謝冬冰,孟建宇,郭玉婷,任霞,李雪
MK801對(duì)結(jié)腸癌細(xì)胞增殖、凋亡和遷移的影響
謝冬冰1,孟建宇2,郭玉婷3Δ,任霞4,李雪1
目的探究N-甲基-D-天冬氨酸受體亞型1(NMDAR1)在結(jié)腸癌細(xì)胞HT29和SW116中的表達(dá),以及NMDAR1拮抗劑MK801對(duì)HT-29和SW116細(xì)胞生長(zhǎng)抑制、凋亡和遷移的影響。方法采用免疫組織化學(xué)法檢測(cè)結(jié)腸癌細(xì)胞HT-29和SW116細(xì)胞表面NMDAR1的表達(dá);應(yīng)用噻唑藍(lán)(MTT)比色法測(cè)定62.5、125.0、250.0、500.0、1 000.0、2 000.0 μmol/L的MK801對(duì)于HT-29和SW116細(xì)胞增殖作用的影響;應(yīng)用流式細(xì)胞術(shù)檢測(cè)2 000 μmol/L的MK801對(duì)HT29和SW116細(xì)胞凋亡的影響;應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)50 μmol/L MK801對(duì)于結(jié)腸癌細(xì)胞HT-29和SW116遷移能力的影響。結(jié)果結(jié)腸癌細(xì)胞HT-29和SW116均表達(dá)NMDAR1,且主要表達(dá)于細(xì)胞質(zhì)中;各濃度的MK801對(duì)HT-29細(xì)胞,以及濃度為500.0、1 000.0、2 000.0 μmol/L的MK801對(duì)SW116細(xì)胞的生長(zhǎng)抑制作用具有時(shí)間效應(yīng)關(guān)系,24、48及72 h各MK801濃度組對(duì)HT-29和SW116細(xì)胞的抑制率隨濃度升高整體呈增強(qiáng)趨勢(shì),但抑制率不呈明顯的劑量效應(yīng)關(guān)系;MK801具有促進(jìn)HT-29和SW116細(xì)胞凋亡的作用,且主要表現(xiàn)誘導(dǎo)細(xì)胞早期凋亡;MK801可抑制HT-29和SW116細(xì)胞遷移。結(jié)論NMDAR1在結(jié)腸癌細(xì)胞胞質(zhì)中表達(dá),且NMDAR1拮抗劑MK801具有抑制腫瘤細(xì)胞生長(zhǎng)、遷移,促進(jìn)其早期凋亡的作用,有望成為新一代抗腫瘤藥物。
結(jié)腸腫瘤;細(xì)胞增殖;細(xì)胞凋亡;細(xì)胞運(yùn)動(dòng);體外研究;N-甲基-D-天冬氨酸受體亞型1;MK801;HT-29;SW116
腦腸肽是指在胃腸道和神經(jīng)系統(tǒng)雙重分布的肽類[1]。谷氨酸是哺乳動(dòng)物中樞神經(jīng)系統(tǒng)主要的興奮性氨基酸,谷氨酸受體可分為離子型受體和G蛋白偶聯(lián)的代謝型受體兩大類[2]。N-甲基-D-天冬氨酸(N-Methyl-D-aspartate,NMDA)受體(NMDAR)為主要的離子型受體,其由NR1、NR2和NR3等亞基組成。NR1是NMDAR1的功能部分,構(gòu)成離子通道[3]。近年來(lái),越來(lái)越多的研究表明,NMDAR不僅在神經(jīng)系統(tǒng)發(fā)育、疼痛感覺(jué)等中發(fā)揮著重要的作用,而且也參與了胃腸道組織細(xì)胞的增殖,被視為一種腦腸肽[4]。研究顯示,NMDAR拮抗劑能抑制腫瘤細(xì)胞的增殖,并誘導(dǎo)腫瘤細(xì)胞的凋亡[5]。然而,有關(guān)NMDAR及其拮抗劑對(duì)于結(jié)腸癌作用的報(bào)道較少。MK801為非選擇性NMDAR1拮抗劑,本文通過(guò)分析MK801對(duì)于結(jié)腸癌細(xì)胞HT-29及SW116增殖、凋亡、遷移作用的影響,探討MK801用于結(jié)腸癌治療的可行性。
1.1 主要試劑與儀器人結(jié)腸癌細(xì)胞株HT-29、SW116(中科院基礎(chǔ)研究所,上海),MK801(MCE公司,美國(guó)),胎牛血清、DMEM培養(yǎng)基(Hyclone公司,美國(guó)),McCOY 5a培養(yǎng)基(吉諾生物醫(yī)藥技術(shù)有限公司),胰蛋白酶-EDTA消化液、噻唑藍(lán)(MTT,Solarbio公司,中國(guó)),Rabbit Anti-NMDAR1、超敏二步法免疫組化試劑盒(武漢博士德生物工程有限公司),流式檢測(cè)凋亡試劑盒AnnexinV-FITC Apoptosis DetectionKit I(BD公司,美國(guó))。
1.2 方法
1.2.1 細(xì)胞培養(yǎng)人結(jié)腸癌HT-29細(xì)胞用McCOY 5a培養(yǎng)基,SW116細(xì)胞用DMEM培養(yǎng)基。培養(yǎng)基中均加入10%胎牛血清,同時(shí)加入100 U/mL青霉素、100 mg/L鏈霉素,接種至細(xì)胞培養(yǎng)瓶后,置于37℃、5%CO2培養(yǎng)箱中培養(yǎng)。取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于后續(xù)實(shí)驗(yàn)。
1.2.2 免疫組織化學(xué)法檢測(cè)HT-29及SW116細(xì)胞表面NMDAR1的表達(dá)分別取對(duì)數(shù)生長(zhǎng)期的HT-29和SW116細(xì)胞分別接種于24孔板,每種細(xì)胞均分為實(shí)驗(yàn)組和對(duì)照組,培養(yǎng)24 h待細(xì)胞貼壁后,吸棄培養(yǎng)液,磷酸鹽緩沖液(PBS)洗細(xì)胞2次,5 min/次,加入4%多聚甲醛固定15 min,吸棄,PBS洗3次;加入0.3%的TritanX-100細(xì)胞打孔,室溫10 min,吸棄,PBS洗3次;加入3%H2O210 min以滅活內(nèi)源性酶,吸棄,PBS洗3次;加入5%BSA室溫封閉30 min,吸棄。實(shí)驗(yàn)組加入NMDAR1抗體,4℃孵育過(guò)夜。對(duì)照組用PBS代替NMDAR1抗體。次日吸棄,PBS洗3次;加入二抗孵育30 min,吸棄,PBS洗4次;加入DAB試劑顯色5 min,PBS終止,流水沖10 min,蘇木精染核30 s,流水沖洗,1%乙醇鹽酸返藍(lán),流水沖10 min,依次過(guò)體積分?jǐn)?shù)85%、90%、95%、100%乙醇,二甲苯2次脫水,封片膠封片。顯微鏡下觀察細(xì)胞染色情況,以細(xì)胞膜及細(xì)胞質(zhì)染色為棕黃色至棕褐色為陽(yáng)性反應(yīng)。
1.2.3 MTT比色法檢測(cè)MK801對(duì)HT-29和SW116細(xì)胞生長(zhǎng)抑制的影響分別取處于指數(shù)生長(zhǎng)期的HT-29和SW116細(xì)胞用胰酶進(jìn)行消化,調(diào)整細(xì)胞濃度為1×105個(gè)/mL,以每孔100 μL接種于96孔板,培養(yǎng)24 h后棄去培養(yǎng)基,分別加入用培養(yǎng)基配置的濃度為62.5、125.0、250.0、500.0、1 000.0、2 000.0 μmol/L的MK801,以不加藥物處理的細(xì)胞為對(duì)照組,繼續(xù)培養(yǎng)24、48、72 h后,每孔加入5 g/L MTT 10 μL,并設(shè)置空白孔,37℃避光孵育4 h,棄去培養(yǎng)液,每孔加入100 μL的二甲基亞砜(DMSO),振蕩15 min,酶標(biāo)儀檢測(cè)波長(zhǎng)為492 nm的光密度(OD)值。計(jì)算細(xì)胞生長(zhǎng)抑制率=(1-實(shí)驗(yàn)組OD值/對(duì)照組OD值)×100%。本實(shí)驗(yàn)每次設(shè)置3個(gè)復(fù)孔,重復(fù)實(shí)驗(yàn)3次。
1.2.4 流式細(xì)胞儀檢測(cè)MK801對(duì)HT-29和SW116細(xì)胞凋亡的影響參照Annexin V細(xì)胞凋亡檢測(cè)試劑盒說(shuō)明書進(jìn)行操作。HT-29和SW116細(xì)胞經(jīng)2 000 μmol/L的MK801處理24 h,以未經(jīng)藥物處理的細(xì)胞為對(duì)照組,每組收集1×106個(gè)細(xì)胞,冷PBS洗滌2次,1 000 r/min離心3 min,棄上清液。將細(xì)胞重懸于500 μL的1×binding buffer,調(diào)整細(xì)胞濃度為1×106個(gè)/mL,取100 μL細(xì)胞置于流式細(xì)胞儀專用試管中,加5 μL AnnexinV FITC,避光室溫反應(yīng)15 min,加入5 μL碘化丙啶(PI)輕輕混勻,再加入400 μL 1×binding buffer,過(guò)濾膜,流式細(xì)胞儀檢測(cè)凋亡情況。實(shí)驗(yàn)重復(fù)3次取平均值。
1.2.5 細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)HT-29和SW116細(xì)胞體外遷移能力調(diào)整細(xì)胞密度為5×106個(gè)/mL,以每孔1 mL接種到12孔板上,培養(yǎng)24 h待細(xì)胞貼壁后,用10 μL槍頭在孔板中央劃1條寬度均一的劃痕,PBS洗2遍以除去劃下的細(xì)胞,加入培養(yǎng)基配置的50 μmol/L的MK801,以不加藥物的培養(yǎng)基為陰性對(duì)照,拍照取樣,記錄劃痕的位置。繼續(xù)培養(yǎng)48 h,取相同位置拍照取樣,用Pro-Plus軟件計(jì)算細(xì)胞遷移前后劃痕距離,并計(jì)算細(xì)胞遷移率=(遷移前距離-遷移后距離)/遷移前距離×100%,實(shí)驗(yàn)重復(fù)3次。
1.3 統(tǒng)計(jì)學(xué)方法采用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,所有計(jì)量資料以表示,2組間比較采用t檢驗(yàn),多組間均數(shù)比較采用單因素方差分析,組間多重比較采用LSD-t法,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 HT-29及SW116細(xì)胞中NMDAR1的表達(dá)情況NMDAR1在結(jié)腸癌HT-29和SW116細(xì)胞中均呈陽(yáng)性表達(dá),且主要表達(dá)于細(xì)胞胞質(zhì)中,表現(xiàn)為細(xì)胞質(zhì)中染色為棕黃色至棕褐色,而對(duì)照組僅表現(xiàn)為核染色,見圖1。
2.2 MK801對(duì)HT-29及SW116細(xì)胞的生長(zhǎng)抑制作用(1)組內(nèi)隨時(shí)間變化:除對(duì)照組外,各MK801濃度組對(duì)HT-29細(xì)胞的生長(zhǎng)抑制率隨時(shí)間延長(zhǎng)呈增強(qiáng)趨勢(shì),而SW116細(xì)胞僅500.0、1 000.0及2 000.0 μmol/L組細(xì)胞生長(zhǎng)抑制率隨時(shí)間延長(zhǎng)呈增強(qiáng)趨勢(shì),細(xì)胞生長(zhǎng)抑制率具有時(shí)間效應(yīng)關(guān)系(P<0.01),見表1、2。(2)各時(shí)點(diǎn)組間比較:24、48及72 h各MK801濃度組對(duì)HT-29和SW116細(xì)胞的生長(zhǎng)抑制率隨濃度升高整體呈增強(qiáng)趨勢(shì),但組間多重比較差異并非全部有統(tǒng)計(jì)學(xué)意義,抑制率不呈明顯的劑量效應(yīng)關(guān)系,見表1、2。
Tab.1The inhibitory effects of MK801 on the growth of HT-29 cells表1 MK801對(duì)HT-29細(xì)胞生長(zhǎng)抑制的作用(n=3,%,)
Tab.1The inhibitory effects of MK801 on the growth of HT-29 cells表1 MK801對(duì)HT-29細(xì)胞生長(zhǎng)抑制的作用(n=3,%,)
**P<0.01,a~f分別表示與對(duì)照組、62.5、125.0、250.0、500.0及1 000.0 μmol/L組比較,P<0.05;A、B分別表示與24、48 h組比較,P<0.05,表2同
組別對(duì)照組實(shí)驗(yàn)組(μmol/L)62.5 125.0 250.0 500.0 1 000.0 2 000.0 F 24 h 0 48 h 0 72 h 0 F 8.40±3.30 24.58±1.66a 37.83±10.12ab 45.36±6.99abcd 75.60±2.74abcde 84.57±1.55abcdef 122.43**27.09±6.30aA 49.29±7.44abA 69.77±3.65abA 89.02±1.22abcdA 90.55±1.35abcdeA 95.68±0.27abcdeA 248.55**46.11±2.29aAB 72.75±0.26abAB 90.88±0.61abcAB 97.03±0.37abcdA 97.79±0.59abcdAB 98.83±0.25abcdAB 4 579.51**57.15**89.71**55.28**137.76**118.54**197.88**
Tab.2The inhibitory effects of MK801 on the growth of SW116 cells表2 MK801對(duì)SW116細(xì)胞生長(zhǎng)抑制的影響(n=3,%,)
Tab.2The inhibitory effects of MK801 on the growth of SW116 cells表2 MK801對(duì)SW116細(xì)胞生長(zhǎng)抑制的影響(n=3,%,)
組別對(duì)照組實(shí)驗(yàn)組(μmol/L)62.5 125.0 250.0 500.0 1 000.0 2 000.0 F 24 h 0 48 h 0 72 h 0 F 4.28±0.76a 13.83±2.05ab 18.91±1.42abc 24.58±2.14abcd 49.09±2.34abcde 78.58±1.08abcdef 901.58**7.81±3.49a 12.97±3.56a 23.16±2.67abc 50.60±2.42abcdA 66.32±4.73abcdeA 79.41±1.64abcdefA 321.24**6.76±1.90a 10.97±2.63a 23.04±2.26abc 45.33±2.88abcAB 80.79±1.66abcdeAB 84.36±1.56abcdeAB 885.579**1.80 0.81 3.68 90.86**73.95**73.95**
2.3 MK801對(duì)HT-29和SW116細(xì)胞凋亡的影響HT-29和SW116細(xì)胞中,實(shí)驗(yàn)組凋亡率均明顯高于對(duì)照組(P<0.01),且主要為早期凋亡,見圖2、表3。
Fig.2The effects of MK801 on apoptotic rates of HT-29 and SW116 cells detected by Annexin V/propidium iodide staining圖2 MK801對(duì)結(jié)腸癌HT-29、SW116細(xì)胞凋亡的影響
Tab.3Effects of MK801 on apoptosis and migration of HT-29 and SW116 cells表3 MK801對(duì)HT-29和SW116細(xì)胞凋亡和遷移能力的影響(n=3,%,)
Tab.3Effects of MK801 on apoptosis and migration of HT-29 and SW116 cells表3 MK801對(duì)HT-29和SW116細(xì)胞凋亡和遷移能力的影響(n=3,%,)
*P<0.05,**P<0.01
遷移率51.92±1.78 37.59±2.79 7.48**組別對(duì)照組實(shí)驗(yàn)組t HT-29凋亡率2.80±0.20 65.67±0.81 129.83**SW116凋亡率1.80±0.20 53.56±0.77 111.78**遷移率25.25±2.77 15.14±3.19 4.14*
2.4 MK801對(duì)HT-29細(xì)胞和SW116細(xì)胞遷移能力的影響HT-29細(xì)胞及SW116細(xì)胞中,實(shí)驗(yàn)組的遷移率均低于對(duì)照組(P<0.05),見表3、圖3。
Fig.3Effects of MK801 on migration of HT-29 and SW116 cells(×100)圖3 MK801對(duì)HT-29和SW116細(xì)胞遷移能力的影響(×100)
結(jié)直腸癌是消化系統(tǒng)常見的惡性腫瘤,美國(guó)2016年的癌癥數(shù)據(jù)顯示,結(jié)直腸癌在癌癥發(fā)病率中位列第4,在癌癥導(dǎo)致的死亡率中位于第2[6]。研究顯示,在我國(guó)結(jié)直腸癌平均每年新發(fā)病例可達(dá)37.6萬(wàn)例,占全部惡性腫瘤發(fā)病的8.7%,居第5位;每年死于該病的患者有19.1萬(wàn)例,占由癌癥導(dǎo)致死亡人數(shù)的6.7%,居第5位[7]。雖然伴隨著分子生物技術(shù)的發(fā)展,對(duì)于結(jié)直腸癌的手術(shù)、化療、放療及分子靶向治療方面取得了很大進(jìn)展,但晚期患者由于發(fā)生多處轉(zhuǎn)移,治愈率仍較低[8-9]。
NMDAR是興奮性神經(jīng)遞質(zhì)谷氨酸的離子型受體,主要存在于中樞神經(jīng)系統(tǒng)中[1]。既往研究表明,NMDAR抑制劑對(duì)于腫瘤細(xì)胞具有抑制作用[10];而在消化道腫瘤的研究中,Watanabe等[11]研究表明,NMDAR可在胃癌細(xì)胞中表達(dá),并且NMDAR拮抗劑能抑制胃癌細(xì)胞的增殖。Kim等[12]研究認(rèn)為NMDAR拮抗劑能抑制食管癌的增殖。
本研究細(xì)胞免疫組織化學(xué)結(jié)果顯示,HT-29細(xì)胞及SW116細(xì)胞胞質(zhì)染色為棕黃色至棕褐色,表明結(jié)腸癌HT-29細(xì)胞及SW116細(xì)胞胞質(zhì)中表達(dá)NMDAR1。細(xì)胞增殖實(shí)驗(yàn)顯示,各濃度的MK801對(duì)HT-29細(xì)胞,以及濃度為500.0、1 000.0、2 000.0 μmol/L的MK801對(duì)SW116細(xì)胞的生長(zhǎng)抑制作用具有時(shí)間效應(yīng)關(guān)系,24、48及72 h各MK801濃度組對(duì)HT-29和SW116細(xì)胞的抑制率隨濃度升高整體呈增強(qiáng)趨勢(shì),但抑制率不呈明顯的劑量效應(yīng)關(guān)系。細(xì)胞凋亡實(shí)驗(yàn)顯示,實(shí)驗(yàn)組的凋亡率明顯高于對(duì)照組,且主要表現(xiàn)為誘導(dǎo)細(xì)胞早期凋亡。細(xì)胞遷移實(shí)驗(yàn)顯示,HT-29細(xì)胞及SW116細(xì)胞中,實(shí)驗(yàn)組的遷移率均低于對(duì)照組,表明MK801可抑制細(xì)胞遷移。該結(jié)果與Watanabe等[11]、Kim等[12]的研究結(jié)果相符。
另有研究認(rèn)為,高濃度的Ca2+能促進(jìn)腫瘤細(xì)胞的增殖,對(duì)于腫瘤細(xì)胞分化至關(guān)重要,而Ca2+對(duì)腫瘤細(xì)胞的形成過(guò)程也起著重要作用,并且影響細(xì)胞遷移[13]。本研究結(jié)果表明,NMDAR1在腫瘤細(xì)胞胞質(zhì)中表達(dá),并且NMDAR1拮抗劑MK801能抑制細(xì)胞的增殖、遷移,并促進(jìn)細(xì)胞早期凋亡,而NMDAR1抑制劑的作用機(jī)制主要是通過(guò)抵抗細(xì)胞內(nèi)的Ca2+濃度,從而降低腫瘤細(xì)胞Ca2+濃度,起到抑制細(xì)胞增殖、遷移,促進(jìn)細(xì)胞凋亡的作用,因此,筆者認(rèn)為MK801對(duì)腫瘤細(xì)胞的作用機(jī)制可能與Ca2+通道或Ca2+調(diào)節(jié)酶有關(guān)。
綜上所述,谷氨酸離子型受體NMDAR1不僅在神經(jīng)系統(tǒng)中表達(dá),在外周組織細(xì)胞中亦有表達(dá),可視為一種腦腸肽[1],而NMDAR1拮抗劑MK801對(duì)腫瘤細(xì)胞具有生長(zhǎng)抑制作用,且可抑制腫瘤細(xì)胞遷移,并促進(jìn)細(xì)胞早期凋亡,故其可能成為新一代的抗腫瘤藥物,為臨床治療提供新途徑。
(圖1見插頁(yè))
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(2016-06-14收稿2016-07-13修回)
(本文編輯陸榮展)
Effects of MK801 on proliferation,apoptosis and migration of colorectal cancer cells
XIE Dongbing1,MENG Jianyu2,GUO Yuting3Δ,REN Xia4,LI Xue1
1 The Second Clinical College of Shandong University of TCM,Jinan 250014,China;2 The Institute of Technology of Shandong University of TCM;3 Department of Gastroenterology,the Second Affiliated Hospital of Shandong University of TCM;4 The Academy of Medical Science of Shandong Province△
ObjectiveTo explore the expression of the N-methyl-D-aspartate receptor type 1(NMDAR1)in the colorectal cancer cells(CRC),and to examine the effects of NMDAR1 antagonists MK801 on proliferation,apoptosis and migration of colorectal cancer cell line HT-29 and SW116 cells.MethodsThe immunocytochemistry method was used to examine the expressions of NMDAR1 in HT-29 and SW116 cells.MTT assay was used to detect the effects of MK801(62.5, 125.0,250.0,500.0,1 000.0,2 000.0 μmol/L)on the proliferation of HT-29 and SW116 cells.The flow cytometry was used to analyze the effect of MK801(2 000 μmol/L)on cell apoptosis of HT-29 and SW116 cells.Wound healing assay was used to detect the effect of MK801(50 μmol/L)on the migration of HT-29 or SW116 cells.ResultsThe NMDAR1 was expressed in both HT-29 and SW116 cells,and mainly in the cytoplasm.MTT assay showed that there were inhibitory effects of different concentrations of MK 801(62.5,125.0,250.0,500.0,1 000.0,2 000.0 μmol/L)on the proliferation of HT-29 cells,and inhibitory effects of different concentrations of MK801(500.0,1 000.0,2 000.0 μmol/L)on SW116 cells,in a time-dependent manner.For 24,48 and 72 h,the inhibitory effects of MK801 on proliferation rates of HT-29 and SW116 cells were increased with the increased concentrations of MK801.MK801 showed an effect to induce cell apoptosis in HT-29 and SW116 cells,and mainly for early apoptosis.Wound healing assay indicated that MK801 inhibited the cell migration of HT-29 and SW116 cells.ConclusionResults suggest that there is expression of NMDAR1 in colorectal cancer cells. The NMDAR1 antagonist MK801 can inhibit the proliferation,induce cells early apoptosis and inhibit cells migration.It maybe a new generation of antitumor drugs.
colonic neoplasms;cell proliferation;apoptosis;cell movement;in vitro;N-methyl-D-aspartate receptor subtype 1;MK801;HT-29;SW116
R735.35
A
10.11958/20160486
山東省自然科學(xué)基金資助項(xiàng)目(Y2008C141)
1山東中醫(yī)藥大學(xué)第二臨床學(xué)院(郵編250014);2山東中醫(yī)藥大學(xué)理工學(xué)院;3山東中醫(yī)藥大學(xué)附屬第二醫(yī)院消化科;4山東省醫(yī)學(xué)科學(xué)院基礎(chǔ)所
謝冬冰(1990),女,碩士在讀,主要從事消化道疾病診治的研究
Δ通訊作者E-mail:zhmqlw@sina.com