Hedgehog通路抑制劑逆轉(zhuǎn)人胰腺癌SW1990細(xì)胞獲得性耐藥的實(shí)驗(yàn)研究

姚捷 安勇 錢建軍 柏斗勝 徐澤寬 苗毅

·論著·

Hedgehog通路抑制劑逆轉(zhuǎn)人胰腺癌SW1990細(xì)胞獲得性耐藥的實(shí)驗(yàn)研究

姚捷 安勇 錢建軍 柏斗勝 徐澤寬 苗毅

目的探討人胰腺癌SW1990耐藥細(xì)胞株中Hedgehog通路成員(Shh、SMO、Gli1、ABCB1、ABCG2)表達(dá)的變化及應(yīng)用Hedgehog信號(hào)通路抑制劑對(duì)其耐藥性的影響。方法建立人胰腺癌SW1990耐吉西他濱細(xì)胞株(SW1990/GZ)。采用Hedgehog信號(hào)通路抑制劑環(huán)巴明作用耐藥細(xì)胞，應(yīng)用實(shí)時(shí)PCR、蛋白質(zhì)印跡法檢測(cè)用藥前后細(xì)胞Shh、SMO、Gli1、ABCB1、ABCG2 mRNA和蛋白的表達(dá)及CD44+CD24+、CD133+細(xì)胞亞群比例。以100 μmol/L吉西他濱作用環(huán)巴明處理后的SW1990/GZ細(xì)胞，應(yīng)用Annexin V加PI雙染法檢測(cè)細(xì)胞凋亡率。結(jié)果SW1990/GZ細(xì)胞中CD44+CD24+細(xì)胞比例從親代的(29.45±1.99)%增加到(93.16±2.46)%、CD133+細(xì)胞比例從(59.37±1.69)%增加到(95.88±1.47)%(P<0.01)；ABCB1、ABCG2、Shh、Gli1 mRNA的表達(dá)分別增加(274.90±31.44)、(3.48±0.33)、(12.07±1.71)、(4.15±0.42)倍(P<0.01)，并檢測(cè)出SMO mRNA表達(dá)。蛋白表達(dá)結(jié)果與mRNA表達(dá)一致。2 μmol/L環(huán)巴明作用SW1990/GZ細(xì)胞1周后，CD44+CD24+比例下降到(36.68±2.44)%、CD133+細(xì)胞比例下降到(62.76±1.28)%(P<0.05)。2、5 μmol/L環(huán)巴明處理SW1990/GZ細(xì)胞后再用100 μmol/L吉西他濱處理細(xì)胞，細(xì)胞的凋亡加死亡率分別為(53.68±5.24)%和(69.99±3.16)%，顯著高于對(duì)照組的(4.55±0.87)%(P<0.01)。結(jié)論人胰腺癌SW1990耐藥細(xì)胞株中高富集腫瘤干細(xì)胞，高表達(dá)Hedgehog信號(hào)通路成員SMO及Gli1。抑制耐藥株的Hedgehog通路，可以降低其腫瘤干細(xì)胞比例，下調(diào)SMO及Gli1表達(dá)，部分恢復(fù)對(duì)吉西他濱的敏感性。

胰腺腫瘤； 腫瘤干細(xì)胞； Hedgehog信號(hào)通路； 吉西他濱； 獲得性耐藥

腫瘤細(xì)胞固有的和獲得性的耐藥機(jī)制是化療后復(fù)發(fā)、轉(zhuǎn)移的主要原因[1-2]。研究表明，人胰腺癌耐吉西他濱細(xì)胞株富集腫瘤干細(xì)胞[3-4]，而腫瘤干細(xì)胞亞群高表達(dá)Hedghog(Hh)通路成員Shh[5]。但人胰腺癌耐藥細(xì)胞株的Hh通路成員表達(dá)狀況及抑制Hh通路對(duì)其的影響尚未知曉。為此，本研究檢測(cè)胰腺癌耐吉西他濱細(xì)胞株(SW1990/GZ)的Hh通路成員及耐藥基因ABC家簇成員的表達(dá)，觀察外源性給予Hh通路抑制劑對(duì)其的影響。

材料和方法

一、SW1990/GZ的建立及分組

采用間歇濃度梯度倍增法誘導(dǎo)建立胰腺癌耐吉西他濱細(xì)胞株SW1990/GZ，具體操作參考文獻(xiàn)[6]。

二、熒光定量PCR

取SW1990/GZ及親本SW1990對(duì)數(shù)生長(zhǎng)期細(xì)胞，應(yīng)用Trizol提取細(xì)胞總RNA。根據(jù)Genebank的人ABCB1(MDR1)、ABCG2(BCRP)、Shh、SMO、Gli1和GAPDH的cDNA序列設(shè)計(jì)引物(5′-3′)。ABCB1上游TGATTGCATTTGGAGGACAA，下游CCAGAAGGCCAGAGCATAAG，產(chǎn)物155 bp；ABCG2上游AGATGGGTTTCCAAGCGTTCAT，下游CCAGTCCCAGTACGACTGTGACG，產(chǎn)物91 bp；Shh上游GTGTACTACGAGTCCAAGGCAC，下游AGGAAGTCGCTGTAGAGCAGC，產(chǎn)物200 bp；SMO上游TTACCTTACGCTGCCACTTCTACG，下游GCCTTGGCAATCATCTTGCTCTTC，產(chǎn)物400 bp；Gli1上游CTCCCGAAGGACAGGTATGTAAC，下游CCCTACTCTTTAGGCACTAGAGTTG，產(chǎn)物248 bp；內(nèi)參GAPDH上游CGACCACTTTGTCAAGCTCA，下游TGTGAGGAGGGGAGATTCAG，產(chǎn)物210 bp。應(yīng)用AMV逆轉(zhuǎn)錄試劑盒逆轉(zhuǎn)錄成cDNA，熒光定量PCR(7500)法檢測(cè)各基因mRNA的表達(dá)。實(shí)驗(yàn)重復(fù)3次，結(jié)果用7500 System Software V2.0分析，運(yùn)用2-ΔΔCT (Livak)法分析實(shí)驗(yàn)數(shù)據(jù)，以SW1990細(xì)胞組為1，計(jì)算mRNA的表達(dá)倍數(shù)。由于SMO mRNA表達(dá)用熒光定量PCR未檢出，改RT-PCR檢測(cè)。

三、蛋白質(zhì)印跡法

取上述兩組細(xì)胞，加入RIPA裂解液裂解細(xì)胞，BCA法測(cè)定蛋白濃度。常規(guī)行蛋白質(zhì)印跡法。ABCG2、ABCB1、SMO和Gli1抗體1∶1000稀釋，辣根過氧化酶標(biāo)記二抗1∶2000稀釋，ECL發(fā)光、顯影。應(yīng)用Bio-Rad公司凝膠成像系統(tǒng)攝取圖像并進(jìn)行灰度分析，以β-actin為內(nèi)參。以SW1990細(xì)胞組為1，計(jì)算蛋白的表達(dá)倍數(shù)。

另用2、5 μmol/L環(huán)巴明處理SW1990/GZ細(xì)胞12、24、48 h，同上述方法測(cè)檢SMO、Gli1、ABCB1、ABCG2 mRNA及蛋白表達(dá)。

四、CD44+CD24+、CD133+細(xì)胞亞群檢測(cè)

SW1990/GZ細(xì)胞按5×105/孔接種于6孔板，在無化療藥物培養(yǎng)液中傳代2次后分為對(duì)照組、DMSO(培養(yǎng)液中單加DMSO)組、2 μmol/L環(huán)巴明處理組，分別繼續(xù)培養(yǎng)0、3、7 d，消化收集細(xì)胞。調(diào)整細(xì)胞濃度為1×106/ml。每組細(xì)胞分為兩管，一管加10 μg/ml的CD44-FITC和10 μg/ml的CD24-PE，另一管加入10 μg/ml的CD133-PE，避光冰上孵育30 min。離心棄上清，細(xì)胞重懸于1 ml PBS，置冰上保存。 加入2 μl/ml PI染液標(biāo)記細(xì)胞5 min后，上流式細(xì)胞儀檢測(cè)CD44+CD24+及CD133+亞群細(xì)胞比例。

五、細(xì)胞凋亡檢測(cè)

SW1990/GZ細(xì)胞在無化療藥物培養(yǎng)液中傳代2次后分為對(duì)照換藥組(24 h后換含100 μmol/L吉西他濱的培養(yǎng)液)、 DMSO換藥組及2、5 μmol/L環(huán)巴明組和2、5 μmol/L環(huán)巴明換藥組。培養(yǎng)48 h后消化收集細(xì)胞，每組分兩檢測(cè)管，一管用Annexin V-FITC 、PI雙染，一管不加Annexin V-FITC、PI作為陰性對(duì)照，上流式細(xì)胞儀檢測(cè)細(xì)胞凋亡及死亡率。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、SW1990/GZ細(xì)胞的ABCB1、ABCG2、Shh、Gli1、SMO mRNA及蛋白表達(dá)

SW1990細(xì)胞ABCB1、ABCG2、Shh、Gli1 mRNA表達(dá)的△CT值分別為-1.41±0.11、-3.93±0.13、-8.05±0.18、-5.48±0.42；SW1990/GZ的△CT值分別為-9.51±0.24、-5.73±0.22、-4.47±0.23、-3.43±0.28，SW1990/GZ細(xì)胞各mRNA表達(dá)分別為親本細(xì)胞的(274.90±31.44)、(3.48±0.33)、(12.07±1.71)、(4.15±0.42)倍，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。親本SW1990無SMO mRNA表達(dá)，SW1990/GZ細(xì)胞表達(dá)SMO mRNA(圖1)。

圖1SW1990細(xì)胞及SW1990/GZ細(xì)胞的SMO mRNA表達(dá)

SW1990/GZ細(xì)胞ABCB1、ABCG2、Gli1蛋白的表達(dá)分別為親本細(xì)胞的(8.28±1.81)、(3.34±0.87)、(4.76±0.12)倍(圖2)。

二、各組SW1990/GZ的CD44+CD24+、CD133+細(xì)胞比例

SW1990/GZ細(xì)胞的對(duì)照組、DMSO組、2 μmol/L環(huán)巴明組CD44+CD24+細(xì)胞比例分別為(93.13±1.19)%、(94.08±2.14)%、(93.16±2.46)%；培養(yǎng)3 d后分別為(92.89±2.98)%、(92.79±2.08)%、(74.05±1.52)%；7 d后分別為(91.89±1.988)%、(92.09±1.89)%、(36.68±2.44)%。

圖2 SW1990/GZ、SW1990細(xì)胞相關(guān)蛋白的表達(dá)

環(huán)巴明處理后SW1990/GZ細(xì)胞的CD44+CD24+細(xì)胞比例顯著下降(P<0.01)。

對(duì)照組、DMSO組、環(huán)巴明組CD133+細(xì)胞比例分別為(94.58±2.34)%、(94.29±2.23)%、(95.88±1.47)%；培養(yǎng)3 d后分別為(91.89±2.78)%、(96.12±2.23)%、(76.52±2.97)%；7 d后為(92.09±2.43)%、(95.67±2.01)%、(62.76±1.28)%，環(huán)巴明處理后SW1990/GZ細(xì)胞的CD133+細(xì)胞比例顯著下降(P<0.01，圖3)。

圖3環(huán)巴明處理后SW1990/GZ細(xì)胞中CD44+CD24+、CD133+細(xì)胞的比例

三、環(huán)巴明對(duì)細(xì)胞SMO、Gli1、ABCB1、ABCG2 mRNA 和蛋白表達(dá)的影響

不同濃度環(huán)巴明處理SW1990/GZ細(xì)胞12、24、48 h后，SMO、Gli1、ABCB1、ABCG2 mRNA及蛋白表達(dá)均顯著下降(表1、圖4)。

表1 環(huán)巴明處理SW1990/GZ細(xì)胞后相關(guān)基因mRNA的表達(dá)

注：與SW1990/GZ組比較，t值為2.82～11.68，aP<0.05

圖40(-)、2(+)、5(++)μmol/L環(huán)巴明處理SW1990/GZ細(xì)胞后相關(guān)蛋白表達(dá)

四、環(huán)巴明處理后SW1990/GZ細(xì)胞對(duì)吉西他濱的耐藥性

以2、5 μmol/L環(huán)巴明處理SW1990/GZ細(xì)胞后12 h再應(yīng)用100 μmol/L吉西他濱處理12 h，細(xì)胞的凋亡加死亡率分別為(53.68±5.24)%和(69.99±3.16)%，顯著高于對(duì)照組、DMSO組及2、5 μmol/L環(huán)巴明組的(4.55±0.87)%、(4.51±0.51)%、(12.81±1.32)%、(24.41±5.18)%(t值分別為10.11、6.62、16.17、35.39，P值均<0.001；圖5)。

討 論

Hedgehog(Hh)信號(hào)通路是胚胎時(shí)期胰腺發(fā)育所必需的信號(hào)通路之一[7]。胰腺癌的發(fā)生從胰腺導(dǎo)管上皮經(jīng)過胰腺上皮內(nèi)瘤變(pancreatic intraepithelial neoplasia, PanIN)逐步發(fā)展而來。研究發(fā)現(xiàn)，在PanIN向胰腺癌發(fā)展的病變組織中Hh信號(hào)通路成員的表達(dá)也急劇上升[8]。本結(jié)果顯示，人胰腺癌SW1990細(xì)胞株表達(dá)Gli,無SMO表達(dá)，和Gao等[9]的研究類似。在持續(xù)地暴露于高濃度的吉

圖5對(duì)照換藥組(C)、DMSO換藥組(V)、2、5 μmol/L環(huán)巴組(1、2)及2、5 μmol/L環(huán)巴明換藥組(3、4)的細(xì)胞凋亡圖

西他濱后建立的SW1990耐藥細(xì)胞株SW1990/GZ的Gli1表達(dá)增加，并出現(xiàn)SMO表達(dá)。SW1990/GZ細(xì)胞大部分為CD44+CD24+和CD133+的細(xì)胞。抑制胰腺癌中Hh通路可以有效地抑制人胰腺癌細(xì)胞的增殖[8]、侵襲和轉(zhuǎn)移能力[6]。應(yīng)用SMO小分子拮抗劑環(huán)巴明處理5株人胰腺癌細(xì)胞株，結(jié)果有2株SMO高表達(dá)的細(xì)胞株凋亡明顯增加，增殖率下降；用對(duì)環(huán)巴明敏感的細(xì)胞株接種裸鼠，并用環(huán)巴明進(jìn)行瘤內(nèi)注射，結(jié)果腫瘤體積明顯縮小。

Mueller等[10]報(bào)道，聯(lián)合應(yīng)用環(huán)巴明和雷帕霉素可以殺死人胰腺癌原代細(xì)胞和L3.6pl細(xì)胞中的CD133+的細(xì)胞亞群。本研究結(jié)果顯示，環(huán)巴明處理SW1990/GZ細(xì)胞后Gli1及SMO表達(dá)顯著下調(diào)，CD44+CD24+和CD133+細(xì)胞比例顯著下降，再應(yīng)用吉西他濱處理后，細(xì)胞凋亡明顯增加，表明環(huán)巴明處理后可以提高耐藥細(xì)胞對(duì)吉西他濱的敏感性，有可能對(duì)臨床胰腺癌患者的治療有潛在的應(yīng)用價(jià)值。

[1] Muerkoster S, Wegehenkel K, Arlt A, et al. Tumor stroma interactions induce chemoresistance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin-1beta. Cancer Res, 2004, 64:1331-1337.

[2] Banerjee D, Mayer-Kuckuk P, Capiaux G, et al. Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. Biochim Biophys Acta, 2002, 1587:164-173.

[3] Shah AN, Summy JM, Zhang J, et al. Development and characterization of gemcitabine-resistant pancreatic tumor cells. Ann Surg Oncol, 2007, 14:3629-3637.

[4] Hong SP, Wen J, Bang S, et al. CD44-positive cells are responsible for gemcitabine resistance in pancreatic cancer cells. Int J Cancer, 2009, 125:2323-2331.

[5] Thayer SP, di Magliano MP, Heiser PW, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature, 2003, 425:851-856.

[6] Feldmann G, Dhara S, Fendrich V, et al. Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers. Cancer Res, 2007, 67:2187-2196.

[7] Toftgard R.Hedgehog signalling in cancer. Cell Mol Life Sci 2000, 57(12):1720-1731.a P, et al. Identification of pancreatic cancer stem cells. Cancer Res, 2007, 67:1030-1037.

[8] Bardeesy N, DePinho RA.Pancreatic cancer biology and genetics. Nat Rev Cancer, 2002, 2:897-909.

[9] Gao J, Li Z, Chen Z, et al. Antisense Smo under the control of the PTCH1 promoter delivered by an adenoviral vector inhibits the growth of human pancreatic cancer. Gene Ther, 2006, 13:1587-1594.

[10] Mueller MT, Hermann PC, Witthauer J, et al. Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer. Gastroenterology, 2009,137:1102-1113.

HedgehogsignalingpathwayinhibitorrevertsacquiredchemoresistanceinhumanpancreaticcancerSW1990cellline

YAOJie,ANYong,QIANJian-jun,BAIDou-sheng,XUZe-kuan,MIAOYi.

DepartmentofHepatobiliary&PancreaticSurgery,NorthernJiangsuPeople′sHospital,Yangzhou225201,China

YAOJie,Email:docyao@126.com

ObjectiveTo investigate the expression levels of hedgehog signaling pathway members (SMO, Gli1, ABCB1, ABCG2) in gemcitabine resistant pancreatic cancer cells and to study the effect of hedgehog signaling pathway inhibitor on acquired chemoresistance.MethodsHuman pancreatic gemcitabine-resistant SW1990 cell line (SW1990/GZ) was established. Hedgehog inhibitor, cyclopamine, was used to treat resistant cells. Different expression levels of SMO, Gli1 and ABCB1, ABCG2 mRNA and proteins, and the percentage of CD44+CD24+and CD133+cells were compared by quantitative RT-PCR and Western blot before and after cyclopamine treatment, respectively. Were treated with 100 μmol/L of gemcitabine SW1990/GZ cells after cyclopamine treatment, and cell apoptosis was determined by Annexin V/PI method.ResultsThe percentage of CD44+CD24+and CD133+cells in SW1990/GZ cells were increased from (29.45±1.99)%, (59.37±1.69)% in parental cells to (93.16±2.46)%, (95.88±1.47)%(P<0.05), respectively. The mRNA expression levels of ABCB1, ABCG2, and Gli1 were increased (274.90±31.44),(3.48±0.33), (4.15±0.42) folds, compared with that of parental cells. The mRNA expression of SMO was also detected in resistant cells. The results of protein expression were consisted with that of mRNA expression. After treatment of 2 μmol/L cyclopamine for 1 week, the percentage of CD44+CD24+and CD133+in SW1990/GZ cells decreased to (36.68±2.44)% and (62.76±1.28)% (P<0.05), respectively. After treatment with 2, 5μmol/L of cyclopamine, SW1990/GZ cells were re-exposed to 100 μmol/L of gemcitabine, and then the cell apoptosis and death rates were (53.68±5.24)% and (69.99±3.16)%, which were significantly higher than those in control group (4.55±0.87)% (P<0.01).ConclusionsGemcitabine-resistant pancreatic cancer SW1990 cells are enriched with cancer stem cells and highly express hedgehog member SMO and Gli1. Inhibition of hedgehog signaling pathway by cyclopamine can reduce the proportion of cancer stem cell in gemcitabine-resistant cells, down-regulate SMO and Gli1 expression levels, partly restore the sensitivity to gemcitabine and reverts the acquired drug resistance.

Pancreatic neoplasms; Tumor stem cells; Hedgehog signaling pathway; Gemcitabine; Acquired chemoresistance

10.3760/cma.j.issn.1674-1935.2012.03.013

225001 江蘇揚(yáng)州，江蘇省蘇北人民醫(yī)院肝膽胰外科(姚捷、錢建軍、柏斗勝)；南京醫(yī)科大學(xué)第一附屬醫(yī)院 胰腺外科中心(安勇、徐澤寬、苗毅)

姚捷，Email:docyao@126.com

2011-10-19)

(本文編輯：呂芳萍)