雷公藤內酯醇對胰腺癌PANC1細胞Toll樣受體4/核因子-κB信號通路的影響

馬建霞 孫運良 王一倩 童依麗 于曉峰

·論著·

雷公藤內酯醇對胰腺癌PANC1細胞Toll樣受體4/核因子-κB信號通路的影響

馬建霞 孫運良 王一倩 童依麗 于曉峰

目的探討雷公藤內酯醇(TP)對人胰腺癌PANC1細胞株侵襲能力的影響及其與Toll樣受體4/核因子-κB(TLR4/NF-κB)信號通路的關系。方法將PANC1細胞分為親本細胞組、TP組、脂多糖(LPS)組和TP+LPS組。TP組培養液中加入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。各組細胞均常規培養24 h。采用實時定量PCR和蛋白質印跡法檢測TLR4、基質金屬蛋白酶-9(MMP-9) mRNA和蛋白的表達，雙熒光素酶報告基因系統檢測NF-κB活性，Transwell小室檢測細胞侵襲能力。結果親本組、LPS組、TP組、TP+LPS組的TLR4 mRNA表達量分別為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；穿膜細胞數分別為(56.8±8.6)、(104.5±12.8)、(32.0±5.7)和(46.8±7.0)個；MMP-9 mRNA表達量分別為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和蛋白表達量與親本組差異無統計學意義，但NF-κB活性、穿膜細胞數、MMP-9 mRNA和蛋白表達量顯著高于親本組(t值分別為8.654、7.593、6.655、4.982，P值均<0.01)。TP組TLR4 mRNA和蛋白表達量、NF-κB活性、穿膜細胞數、MMP-9 mRNA和蛋白表達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和蛋白表達量、NF-κB活性、穿膜細胞數、MMP-9 mRNA和蛋白表達量均顯著低于LPS組(t值分別為-4.437、-14.805、-10.506、-9.700、-9.055、-8.932，P值均<0.01)。結論TP具有抑制胰腺癌細胞侵襲的作用，其機制與抑制TLR4/NF-κB信號通路、下調MMP-9的表達有關。

胰腺腫瘤； Toll樣受體4； 雷公藤內酯； 基質金屬蛋白酶類

雷公藤內酯醇(triptolide，TP)是從衛茅科植物雷公藤中抽提到的二萜內酯化合物。近年來研究發現，TP可抑制結腸癌、前列腺癌等多種腫瘤細胞生長、侵襲和轉移[1-3]，但其作用機制尚未完全闡明。Toll樣受體4(Toll-like receptor 4, TLR4)是最早發現的、同時也是研究最為廣泛的Toll樣受體家族成員。研究已證實，TLR4在多種腫瘤細胞中呈高表達，參與腫瘤的發生與發展[4]。TLR4在其配體脂多糖(1ipopolysaccharide，LPS)的刺激下，可激活核因子-κB (NF-κB)，誘導腫瘤細胞分泌多種細胞因子和炎癥介質，促進腫瘤的侵襲和轉移[5]。因此，本研究應用TP、LPS干預胰腺癌PANC1細胞，觀察TP對胰腺癌細胞侵襲能力的影響，分析其與TLR4/NF-κB信號通路的關系。

材料和方法

一、材料

人胰腺癌細胞株PANC1購自美國ATCC公司。TP購自上海融禾醫藥科技發展有限公司(純度≥98.0%)。LPS為Sigma公司產品。逆轉錄試劑盒和實時PCR試劑盒均為TakaRa公司產品。兔抗人TLR4多抗、山羊抗人MMP-9多抗購自Santa Cruz公司。pNF-κB-luc報告基因質粒購自碧云天生物技術研究所，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．細胞培養和分組：PANC1細胞常規培養、傳代。取對數生長期細胞分為親本細胞組、TP組、LPS組和TP+LPS組。親本組為常規培養液，TP組在培養液中加入終濃度為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。各組細胞均常規培養24 h。

2．TLR4、MMP-9蛋白檢測：收集各組細胞，提取細胞總蛋白，采用蛋白質印跡法檢測TLR4、MMP-9蛋白，以β-actin為內參，最后ECL發光，暗室顯影、定影。用凝膠圖像軟件分析系統掃描，以目的條帶與內參條帶灰度比值為蛋白相對表達量。

3．TLR4、MMP-9 mRNA檢測：收集各組細胞，用Trizol抽提細胞總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服務有限公司合成。按試劑盒說明逆轉錄合成cDNA，再行實時定量PCR。通過公式2-△△Ct計算mRNA表達量。

4．NF-κB活性檢測：收集各組細胞，以1×105/孔細胞接種于24孔板，通過Lipofectamine 2000將pNF-κB-luc質粒0.5 μg及0.1 μg pRL-TK熒光素酶表達質粒同時轉染PANC1細胞，繼續培養24 h后收集轉染細胞，使用雙熒光素酶報告基因系統檢測NF-κB的相對活性，按說明書操作。

5．細胞侵襲力檢測：采用Transwell小室檢測。用無血清DMEM培養液稀釋Matrigel (1∶8)，每個Transwell小室加入50 μl(低溫條件下進行)， 37℃放置l h使Matrigel充分聚合。Transwell下室加入600 μl含10% FBS的DMEM培養液。用無血清的DMEM培養液將各組PANC1細胞制備成5×105/ml細胞懸液，取200 μl接種至Transwell上室，常規培養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%結晶紫染色10 min，去除多余結晶紫染液，顯微鏡下隨機選取5個200倍視野，計算穿膜細胞數，取均值。

三、統計學處理

結 果

一、PANC1細胞TLR4 mRNA和蛋白表達

親本組、LPS組、TP組、TP+LPS組的TLR4 mRNA表達量分別為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細胞TLR4蛋白的表達

二、PANC1細胞的NF-κB活性

親本組、LPS組、TP組、TP+LPS組的NF-κB相對活性分別為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細胞的侵襲能力

親本組、LPS組、TP組、TP+LPS組的穿膜細胞數分別為(56.8±8.6)、(104.5±12.8)、(32.0±5.7)和(46.8±7.0)個(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)穿膜細胞(結晶紫染色 ×200)

四、PANC1細胞MMP-9 mRNA和蛋白表達

親本組、LPS組、TP組、TP+LPS組的MMP-9 mRNA表達量分別為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細胞MMP-9蛋白表達

討 論

Toll樣受體(Toll-like receptors, TLRs)是人們在研究果蠅胚胎發育過程中發現的dToll 基因所編碼的一種跨膜受體蛋白， 隨后在人類細胞表面鑒定出與其同源的Toll樣蛋白，并命名為TLR4。TLR4主要表達于巨噬細胞、樹突狀細胞、淋巴細胞及自然殺傷細胞。在外源性LPS或內源性配體的刺激下，TLR4能促進髓樣分化蛋白88的活化，激活NF-κB，促進炎性因子釋放，誘導機體免疫應答效應[6]。

近年來的研究發現，TLR4在腫瘤細胞普遍高表達，與腫瘤細胞的免疫逃逸、侵襲和轉移密切相關[7-9]。研究證實，經LPS刺激后，TLR4可激活NF-κB信號通路，增強腫瘤細胞的侵襲能力，而沉默TLR4基因或使用NF-κB信號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的表達尚存在爭議。王玢等[12]的研究表明，在LPS刺激的初期，TLR4表達增加，但后期則逐漸下降。Sun等[5]報道，LPS作用后TLR4 mRNA表達無明顯變化，但細胞膜表面TLR4分布卻增加，提示LPS能促進TLR4的膜轉位。本研究結果顯示，LPS刺激后PANC1細胞的NF-κB活性明顯升高，侵襲能力顯著增加，但TLR4基因表達無明顯變化。

MMP-9可降解、破壞靠近腫瘤表面的細胞外基質，使腫瘤細胞沿著缺失的基底膜向周圍組織浸潤。Xu等[8]報道， TLR4/NF-κB信號通路的激活可誘導MMP-9表達，促進肺癌細胞的侵襲。本研究結果顯示，LPS刺激后PANC1細胞的MMP-9基因表達均顯著上調，表明MMP-9是TLR4/NF-κB信號通路促進胰腺癌侵襲和轉移重要的下游蛋白。

由于TLR4/NF-κB信號通路與腫瘤的發生與發展密切相關，近年來受到了學者的廣泛關注[2,13]。我國學者首先發現TP對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的多種腫瘤細胞的增殖、促進腫瘤細胞凋亡，并能夠抑制腫瘤細胞的侵襲和轉移[14-16]。但其抗腫瘤機制尚未完全闡明。本研究結果表明，TP可抑制TLR4的表達，降低NF-κB活性，抑制胰腺癌細胞的侵襲能力，并下調MMP-9的表達；經TP預處理后，能夠明顯逆轉LPS所引起的癌細胞侵襲能力的增強和MMP-9的表達，提示TP可通過抑制TLR4/NF-κB信號通路、下調MMP-9的表達，從而抑制胰腺癌細胞的侵襲能力。

[1] Johnson SM, Wang X, Evers BM.Triptolide inhibits proliferation and migration of colon cancer cells by inhibition of cell cycle regulators and cytokine receptors. J Surg Res, 2011, 168:197-205.

[2] Zhu W, Ou Y, Li Y, et al. A small-molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway. Mol Pharmacol, 2009,75:812-819.

[3] Tang XY, Zhu YQ, Tao WH, et al. Synergistic effect of triptolide combined with 5-fluorouracil on colon carcinoma. Postgrad Med J, 2007, 83:338-343.

[4] Rakoff-Nahoum S, Medzhitov R.Toll-like receptors and cancer. Nat Rev Cancer, 2009, 9:57-63.

[5] Sun Q, Liu Q, Zheng Y, et al. Rapamycin suppresses TLR4-triggered IL-6 and PGE(2) production of colon cancer cells by inhibiting TLR4 expression and NF-kappaB activation. Mol Immunol, 2008,45:2929-2936.

[6] O′Neill LA, Bryant CE, Doyle SL.Therapeutic targeting of Toll-like receptors for infectious and inflammatory diseases and cancer. Pharmacol Rev, 2009, 61:177-197.

[7] Hsu RY, Chan CH, Spicer JD, et al.LPS-induced TLR4 signaling in human colorectal cancer cells increases beta1 integrin-mediated cell adhesion and liver metastasis. Cancer Res, 2011, 71:1989-1998.

[8] Xu Z, Ren T, Xiao C, et al. Nickel promotes the invasive potential of human lung cancer cells via TLR4/MyD88 signaling. Toxicology, 2011, 285:25-30.

[9] Szczepanski MJ, Czystowska M, Szajnik M, et al.Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res, 2009, 69:3105-3113.

[10] Hua D, Liu MY, Cheng ZD, et al.Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion, survival, and tumorigenicity. Mol Immunol, 2009, 46: 2876-2884.

[11] Ikebe M, Kitaura Y, Nakamura M, et al. Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway. J Surg Oncol, 2009, 100:725-731.

[12] 王玢，黃玉政，周永華，等.LPS刺激誘導人卵巢癌細胞株Toll樣受體4表達及細胞免疫調節.中華微生物和免疫學雜志，2010，30：154-158.

[13] Jung ID, Jeong SK, Lee CM, et al. Enhanced efficacy of therapeutic cancer vaccines produced by co-treatment with Mycobacterium tuberculosis heparin-binding hemagglutinin, a novel TLR4 agonist. Cancer Res, 2011, 71:2858-2870.

[14] Mujumdar N, Mackenzie TN, Dudeja V, et al. Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways. Gastroenterology, 2010, 139:598-608.

[15] Li J, Zhu W, Leng T, et al. Triptolide-induced cell cycle arrest and apoptosis in human renal cell carcinoma cells. Oncol Rep, 2011, 25:979-987.

[16] Phillips PA, Dudeja V, McCarroll JA, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res, 2007, 67:9407-9416.

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

復旦大學上海醫學院青年骨干科研啟動基金(11L-3)

200338 上海，上海復旦大學附屬華東醫院消化內科(馬建霞、王一倩、童依麗、于曉峰)；贛榆縣人民醫院消化內科(孫運良)

于曉峰，Email：yuxiaofeng252@yahoo.cn