蔡篤雄，蘇穎潔，湯凈(海南醫(yī)學(xué)院第一附屬醫(yī)院消化內(nèi)科，海南?？?70102)

鞘鞍醇激酶-1對(duì)胰腺癌SW1990細(xì)胞增殖和凋亡的影響

蔡篤雄，蘇穎潔，湯凈(海南醫(yī)學(xué)院第一附屬醫(yī)院消化內(nèi)科，海南?？?70102)

目的探討鞘鞍醇激酶-1(SPK1)對(duì)胰腺癌SWl990細(xì)胞增殖和凋亡的影響。方法培養(yǎng)的胰腺癌SW1990細(xì)胞株，實(shí)驗(yàn)分為DMS組、PMA組和對(duì)照組，各組細(xì)胞接種于孔板中，每組設(shè)3個(gè)復(fù)孔，每個(gè)實(shí)驗(yàn)至少重復(fù)3次。DMS組加入N,N-二甲基鞘氨醇(DMS，50μmol/L)，PMA組加入佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA，100 nmol/L)，對(duì)照組按常規(guī)培養(yǎng)，采用MTT法和克隆形成實(shí)驗(yàn)檢測細(xì)胞生長增殖的變化，流式細(xì)胞術(shù)檢測細(xì)胞凋亡，RT-PCR檢測SPK1 mRNA的表達(dá)，Western blot檢測SPK1蛋白的表達(dá)。結(jié)果PMA組細(xì)胞的增殖活力[OD值：(1.37±0.03)，細(xì)胞克隆數(shù)目：(292.45±10.68)]較對(duì)照組[OD:(1.00±0.01)，細(xì)胞克隆數(shù)目：(215.34±12.23)]顯著增加，DMS組細(xì)胞的增殖活力[OD值：(0.65±0.02)，細(xì)胞克隆數(shù)目：(130.56±15.6)]較對(duì)照組顯著降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。PMA組細(xì)胞凋亡率[(9.7±0.62)%]較對(duì)照組[(16.71±1.53)%]明顯下降，DMS組細(xì)胞凋亡率[(31.7± 1.32)%]較對(duì)照組明顯升高，差異均有統(tǒng)計(jì)學(xué)意義(P<0.01)，PMA組SPK1 mRNA表達(dá)水平(0.202±0.013)及蛋白表達(dá)水平(0.258±0.015)較對(duì)照組[SPK1 mRNA：(0.148±0.006)，蛋白：(0.182±0.044)]顯著增加，而DMS組SPK1 mRNA表達(dá)水平(0.112±0.022)及SPK1蛋白表達(dá)水平(0.101±0.034)較對(duì)照組顯著降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論SPK1與胰腺癌細(xì)胞的增殖和凋亡密切相關(guān)，SPK1的激活可促進(jìn)胰腺癌細(xì)胞的增殖并抑制細(xì)胞的凋亡。

鞘鞍醇激酶-1；胰腺癌；增殖；凋亡

近年來，分子靶向治療成為胰腺癌治療研究中的熱點(diǎn)，目前國內(nèi)外許多學(xué)者都在探求基因水平的調(diào)控方法和尋找治療的新靶點(diǎn)，以期在胰腺癌的治療上取得突破[1]。近年研究證明鞘氨醇激酶1(sphingosine kinase 1，SPK1)是細(xì)胞增殖及存活的重要調(diào)控因子，SPK1信號(hào)途徑與細(xì)胞的凋亡、增殖和遷移密切相關(guān)[2-3]。本研究旨在研究SPK1對(duì)胰腺癌細(xì)胞增殖和凋亡的影響，探討SPK1是否可能成為胰腺癌分子靶向治療的一個(gè)新型分子，為胰腺癌的基因治療尋找新靶點(diǎn)。

1 材料與方法

1.1 材料人胰腺癌細(xì)胞株SWl990購自中國科學(xué)院上海生命科學(xué)研究院，佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA)購自美國Sigma公司，N,N-二甲基鞘氨醇(DMS)購自美國Sigma公司，四氮唑藍(lán)(MTT)、DMEM完全培養(yǎng)基、1640培養(yǎng)液及胎牛血清購自美國Gibco公司。

1.2 方法

1.2.1 MTT法檢測細(xì)胞增殖活力人胰腺癌SWl990細(xì)胞采用100 mL/L胎兒血清的DMEM完全培養(yǎng)基在5%CO2，37℃的培養(yǎng)箱培養(yǎng)，取對(duì)數(shù)生長期細(xì)胞，按實(shí)驗(yàn)分組分別加入100 nmol/L的PMA(PMA組)及50μmol/L的DMS(DMS組)，對(duì)照組的細(xì)胞用含100 mmol/L胎牛血清的培養(yǎng)基培養(yǎng)，每組設(shè)3個(gè)復(fù)孔，采用MTT法檢測，所有步驟均參照試劑盒說明書進(jìn)行,最后在酶聯(lián)免疫檢測儀570 nm處測量各孔的吸光值(OD值)，反映細(xì)胞增殖活力。

1.2.2 克隆形成實(shí)驗(yàn)檢測細(xì)胞增殖活性將各組細(xì)胞分別培養(yǎng)，收集各組對(duì)數(shù)生長期細(xì)胞，調(diào)整細(xì)胞懸液濃度500個(gè)/mL，分于6個(gè)孔板，每組設(shè)3個(gè)復(fù)孔，培養(yǎng)10～14 d，最后觀察克隆出現(xiàn)日期及生長情況，大于50個(gè)細(xì)胞的集落算一個(gè)克隆，計(jì)數(shù)每個(gè)孔的克隆數(shù)并拍照。

1.2.3 AnnexinV-FITC/PI染色流式細(xì)胞技術(shù)檢測細(xì)胞凋亡率取生長狀態(tài)良好的對(duì)數(shù)生長期的細(xì)胞，接種于6個(gè)孔板，每組接種3孔，各組細(xì)胞經(jīng)處理后采用0.25%胰酶消化單層細(xì)胞，800 r/min離心，冷磷酸鹽緩沖液(PBS)洗兩次，離心棄上清，最后用1× annexin-binding buffer重懸細(xì)胞，將細(xì)胞密度調(diào)整為約1×106細(xì)胞/mL，取100μL體積為待測樣本，加5μL Alexa Fluor?488 annexin V樣本中。室溫卵育細(xì)胞15 min后，加400μL 1×annexin-binding buffer，及1μL 100 μg/mL PI到待測細(xì)胞樣本，混勻。流式細(xì)胞儀檢測細(xì)胞凋亡率。

1.2.4 PMA和DMS對(duì)SPK1 mRNA表達(dá)的影響各組細(xì)胞繼續(xù)培養(yǎng)24 h，提取細(xì)胞總RNA，采用RT-PCR法；引物序列如下：SPKl：正義：5'-CCGACGAGGACTITGTGCTAGT-3'，反義：5'-GCCTGTCCC CCCAAAGCAIAGC-3'；GAPDH：正義：5'-AATCCCA TCACCATCTTCCA-3'，反義：5'-CCTGCTrCACCAC CTTCTTG-3'。PCR產(chǎn)物電泳后用凝膠成相儀掃描分析。以目的條帶SPKl與GAPDH的灰度比值表示SPK1 mRNA的相對(duì)表達(dá)量。

1.2.5 PMA和DMS對(duì)SPK1蛋白表達(dá)的影響各組細(xì)胞培養(yǎng)24 h后提取總蛋白，采用Western blot檢測SPK1蛋白的表達(dá)，所有步驟均參照試劑盒說明書進(jìn)行，用Kodak攝像系統(tǒng)拍照。Image J軟件分析蛋白的灰度值，以SPKl與β-actin的灰度比值表示SPKl蛋白的相對(duì)表達(dá)水平。

2 結(jié)果

2.1 PMA和DMS對(duì)SWl990細(xì)胞增殖活力的影響對(duì)照組、PMA組及DMS組OD值分別為：(1.00± 0.01)、(1.37±0.03)和(0.65±0.02)，PMA組OD值明顯高于對(duì)照組(t=3.75，P<0.05)，DMS組OD值明顯低于對(duì)照組，差異均具有統(tǒng)計(jì)學(xué)意義(t=4.12，P<0.05)(表1，圖1)。對(duì)照組、PMA組及DMS組克隆形成數(shù)目分別為：(215.34±12.23)、(292.45±10.68)、(130.56±15.6)，PMA組的克隆形成數(shù)目明顯高于對(duì)照組(t=2.62，P< 0.05)，DMS組的克隆形成數(shù)目明顯低于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(t=2.84，P<0.05)，見表1、圖2。

2.2 PMA和DMS對(duì)SWl990細(xì)胞凋亡的影響對(duì)照組、PMA組、DMS組細(xì)胞凋亡率分別為(16.71± 1.53)%、(9.7±0.62)%和(31.7±1.32)%。PMA組細(xì)胞凋亡率明顯低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(t=13.04，P< 0.01)，DMS細(xì)胞凋亡率明顯高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(t=14.12，P<0.01)，見表1、圖3。

2.3 各組SPK1 mRNA的表達(dá)對(duì)照組、PMA組及DMS組的SPK1 mRNA表達(dá)強(qiáng)度分別為(0.148± 0.006)，(0.202±0.013)，(0.112±0.022），PMA組SPK1 mRNA表達(dá)較對(duì)照組顯著增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(t= 5.46，P<0.05)，而DMS組SPK1 mRNA表達(dá)較對(duì)照組明顯降低，差異有統(tǒng)計(jì)學(xué)意義(t=5.75，P<0.05)，見表1、圖4。

圖1 各組細(xì)胞的增殖活性與對(duì)照組比較，aP<0.05。

圖2 各組細(xì)胞的克隆形成數(shù)目與對(duì)照組比較，aP<0.05。

圖3 PMA和DMS對(duì)SWl990細(xì)胞凋亡的影響

表1 各組細(xì)胞OD值、細(xì)胞克隆形成數(shù)目、細(xì)胞凋亡率、SPK1/GADPH及SPK1/β-actin比較

表1 各組細(xì)胞OD值、細(xì)胞克隆形成數(shù)目、細(xì)胞凋亡率、SPK1/GADPH及SPK1/β-actin比較

注：與對(duì)照組比較，aP<0.05，bP<0.01。

組別對(duì)照組PMA組DMS組OD值1.00±0.01 1.37±0.03a0.65±0.02a細(xì)胞克隆形成數(shù)目215.34±12.23 292.45±10.68a130.56±15.6a細(xì)胞凋亡率(%) 16.71±1.53 9.70±0.62b31.74±1.32bSPK1/GADPH 0.148±0.006 0.202±0.013a0.112±0.022aSPK1/β-actin 0.182±0.044 0.258±0.015a0.101±0.034a

圖4 各組SPK1 mRNA的表達(dá)

2.4 各組SPK1蛋白的表達(dá)對(duì)照組、PMA組及DMS組SPK1蛋白表達(dá)強(qiáng)度分別為(0.182±0.044)、(0.258±0.015)、(0.101±0.034)，PMA組SPK1蛋白表達(dá)較對(duì)照組顯著增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(t=6.16，P< 0.05)，而DMS組SPK1蛋白表達(dá)較對(duì)照組明顯降低，差異有統(tǒng)計(jì)學(xué)意義(t=-5.83，P<0.05)，見表1、圖5。

圖5 各組SPK1蛋白的表達(dá)

3 討論

近年研究證明細(xì)胞膜鞘磷脂的衍生物包括神經(jīng)酰胺(Cer)、鞘氨醇(Sph)、1-磷酸鞘氨醇(S1P)等多種代謝產(chǎn)物,在腫瘤發(fā)生發(fā)展過程中發(fā)揮著極為重要的作用。研究證實(shí)Cer是一種細(xì)胞增殖負(fù)調(diào)控因子，抑制細(xì)胞生長、促進(jìn)細(xì)胞凋亡，而其轉(zhuǎn)化生成的代謝物S1P則抑制細(xì)胞凋亡、促進(jìn)細(xì)胞增殖[4]。鞘氨醇激酶(SPK)是細(xì)胞內(nèi)合成S1P的主要限速酶，其中SPK1的作用最為關(guān)鍵。SPK1的活性受抑會(huì)導(dǎo)致細(xì)胞內(nèi)Cer、Sph水平升高，S1P水平降低，導(dǎo)致細(xì)胞生長抑制或誘導(dǎo)凋亡的產(chǎn)生，Li等[5]研究發(fā)現(xiàn)應(yīng)用SPK1抑制劑可抑制人胃癌細(xì)胞的生長并促進(jìn)細(xì)胞的凋亡，因此，SPK1的表達(dá)及活性高低直接決定著細(xì)胞的命運(yùn)，SPK1也是細(xì)胞增殖及存活的重要調(diào)控因子。

近年來，有關(guān)SPK1在腫瘤方面的研究越來越多，目前國內(nèi)外研究主要集中在胃癌、結(jié)腸癌、鼻咽癌、肝癌、白血病等方面[6-9]，但有關(guān)SPK1與胰腺癌的研究較少，Aoki等[10]研究發(fā)現(xiàn)SPK1的激活可通過促進(jìn)S1P的生成從而促進(jìn)胰腺癌細(xì)胞的腹膜轉(zhuǎn)移。Gong等[11]研究發(fā)現(xiàn)應(yīng)用SPK1抑制劑可通過促進(jìn)神經(jīng)酰胺的生成從而促進(jìn)胰腺癌細(xì)胞的凋亡，Guillermet-Guibert等[12]研究發(fā)現(xiàn)，應(yīng)用SPK1抑制劑可通過上調(diào)神經(jīng)酰胺表達(dá)水平或降低SPK1活性而增加吉西他濱對(duì)胰腺癌細(xì)胞的化療敏感性，因而提示SPK1可能成為一種腫瘤靶向治療的新型分子。

本研究通過運(yùn)用PMA和DMS來調(diào)節(jié)SPK1的活性和表達(dá)，研究SPK1對(duì)胰腺癌SWl990細(xì)胞增殖和凋亡的影響，發(fā)現(xiàn)PMA組細(xì)胞的增殖活力顯著增加，細(xì)胞凋亡率明顯下降，SPK1 mRNA及SPK1蛋白表達(dá)水平顯著增加，DMS組細(xì)胞的增殖活力顯著降低，細(xì)胞凋亡率明顯升高，SPK1 mRNA及SPK1蛋白表達(dá)水平顯著降低，表明激活SPK1能顯著促進(jìn)胰腺癌細(xì)胞的增殖并抑制細(xì)胞的凋亡，抑制SPK1顯著抑制胰腺癌細(xì)胞的增殖并促進(jìn)細(xì)胞的凋亡，SPK1可調(diào)控胰腺癌SWl990細(xì)胞的增殖和凋亡。

綜上所述，SPK1與胰腺癌細(xì)胞的增殖和凋亡密切相關(guān)，SPK1的激活能顯著促進(jìn)胰腺癌細(xì)胞的增殖并抑制細(xì)胞的凋亡，而抑制SPK1顯著抑制胰腺癌細(xì)胞的增殖并促進(jìn)細(xì)胞的凋亡，通過調(diào)控SPK1活性可影響胰腺癌SWl990細(xì)胞的增殖和凋亡。因此，SPK1有可能成為胰腺癌分子靶向治療的一個(gè)新型分子。

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Effect of sphingosine kinase 1 on the proliferation and apoptosis in human pancreatic cancer cell line SW1990.

CAI Du-xiong,SU Ying-jie,TANG Jing.Department of Gastroenterology,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,Hainan,CHINA

ObjectiveTo investigate the effect of sphingosine kinase 1(SPK1)on the proliferation and apoptosis of pancreatic cancer line SW1990.MethodsCultured SW1990 cell were divided into three groups:PMA group, DMS group and the control group.The cells of each group were seeded in the orifice plate,with 3 wells in each group,at least 3 replicates for each experiment.The cells of the PMA group were treated with 100 nmol/L of phorbol 12-myristate13-acetate(PMA);the DMS group was treated with 50μmol/L N,N-dimethylsphingosine(DMS);while the control group was cultured routinely.After the treatment,cell proliferation was determined by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay and colony formation assay,cell apoptosis was detected by flow cytometry,and mRNA and protein expression of SPK1 were detected by RT-PCR and Western blot.ResultsThe cell viability in PMAgroup was the OD value of(1.37±0.03)and the number of cell clones of(292.45±10.68),which was significantly higher than the OD value of(1.00±0.01)and the number of cell clones of(215.34±12.23)in the control group(P< 0.05).The cell viability in DMS group was the OD value of(0.65±0.02)and the number of cell clones of(130.56±15.6), which was significantly higher than that in the control group(P<0.05).The apoptosis rate in PMA group was(9.7± 0.62)%,which was significantly lower than(16.71±1.53)%in the control group,and the apoptosis rate in DMS group was(31.7±1.32)%,which was significantly higher than that in the control group(P<0.01).The mRNA and protein expression of SPK1 in PMA group were respectively(0.202±0.013)and(0.258±0.015),which were significantly higher than(0.148±0.006)and(0.182±0.044)in the control group,and the mRNA and protein expression of SPK1 in DMS group were(0.112±0.022)and(0.101±0.034),which were significantly lower than those in the control group(P<0.05).ConclusionSPK1 is closely related to the proliferation and apoptosis of pancreatic cancer cells.The activation of SPK1 can promote the proliferation of pancreatic cancer cells and inhibit the apoptosis.

Sphingosine kinase 1(SPK1);Pancreatic cancer;Proliferation;Apoptosis

10.3969/j.issn.1003-6350.2017.10.002

R735.9

A

1003—6350（2017）10—1552—04

2017-01-06)