基質(zhì)(ECM)的粘連中起到分子支架的作用，同時(shí)還參與多種信號(hào)轉(zhuǎn)導(dǎo)途徑，在細(xì)胞黏附、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞周期等多方面影響腫瘤的形成、增殖、侵襲和轉(zhuǎn)移。本實(shí)驗(yàn)檢測(cè)ILK在胰腺癌組織中的表達(dá)，并分析其與臨床病理參數(shù)的關(guān)系。

10.3760/cma.j.issn.1674-1935.2010.01.010

遼寧省教育廳2009高等學(xué)?？蒲许?xiàng)目(2009A783)

110001 沈陽，中國醫(yī)科大學(xué)附屬第一醫(yī)院普通外科教研室胃腸胰外科

宋少偉，Email：songsw10@hotmail.com