吉西他濱對人胰腺癌SW1990和BxPC3細胞株Notch信號通路的誘導作用

程憲永 熊光蘇 李祥蘇 吳叔明

·論著·

吉西他濱對人胰腺癌SW1990和BxPC3細胞株Notch信號通路的誘導作用

程憲永 熊光蘇 李祥蘇 吳叔明

目的觀察吉西他濱對人胰腺癌細胞(SW1990和BxPC3)Notch信號通路活性的影響，探討其與胰腺癌對吉西他濱化療耐藥的關系。方法不同濃度吉西他濱處理人胰腺癌SW1990和BxPC3細胞株48 h，實時定量PCR檢測Notch信號通路受體Notch1、2、3、4，配體Jagged1、2和下游靶基因Hes1 mRNA的表達，Western blotting測定細胞Hes1蛋白表達。結果2 μmol/L吉西他濱作用胰腺癌細胞株48 h，SW1990細胞的Notch1、2、3、Jagged1、2和Hes1 mRNA表達量分別為8.26±0.48、39.12±4.87、0.84±0.06、105.8±17.92、6.59±0.32和17.30±2.96，均較未處理細胞的1.02±0.15、15.25±1.28、0.12±0.02、32.66±1.98、1.88±0.29和5.02±0.64明顯升高(P<0.05或P<0.01)；BxPC3細胞上述各項表達量分別為7.87±0.59、109.4±10.98、0.74±0.19、62.73±13.50、2.09±0.16和15.38±1.06，也均較未處理細胞的1.14±0.43、58.96±2.63、0.10±0.02、16.95±3.79、0.98±0.02和2.04±0.16，明顯升高(P<0.05或P<0.01)。1、2 μmol/L吉西他濱作用胰腺癌細胞株48 h，SW1990細胞Hes1蛋白表達量分別為0.30±0.03、0.42±0.03；BxPC3細胞分別為0.33±0.02、0.45±0.03，均較未處理細胞顯著增高(0.13±0.01、F=33.71；0.09±0.02、F=38.54，P值均<0.01)。結論吉西他濱可明顯激活SW1990和BxPC3細胞的Notch信號通路，這可能是胰腺癌細胞獲得化療耐受性的機制之一。

胰腺腫瘤； 吉西他濱； Notch受體； 信號轉導

吉西他濱單藥化療是晚期胰腺癌標準的一線治療，但療效較差，耐藥是化療失敗的主要原因之一[1]。研究發現，Notch信號的異常與多種腫瘤的發生有關，其中包括胰腺癌[2]。并且，Notch的激活與多種腫瘤耐藥性的產生有關，阻斷Notch信號可以明顯增強化療藥物的敏感性[3-4]。Hes1是Notch信號通路特異的下游靶基因，可以作為Notch活性的標志[5]。最近文獻報道，吉西他濱抵抗胰腺癌細胞株較敏感株Notch信號分子Notch2和Jagged1明顯上調[6]，提示Notch的激活可能與吉西他濱的耐藥有關。本研究應用吉西他濱處理胰腺癌細胞株，觀察Notch信號通路各分子表達水平的變化，探討Notch的活性與化療耐藥的關系。

材料和方法

一、細胞培養

胰腺癌細胞株SW1990和BxPC3由本實驗室保存，常規培養傳代。

二、Notch信號通路各分子mRNA表達的檢測

采用實時定量PCR方法。應用0、2μmol/L吉西他濱(江蘇豪森藥業公司)處理SW1990和BxPC3細胞48 h后收集細胞，采用Trizol法抽提總RNA。引物序列見表1，由上海生工生物工程技術服務公司合成。先逆轉錄為cDNA， PCR反應：95℃ 30s，95℃ 5 s、60℃ 31 s，40個循環，95℃ 15 s， 60℃ 60，95℃ 15 s，60℃ 15 s；以SDS2.3軟件分析Ct值。根據2-△△Ct法計算mRNA表達量，△△Ct=(Ct目的基因-Ct內參基因)實驗組-(Ct目的基因-Ct內參基因)對照組。

三、Hes1蛋白表達檢測

采用Western blotting方法檢測。應用0、1、2 μmol/L吉西他濱處理SW1990和BxPC3細胞48 h后采用細胞裂解液冰浴裂解收集細胞，BCA法測蛋白濃度。鼠抗人Hes1單克隆抗體購自Abnova公司；鼠抗α-tublin抗體和辣根過氧化物酶標記羊抗鼠二抗購自美國Sigma公司。最后DAB顯色，掃描儀掃描，以目的條帶與內參條帶灰度值比表示目的蛋白相對表達量，實驗重復3次。

四、統計學分析

表1 Q-PCR所用引物序列

結 果

一、Notch信號通路mRNA表達的變化

SW1990和BxPC3細胞都不同程度表達Notch信號通路受體(Notch1、2、3)、配體(Jagged1、2)和下游靶基因Hes1。其中Notch2及Jagged1表達水平最高；Notch3表達很微弱，Notch4未見表達。吉西他濱處理細胞48 h，各分子mRNA表達水平均較未處理細胞明顯升高(P<0.05或P<0.01，表2)。

二、Hes1蛋白表達的變化

應用1、2 μmol/L吉西他濱處理細胞48 h后，SW1990的Hes1蛋白表達量分別為0.30±0.03、0.42±0.03；BxPC3分別為0.33±0.02、0.45±0.03，均較未處理細胞的0.13±0.01和0.09±0.02顯著增高(F=33.71，F=38.54，P值均<0.01；圖1)，并均與吉西他濱濃度呈正相關(P<0.01)。

討 論

Notch信號通路在胰腺發育和成熟胰腺組織穩態中發揮重要作用[7]。人胰腺癌和動物模型的癌及癌前病變組織均有Notch受體、配體以及下游靶基因的中到高度表達[8-9]。用γ分泌酶抑制劑阻斷Notch信號通路，可以明顯抑制小鼠移植瘤的生成，并可阻斷癌前病變向胰腺癌的進展[5]。

圖1SW1990和BxPC3細胞經吉西他濱處理前后Hes1蛋白表達的變化

表2 吉西他濱處理前后SW1990和BxPC3細胞Notch信號通路各基因mRNA表達的變化

注：與處理前比較，aP<0.05，bP<0.01

Mungamuri等[10]首先發現Notch激活可引發下游促生存信號PI3K/Akt、Bcl-Xl和Survivin等的表達，使腫瘤細胞免于化療藥物誘導的凋亡。阻斷Notch信號通路可以增加結腸癌、T淋巴細胞白血病、黑色素瘤細胞對化療藥物的敏感性[3-4]。而一些化療藥如奧沙利鉑、5-FU、SN-38都可以激活結腸癌細胞Notch信號通路[3]。

最近有人發現，吉西他濱抵抗胰腺癌細胞株較敏感株Notch信號分子Notch2和Jagged1明顯上調[6]，本研究結果顯示，吉西他濱干預胰腺癌細胞株48 h， Notch信號通路受體、配體和靶基因Hes1的mRNAs表達水平均明顯上調，Hes1蛋白的表達也明顯上升，且與吉西他濱濃度呈正相關。因此，我們認為Notch的激活可能在胰腺癌對化療耐藥產生中起著重要作用，提示針對Notch信號通路的靶向干預治療可能有助于提高胰腺癌的化療敏感性。

[1] Fryer RA,Galustian C,Dalgelish AG. Recent advances and developments in treatment strategies against pancreatic cancer.Curr Clin Pharmacol,2009,4:102-112.

[2] Miyamoto Y,Maitra A,Ghosh B,et al.Notch mediates TGF alpha induced changes in epithelial differentiation during pancreatic tumorigenesis.Cancer Cell,2003,3:565-576.

[3] Meng RD,Shelton CC,Li YM,et al.gamma-Secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity.Cancer Res,2009,69:573-582.

[4] Real PJ,Tosello V,Palomero T,et al. Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia.Nat Med,2009,15:50-58.

[5] Plentz R,Park JS,Rhim AD,et al. Inhibition of gamma-secretase activity inhibits tumor progression in a mouse model of pancreatic ductal adenocarcinoma.Gastroenterology,2009,136:1741-1749.

[6] Wang Z,Li Y,Kong D,et al. Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway.Cancer Res,2009,69:2400-2407.

[7] Nakhai H,Siveke JT,Klein B,et al.Conditional ablation of Notch signaling in pancreatic development.Development,2008,135:2757-2765.

[8] Habbe N,Shi G,Meguid RA,et al.Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.Proc Natl Acad Sci USA,2008,105:18913-18918.

[9] De La O JP,Emerson LL,Goodman JL,et al.Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.Proc Natl Acad Sci USA,2008,105:18907-18912.

[10] Mungamuri SK,Yang X,Thor AD,et al.Survival signaling by Notch1:mammalian target of rapamycin(mTOR)-dependent inhibition of p53.Cancer Res,2006,66:4715-4724.

2009-11-09)

(本文編輯：呂芳萍)

GemcitabineinducesNotchsignalingpathwayactivationinpancreaticcancercelllinesSW1990andBxPC3

CHENGXian-yong,XIONGGuang-su,LIXiang-su,WUShu-ming.

DepartmentofGastroenterology,ShanghaiInstituteofDigestiveDisease，RenjiHospital,MedicalSchool，ShanghaiJiaotongUniversity,Shanghai200001,China

WUShu-ming,Email:wushuming@vip.sina.com

ObjectiveTo investigate the changes of Notch signaling pathway activity in human pancreatic cancer cell lines(SW1990,BxPC3)after gemcitabine induction, and to study its relationship with pancreatic cancer resistant to gemcitabine chemotherapy.MethodsThe pancreatic cancer cell lines SW1990 and BxPC3 were cultured with different concentrations of gemcitabine for 48 hours. The Notch signaling pathway receptors (Notch1, Notch2, Notch3, Notch4), ligands (Jagged1, Jagged2) and downstream target Hes1 mRNAs expression were detected by quantitative real-time PCR (Q-PCR). Protein levels of Hes1 were determined by Western blotting.ResultsAfter treatment with 2 μmol/L gemcitabine for 48 hours, the expression of Notch1, Notch2, Notch3, Jagged1, Jagged2 and Hes1 mRNAs in SW1990 cells were 8.26±0.48, 39.12±4.87, 0.84±0.06, 105.8±17.92, 6.59±0.32 and 17.30±2.96, which were significantly elevated when compared with those without gemcitabine treatment (1.02±0.15, 15.25±1.28, 0.12±0.02, 32.66±1.98, 1.88±0.29 and 5.02±0.64,P<0.05 orP<0.01); the expression in BxPC3 cells was 7.87±0.59, 109.4±10.98, 0.74±0.19, 62.73±13.50, 2.09±0.16 and 15.38±1.06, which were significantly elevated when compared with those without gemcitabine treatment (1.14±0.43, 58.96±2.63,0.10±0.02, 16.95±3.79, 0.98±0.02 and 2.04±0.16,P<0.05 orP<0.01). The expressions of Hes1 protein in SW1990 cells after 1, 2 μmol/L gemcitabine treatment for 48 h were 0.30±0.03, 0.42±0.03; and the expressions in BxPC3 cells were 0.33±0.02, 0.45±0.03, which were significantly increased when compared with those without gemcitabine treatment (0.13±0.01,F=33.71,0.09±0.02,F=38.54,P<0.01).ConclusionsThe Notch signaling pathway is significantly activated in pancreatic cancer cells SW1990 and BxPC3 by gemcitabine, which may be one of the mechanisms of chemoresistance.

Pancreatic neoplasms; Gemcitabine; Notch receptors; Signal transducing

10.3760/cma.j.issn.1674-1935.2010.05.011

200001 上海，上海交通大學醫學院附屬仁濟醫院，上海市消化疾病研究所

吳叔明，Email:wushuming@vip.sina.com