雷公藤內(nèi)酯醇對(duì)胰腺癌PANC1細(xì)胞Toll樣受體4/核因子-κB信號(hào)通路的影響

馬建霞 孫運(yùn)良 王一倩 童依麗 于曉峰

·論著·

雷公藤內(nèi)酯醇對(duì)胰腺癌PANC1細(xì)胞Toll樣受體4/核因子-κB信號(hào)通路的影響

馬建霞 孫運(yùn)良 王一倩 童依麗 于曉峰

目的探討雷公藤內(nèi)酯醇(TP)對(duì)人胰腺癌PANC1細(xì)胞株侵襲能力的影響及其與Toll樣受體4/核因子-κB(TLR4/NF-κB)信號(hào)通路的關(guān)系。方法將PANC1細(xì)胞分為親本細(xì)胞組、TP組、脂多糖(LPS)組和TP+LPS組。TP組培養(yǎng)液中加入50 ng/ml的TP，LPS組加入1 μg/ml的LPS，TP+LPS組先用50 ng/ml的TP處理2 h，再加入1 μg/ml的LPS。各組細(xì)胞均常規(guī)培養(yǎng)24 h。采用實(shí)時(shí)定量PCR和蛋白質(zhì)印跡法檢測(cè)TLR4、基質(zhì)金屬蛋白酶-9(MMP-9) mRNA和蛋白的表達(dá)，雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)NF-κB活性，Transwell小室檢測(cè)細(xì)胞侵襲能力。結(jié)果親本組、LPS組、TP組、TP+LPS組的TLR4 mRNA表達(dá)量分別為0.41±0.06、0.46±0.10、0.20±0.04和0.25±0.06，TLR4蛋白表達(dá)量分別為0.55±0.06、0.60±0.03、0.18±0.04和0.13±0.00；NF-κB活性分別為13.0±3.0、31.6±4.3、7.3±1.5和10.8±2.1；穿膜細(xì)胞數(shù)分別為(56.8±8.6)、(104.5±12.8)、(32.0±5.7)和(46.8±7.0)個(gè)；MMP-9 mRNA表達(dá)量分別為0.36±0.05、0.58±0.07、0.18±0.03和0.30±0.004，MMP-9蛋白表達(dá)量分別為0.31±0.04、0.53±0.08、0.11±0.02和0.15±0.00。 LPS組TLR4 mRNA和蛋白表達(dá)量與親本組差異無統(tǒng)計(jì)學(xué)意義，但NF-κB活性、穿膜細(xì)胞數(shù)、MMP-9 mRNA和蛋白表達(dá)量顯著高于親本組(t值分別為8.654、7.593、6.655、4.982，P值均<0.01)。TP組TLR4 mRNA和蛋白表達(dá)量、NF-κB活性、穿膜細(xì)胞數(shù)、MMP-9 mRNA和蛋白表達(dá)量均顯著低于親本組(t值分別為-7.609、-9.948、 -4.176、-5.915、-8.179、-9.948，P值均<0.01)。TP+LPS組TLR4 mRNA和蛋白表達(dá)量、NF-κB活性、穿膜細(xì)胞數(shù)、MMP-9 mRNA和蛋白表達(dá)量均顯著低于LPS組(t值分別為-4.437、-14.805、-10.506、-9.700、-9.055、-8.932，P值均<0.01)。結(jié)論TP具有抑制胰腺癌細(xì)胞侵襲的作用，其機(jī)制與抑制TLR4/NF-κB信號(hào)通路、下調(diào)MMP-9的表達(dá)有關(guān)。

胰腺腫瘤； Toll樣受體4； 雷公藤內(nèi)酯； 基質(zhì)金屬蛋白酶類

雷公藤內(nèi)酯醇(triptolide，TP)是從衛(wèi)茅科植物雷公藤中抽提到的二萜內(nèi)酯化合物。近年來研究發(fā)現(xiàn)，TP可抑制結(jié)腸癌、前列腺癌等多種腫瘤細(xì)胞生長(zhǎng)、侵襲和轉(zhuǎn)移[1-3]，但其作用機(jī)制尚未完全闡明。Toll樣受體4(Toll-like receptor 4, TLR4)是最早發(fā)現(xiàn)的、同時(shí)也是研究最為廣泛的Toll樣受體家族成員。研究已證實(shí)，TLR4在多種腫瘤細(xì)胞中呈高表達(dá)，參與腫瘤的發(fā)生與發(fā)展[4]。TLR4在其配體脂多糖(1ipopolysaccharide，LPS)的刺激下，可激活核因子-κB (NF-κB)，誘導(dǎo)腫瘤細(xì)胞分泌多種細(xì)胞因子和炎癥介質(zhì)，促進(jìn)腫瘤的侵襲和轉(zhuǎn)移[5]。因此，本研究應(yīng)用TP、LPS干預(yù)胰腺癌PANC1細(xì)胞，觀察TP對(duì)胰腺癌細(xì)胞侵襲能力的影響，分析其與TLR4/NF-κB信號(hào)通路的關(guān)系。

材料和方法

一、材料

人胰腺癌細(xì)胞株P(guān)ANC1購自美國ATCC公司。TP購自上海融禾醫(yī)藥科技發(fā)展有限公司(純度≥98.0%)。LPS為Sigma公司產(chǎn)品。逆轉(zhuǎn)錄試劑盒和實(shí)時(shí)PCR試劑盒均為TakaRa公司產(chǎn)品。兔抗人TLR4多抗、山羊抗人MMP-9多抗購自Santa Cruz公司。pNF-κB-luc報(bào)告基因質(zhì)粒購自碧云天生物技術(shù)研究所，Dual-luciferase reporter assay system購自Promega公司。Transwell小室購自Coster公司，Matrigel購自BD公司。

二、方法

1．細(xì)胞培養(yǎng)和分組：PANC1細(xì)胞常規(guī)培養(yǎng)、傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞分為親本細(xì)胞組、TP組、LPS組和TP+LPS組。親本組為常規(guī)培養(yǎng)液，TP組在培養(yǎng)液中加入終濃度為50 ng/ml的TP，LPS組加入終濃度為1 μg/ml的LPS，TP+LPS組先用50 ng/ml的TP處理2 h，再加入終濃度為1 μg/ml的LPS。各組細(xì)胞均常規(guī)培養(yǎng)24 h。

2．TLR4、MMP-9蛋白檢測(cè)：收集各組細(xì)胞，提取細(xì)胞總蛋白，采用蛋白質(zhì)印跡法檢測(cè)TLR4、MMP-9蛋白，以β-actin為內(nèi)參，最后ECL發(fā)光，暗室顯影、定影。用凝膠圖像軟件分析系統(tǒng)掃描，以目的條帶與內(nèi)參條帶灰度比值為蛋白相對(duì)表達(dá)量。

3．TLR4、MMP-9 mRNA檢測(cè)：收集各組細(xì)胞，用Trizol抽提細(xì)胞總RNA。TLR4引物上游 5′-CTGCAATGGATCAAGGACCA-3′，下游 5′-TCCCACTCC-AGGTAAGTGTT-3′；MMP-9引物上游5′-GACTCGGTCTTTGAGGAGCG-3′，下游5′-AACTCACGCGCC-AGTAGAA-3′；內(nèi)參β-actin上游5′-CATCCTGCGTCTGGACCT-3′，下游 5′-TCAGGAGGAGCAATGATCTTG-3′。引物由上海生工生物工程技術(shù)服務(wù)有限公司合成。按試劑盒說明逆轉(zhuǎn)錄合成cDNA，再行實(shí)時(shí)定量PCR。通過公式2-△△Ct計(jì)算mRNA表達(dá)量。

4．NF-κB活性檢測(cè)：收集各組細(xì)胞，以1×105/孔細(xì)胞接種于24孔板，通過Lipofectamine 2000將pNF-κB-luc質(zhì)粒0.5 μg及0.1 μg pRL-TK熒光素酶表達(dá)質(zhì)粒同時(shí)轉(zhuǎn)染PANC1細(xì)胞，繼續(xù)培養(yǎng)24 h后收集轉(zhuǎn)染細(xì)胞，使用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)NF-κB的相對(duì)活性，按說明書操作。

5．細(xì)胞侵襲力檢測(cè)：采用Transwell小室檢測(cè)。用無血清DMEM培養(yǎng)液稀釋Matrigel (1∶8)，每個(gè)Transwell小室加入50 μl(低溫條件下進(jìn)行)， 37℃放置l h使Matrigel充分聚合。Transwell下室加入600 μl含10% FBS的DMEM培養(yǎng)液。用無血清的DMEM培養(yǎng)液將各組PANC1細(xì)胞制備成5×105/ml細(xì)胞懸液，取200 μl接種至Transwell上室，常規(guī)培養(yǎng)24 h。取出Transwell小室，用棉簽拭去膜表面的Matrigel，PBS輕輕沖洗，晾干，然后用0.1%結(jié)晶紫染色10 min，去除多余結(jié)晶紫染液，顯微鏡下隨機(jī)選取5個(gè)200倍視野，計(jì)算穿膜細(xì)胞數(shù)，取均值。

三、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、PANC1細(xì)胞TLR4 mRNA和蛋白表達(dá)

親本組、LPS組、TP組、TP+LPS組的TLR4 mRNA表達(dá)量分別為0.41±0.06、0.46±0.10、0.20±0.04和0.25±0.06；TLR4蛋白表達(dá)量分別為0.55±0.06、0.60±0.03、0.18±0.04和0.13±0.00(圖1)。LPS組TLR4 mRNA和蛋白表達(dá)量與親本組無顯著差異(t值分別為0.908、0.947，P值均>0.05)，TP組較親本組的表達(dá)量顯著減少(t值分別為-7.609、-9.948，P值均<0.01)，TP+LPS組的表達(dá)量較LPS組顯著減少(t值分別為-4.437、-14.805，P值均<0.01)。

圖1 各組PANC1細(xì)胞TLR4蛋白的表達(dá)

二、PANC1細(xì)胞的NF-κB活性

親本組、LPS組、TP組、TP+LPS組的NF-κB相對(duì)活性分別為13.0±3.0、31.6±4.3、7.3±1.5和10.8±2.1。LPS組的NF-κB相對(duì)活性較親本組顯著升高(t=8.654，P<0.01)，TP組較親本組顯著降低(t=-4.176，P<0.01)，TP+LPS組較LPS組顯著下降(t=-10.506，P<0.01)。

三、PANC1細(xì)胞的侵襲能力

親本組、LPS組、TP組、TP+LPS組的穿膜細(xì)胞數(shù)分別為(56.8±8.6)、(104.5±12.8)、(32.0±5.7)和(46.8±7.0)個(gè)(圖2)。LPS組顯著多于親本組(t=7.593，P<0.01)，TP組顯著少于親本組(t=-5.915，P<0.01)，TP+LPS組顯著少于LPS組(t=-9.700，P<0.01)。

圖2親本組(a)、TP組(b)、LPS組(c)、TP+LPS組(d)穿膜細(xì)胞(結(jié)晶紫染色 ×200)

四、PANC1細(xì)胞MMP-9 mRNA和蛋白表達(dá)

親本組、LPS組、TP組、TP+LPS組的MMP-9 mRNA表達(dá)量分別為0.36±0.05、0.58±0.07、0.18±0.03和0.30±0.00；MMP-9蛋白表達(dá)量分別為0.31±0.04、0.53±0.08、0.11±0.02和0.15±0.00(圖3)。LPS組顯著高于親本組(t值分別為6.655、4.982，P值均<0.01)，TP組顯著低于親本組(t值分別為-8.179、-9.948，P值均<0.01)，TP+LPS組顯著低于LPS組(t值分別為-9.055、-8.932，P值均<0.01)。

圖3 各組PANC1細(xì)胞MMP-9蛋白表達(dá)

討 論

Toll樣受體(Toll-like receptors, TLRs)是人們?cè)谘芯抗壟咛グl(fā)育過程中發(fā)現(xiàn)的dToll 基因所編碼的一種跨膜受體蛋白， 隨后在人類細(xì)胞表面鑒定出與其同源的Toll樣蛋白，并命名為TLR4。TLR4主要表達(dá)于巨噬細(xì)胞、樹突狀細(xì)胞、淋巴細(xì)胞及自然殺傷細(xì)胞。在外源性LPS或內(nèi)源性配體的刺激下，TLR4能促進(jìn)髓樣分化蛋白88的活化，激活NF-κB，促進(jìn)炎性因子釋放，誘導(dǎo)機(jī)體免疫應(yīng)答效應(yīng)[6]。

近年來的研究發(fā)現(xiàn)，TLR4在腫瘤細(xì)胞普遍高表達(dá)，與腫瘤細(xì)胞的免疫逃逸、侵襲和轉(zhuǎn)移密切相關(guān)[7-9]。研究證實(shí)，經(jīng)LPS刺激后，TLR4可激活NF-κB信號(hào)通路，增強(qiáng)腫瘤細(xì)胞的侵襲能力，而沉默TLR4基因或使用NF-κB信號(hào)通路的阻斷劑均可降低LPS的這一作用[10-11]。但LPS是否能刺激TLR4的表達(dá)尚存在爭(zhēng)議。王玢等[12]的研究表明，在LPS刺激的初期，TLR4表達(dá)增加，但后期則逐漸下降。Sun等[5]報(bào)道，LPS作用后TLR4 mRNA表達(dá)無明顯變化，但細(xì)胞膜表面TLR4分布卻增加，提示LPS能促進(jìn)TLR4的膜轉(zhuǎn)位。本研究結(jié)果顯示，LPS刺激后PANC1細(xì)胞的NF-κB活性明顯升高，侵襲能力顯著增加，但TLR4基因表達(dá)無明顯變化。

MMP-9可降解、破壞靠近腫瘤表面的細(xì)胞外基質(zhì)，使腫瘤細(xì)胞沿著缺失的基底膜向周圍組織浸潤(rùn)。Xu等[8]報(bào)道， TLR4/NF-κB信號(hào)通路的激活可誘導(dǎo)MMP-9表達(dá)，促進(jìn)肺癌細(xì)胞的侵襲。本研究結(jié)果顯示，LPS刺激后PANC1細(xì)胞的MMP-9基因表達(dá)均顯著上調(diào)，表明MMP-9是TLR4/NF-κB信號(hào)通路促進(jìn)胰腺癌侵襲和轉(zhuǎn)移重要的下游蛋白。

由于TLR4/NF-κB信號(hào)通路與腫瘤的發(fā)生與發(fā)展密切相關(guān)，近年來受到了學(xué)者的廣泛關(guān)注[2,13]。我國學(xué)者首先發(fā)現(xiàn)TP對(duì)白血病具有治療作用。之后的研究表明，TP具有廣譜抗腫瘤作用，它可抑制包括胰腺癌在內(nèi)的多種腫瘤細(xì)胞的增殖、促進(jìn)腫瘤細(xì)胞凋亡，并能夠抑制腫瘤細(xì)胞的侵襲和轉(zhuǎn)移[14-16]。但其抗腫瘤機(jī)制尚未完全闡明。本研究結(jié)果表明，TP可抑制TLR4的表達(dá)，降低NF-κB活性，抑制胰腺癌細(xì)胞的侵襲能力，并下調(diào)MMP-9的表達(dá)；經(jīng)TP預(yù)處理后，能夠明顯逆轉(zhuǎn)LPS所引起的癌細(xì)胞侵襲能力的增強(qiáng)和MMP-9的表達(dá)，提示TP可通過抑制TLR4/NF-κB信號(hào)通路、下調(diào)MMP-9的表達(dá)，從而抑制胰腺癌細(xì)胞的侵襲能力。

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TriptolideinhibitedtheinvasionabilityofpancreaticcancercellsthroughTLR4/NF-κBsignalingpathway

MAJian-Xia,SUNYun-liang,WANGYi-Qian,TONGYi-li,YUXiao-Feng.

DepartmentofGastroenterology,HuadongHospital,FudanUniversity,Shanghai200338,China

Correspondingauthor:YUXiao-Feng,Email：yuxiaofeng252@yahoo.cn

ObjectiveTo investigate the role of TLR4/NF-κB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide (TP).MethodsPANC1 cells were divided into parental cells group, TP group, lipopolysaccharide (LPS) group and TP+LPS group. 50 ng/ml of TP was added in culture medium in TP group, and 1 μg/ml of LPS was added in culture medium in LPS group, while 50 ng/ml of TP was pretreated for 2 h and 1 μg/ml of LPS was added in culture medium in TP+LPS group. All the cells were cultured for 24 h. The TLR4 and matrix metalloproteinase-9(MMP-9) mRNA and protein expression were evaluated by real-time PCR and Western blot. The NF-κB activity was determined by dual-luciferase reporter assay system. The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamber assay.ResultsThe TLR4 mRNA expressions in parental cells group, TP group, LPS group and TP+LPS group were 0.41±0.06,0.46±0.10,0.20±0.04, 0.25±0.06; the TLR4 protein expressions were 0.55±0.06,0.55±0.06,0.18±0.04,0.13±0.00; the activities of NF-κB were 13.0±3.0,31.6±4.3,7.3±1.5and 10.8±2.1, and the numbers of invasion cell were (56.8±8.6), (104.5±12.8), (32.0±5.7) and (46.8±7.0); the MMP-9 mRNA expressions were 0.36±0.05,0.58±0.07,0.18±0.03,0.30±0.004; the MMP-9 protein expressions were 0.31±0.04,0.53±0.08,0.11±0.02,0.15±0.00. In LPS group, TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group, but the activities of NF-κB, the numbers of invasion cell, MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group (t=8.654, 7.593, 6.655, 4.982,P<0.01). TLR4 mRNA and protein expressions, activities of NF-κB, the numbers of invasion cell, MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group (t=-7.609,-9.948, -4.176, -5.915, -8.179,-9.948,P<0.01). TLR4 mRNA and protein expressions, activities of NF-κB, the numbers of invasion cell, MMP 9 mRNA and protein expressions in TP+LPS group were significantly lower than those in LPS group (t=-4.437,-14.805,-10.506, -9.700, -9.055, -8.932,P<0.01).ConclusionsTP can inhibit pancreatic cancer cell invasion, and the mechanism is related to the inhibition of TLR4/NF-κB signaling pathway and down-regulation of MMP-9 expression.

Pancreatic neoplasms; Toll-like receptors 4; Triptolide; Matrix metalloproteinases

2013-01-15)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.02.010

復(fù)旦大學(xué)上海醫(yī)學(xué)院青年骨干科研啟動(dòng)基金(11L-3)

200338 上海，上海復(fù)旦大學(xué)附屬華東醫(yī)院消化內(nèi)科(馬建霞、王一倩、童依麗、于曉峰)；贛榆縣人民醫(yī)院消化內(nèi)科(孫運(yùn)良)

于曉峰，Email：yuxiaofeng252@yahoo.cn