花姜酮對(duì)胰腺癌PANC1細(xì)胞株增殖和凋亡的影響

陳曉燕 王衛(wèi)星 丁佑銘 殷濤 阿布力克木·阿布力孜

·論著·

花姜酮對(duì)胰腺癌PANC1細(xì)胞株增殖和凋亡的影響

陳曉燕 王衛(wèi)星 丁佑銘 殷濤 阿布力克木·阿布力孜

目的探討花姜酮對(duì)胰腺癌PANC1細(xì)胞增殖和凋亡的影響，探討其作用機(jī)制。方法應(yīng)用3.75、7.5、15、30、60 μg/ml的花姜酮處理PANC1細(xì)胞，以未處理細(xì)胞作為對(duì)照。CCK-8法檢測(cè)細(xì)胞增殖抑制率，Hoechst33342染色觀察細(xì)胞形態(tài)，流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率，Western blotting法檢測(cè)細(xì)胞磷酸化STAT1(p-STAT1)、Bax和Bcl-2蛋白的表達(dá)。結(jié)果花姜酮呈時(shí)間-劑量依賴性抑制PANC1細(xì)胞的生長(zhǎng)，15 μg/ml花姜酮作用48 h后，細(xì)胞增殖抑制率達(dá)(72.8±2.72)%，并可觀察到典型的細(xì)胞凋亡形態(tài)學(xué)改變，細(xì)胞凋亡率達(dá)14.2%；同時(shí)，PANC1細(xì)胞p-STAT1和Bax蛋白表達(dá)明顯增加(0.654±0.048對(duì)0.074±0.011，0.577±0.044對(duì)0.218±0.027，P<0.05)，Bcl-2蛋白表達(dá)明顯下降(0.162±0.029 對(duì) 0.459±0.034,P<0.05)。結(jié)論花姜酮可能通過上調(diào)STAT1活性，升高Bax/Bcl-2比率，從而誘導(dǎo)PANC1細(xì)胞的凋亡和抑制細(xì)胞增殖。

胰腺腫瘤； 轉(zhuǎn)錄激活因子1； 花姜酮； 細(xì)胞凋亡

花姜酮是一種從球姜的根莖中分離出的倍半萜類化合物，已被證明對(duì)結(jié)腸癌、皮膚癌均具有好的化學(xué)預(yù)防作用[1]。聯(lián)合應(yīng)用花姜酮和順鉑也可延緩宮頸上皮瘤樣病變向?qū)m頸癌發(fā)展的進(jìn)程[2]。新近研究發(fā)現(xiàn)，花姜酮參與調(diào)控胰腺癌細(xì)胞內(nèi)某些信號(hào)轉(zhuǎn)導(dǎo)通路。信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子1(signal transducer and activator of transcription 1，STAT1)信號(hào)通路是近年來發(fā)現(xiàn)的比較重要的信號(hào)通路，具有調(diào)控細(xì)胞生長(zhǎng)、分化等多種生理功能。本研究觀察花姜酮對(duì)PANC1細(xì)胞的增殖、凋亡以及STAT1活性的影響，探討其可能的機(jī)制。

材料與方法

一、細(xì)胞培養(yǎng)

胰腺癌細(xì)胞系PANC1由中國(guó)科學(xué)院上海細(xì)胞生物研究所細(xì)胞庫(kù)提供。常規(guī)培養(yǎng)傳代。取對(duì)數(shù)生長(zhǎng)期的PANC1細(xì)胞進(jìn)行實(shí)驗(yàn)。每個(gè)濃度設(shè)5個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

二、CCK-8法檢測(cè)細(xì)胞增殖抑制率

CCK-8細(xì)胞計(jì)數(shù)試劑盒購(gòu)自日本同仁化學(xué)公司。以每孔5×107個(gè)細(xì)胞接種于96孔板，培養(yǎng)24 h。換含15%胎牛血清的DMEM培養(yǎng)48 h。分別加入0、3.75、7.5、15、30、60 μg/ml的花姜酮繼續(xù)培養(yǎng)12、24、48 h，以無細(xì)胞孔作為空白對(duì)照。按說明書操作。抑制率=(空白對(duì)照A450值-實(shí)驗(yàn)組A450值)/(空白對(duì)照A450值-對(duì)照組A450值)×100%。

三、Hoechst33342染色觀察細(xì)胞核形態(tài)的變化

細(xì)胞接種于24孔細(xì)胞培養(yǎng)板培養(yǎng)24 h，加入0、7.5、15 μg/ml花姜酮繼續(xù)培養(yǎng)48 h。吸棄半量培養(yǎng)液，加入Hoechst33342，終濃度為10 μg/ml，室溫避光作用10 min，棄上清培養(yǎng)液。于熒光倒置顯微鏡下觀察、攝片。

四、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率

細(xì)胞接種于6孔板培養(yǎng)24 h，加入0、7.5、15 μg/ml花姜酮繼續(xù)培養(yǎng)48 h。收集各孔細(xì)胞，分別制成單細(xì)胞懸液，加入5 μl Annexin V-FITC和10 μl PI，室溫避光5 min，然后上流式細(xì)胞儀檢測(cè)。

五、Western blotting檢測(cè)細(xì)胞p-STAT1、 Bax和Bcl-2蛋白表達(dá)水平

分別收集0、7.5、15 μg/ml花姜酮處理48 h的細(xì)胞，提取蛋白。取100 μg常規(guī)行Western blotting，最后ECL化學(xué)發(fā)光，X線膠片曝光，凝膠成像系統(tǒng)掃描，以目的條帶與actin條帶灰度值的比值代表蛋白的表達(dá)水平。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、花姜酮對(duì)PANC1細(xì)胞增殖的抑制作用

花姜酮呈濃度和時(shí)間依賴性抑制PANC1細(xì)胞的增殖 (表1)。

表1 花姜酮對(duì)PANC1細(xì)胞增殖的影響

二、花姜酮對(duì)PANC1細(xì)胞凋亡的影響

PANC1細(xì)胞核經(jīng)Hoechst33258染色呈圓形，淡藍(lán)色，內(nèi)有較深的藍(lán)色顆粒。花姜酮處理后，細(xì)胞數(shù)減少，出現(xiàn)較多核固縮或呈分葉狀的凋亡細(xì)胞。隨著藥物濃度的升高，凋亡細(xì)胞的數(shù)也增加(圖1)。對(duì)照組PANC1細(xì)胞凋亡率1.1%，7.5 μg/ml和15 μg/ml花姜酮處理組細(xì)胞凋亡率分別為3.9%和14.2%，三組間差異顯著(P<0.01，圖1)。

圖10(a，d)、7.5(b，e)、15(c，f) μg/ml花姜酮處理48 h后PANC1細(xì)胞的形態(tài)改變(Hoechst33258染色 ×200)及細(xì)胞凋亡率(流式細(xì)胞儀)

三、花姜酮對(duì)PANC1細(xì)胞p-STAT1、 Bax和Bcl-2蛋白表達(dá)的影響

花姜酮處理后細(xì)胞p-STAT1和Bax蛋白表達(dá)水平明顯升高；而Bcl-2蛋白表達(dá)水平明顯降低(P<0.05,表2、圖2)。

表2 各組p-STAT1、Bax、Bcl-2蛋白的表達(dá)

注：與對(duì)照組比較，aP<0.05；與7.5 μg/ml組比較，bP<0.05

圖2不同濃度花姜酮處理48 h后PANC1細(xì)胞p-STAT1、Bax和Bcl-2蛋白的表達(dá)(Western blotting)

討 論

花姜酮具有抗炎、抗腫瘤等多種生物活性，本研究結(jié)果也證明花姜酮能夠明顯抑制胰腺癌細(xì)胞的生長(zhǎng)。有研究證實(shí)，花姜酮能通過抑制NF-κB及其相關(guān)基因的表達(dá)，從而起到預(yù)防和治療癌癥的作用[3]。Sakinah等[4]發(fā)現(xiàn)，花姜酮能夠通過調(diào)節(jié)Bax/Bcl-2比率來誘導(dǎo)肝癌細(xì)胞凋亡[5]。

STAT是一組能與DNA結(jié)合的蛋白質(zhì)，介導(dǎo)多種細(xì)胞因子和生長(zhǎng)因子向細(xì)胞核內(nèi)傳導(dǎo)，影響靶基因的轉(zhuǎn)錄，調(diào)節(jié)人體免疫反應(yīng)、炎癥反應(yīng)和細(xì)胞的生長(zhǎng)、分化等[5]。大量研究證明，通過增強(qiáng)或抑制STAT的活性，可影響多種癌細(xì)胞系的增殖和凋亡[6-8]。其成員STAT1在多種腫瘤組織中呈現(xiàn)低表達(dá)趨勢(shì)[9-10]。通過藥物刺激上調(diào)STAT1水平能起到促進(jìn)胰腺癌細(xì)胞凋亡和增強(qiáng)癌細(xì)胞化療敏感性的作用[11-12]。本研究結(jié)果顯示，隨著花姜酮濃度的升高，發(fā)生凋亡的細(xì)胞明顯增多，同時(shí)磷酸化STAT1和Bax蛋白表達(dá)顯著增強(qiáng)， Bcl-2表達(dá)降低。因此我們推測(cè)，花姜酮通過激活STAT1信號(hào)轉(zhuǎn)導(dǎo)通路進(jìn)而影響凋亡相關(guān)蛋白Bax和Bcl-2的表達(dá)，促進(jìn)胰腺癌細(xì)胞凋亡。

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2010-03-17)

(本文編輯：屠振興)

EffectsofzerumboneontheproliferationandapoptosisofhumanpancreaticcancercelllinePANC1

CHENXiao-yan,WANGWei-xing,DINGYou-ming,YINTao,ABLIZAblikim.

DepartmentofHepatobiliaryandLaparoscopicSurgery,RenminHospital，WuhanUniversity,Wuhan430060,China

WANGWei-xing,Email:sate.llite@163.com

ObjectiveTo investigate the effect of zerumbone on the proliferation and apoptosis of human pancreatic cancer cell line PANC1 and its possible mechanism.MethodsZerumbone of various concentrations (3.75, 7.5, 15, 30, 60 μg/ml) were used to treat PANC1, and cells without treatment were used as control. CCK-8 assay was used to detect the inhibitory rate of cell proliferation. Cell apoptosis analysis was determined by using Hoechst 33342 staining and flow cytometry. Western blotting was performed to evaluate the phosphorylation Stat1 (p-STAT1), and Bax and Bcl2 protein expression.ResultsZerumbone caused a time- and dose-dependent reduction of cell viability in PANC1 cells. After 48h treatment of Zerumbone of 15 μg/ml, cells inhibitory rate was increased to (72.8±2.72)%, and classic apoptosis morphology was observed, with apoptosis rate was 14.2%. At the same time, p-STAT1, and Bax protein expression was significantly increased (0.654±0.048vs0.074±0.011, 0.577±0.044vs0.218±0.027,P<0.05); Bcl-2 protein expression was significantly decreased (0.162±0.029vs0.459±0.034,P<0.05).ConclusionsZerumbone may inhibit the proliferation of PANC1 cells and inducing cell apoptosis, which may be related to the up-regulation of STAT1′s activity and Bcl-2/Bax ratio.

Pancreatic neoplasms; Activator transcription factor 1; Zerumbone; Apoptosis

10.3760/cma.j.issn.1674-1935.2010.06.014

430060 武漢，武漢大學(xué)人民醫(yī)院肝膽外科

王衛(wèi)星，Email: sate.llite@163.com