夏 紅,蘇 堅(jiān),2,趙曉紅,3,劉 芳,蘇 波,凌 暉,曾 希,蘇 琦
1.湖南省胃癌研究中心,湖南省高校腫瘤細(xì)胞與分子病理學(xué)重點(diǎn)實(shí)驗(yàn)室,南華大學(xué)腫瘤研究所,湖南 衡陽 421001;2.南華大學(xué)附屬第二醫(yī)院病理科,湖南 衡陽 421001;3.海南省婦幼保健院婦產(chǎn)科,海南 ???570206
RORα高表達(dá)對二烯丙基二硫抑制人胃癌MGC803細(xì)胞上皮-間質(zhì)轉(zhuǎn)化的影響
夏 紅1,蘇 堅(jiān)1,2,趙曉紅1,3,劉 芳1,蘇 波1,凌 暉1,曾 希1,蘇 琦1
1.湖南省胃癌研究中心,湖南省高校腫瘤細(xì)胞與分子病理學(xué)重點(diǎn)實(shí)驗(yàn)室,南華大學(xué)腫瘤研究所,湖南 衡陽 421001;2.南華大學(xué)附屬第二醫(yī)院病理科,湖南 衡陽 421001;3.海南省婦幼保健院婦產(chǎn)科,海南 海口 570206
背景與目的:二烯丙基二硫(diallyl disulfide,DADS)具有抗腫瘤的作用。該研究在DADS上調(diào)人胃癌MGC803細(xì)胞RORα的基礎(chǔ)上,觀察DADS與RORα高表達(dá)對MGC803細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(epithelialmesenchymal transformation,EMT)的影響。方法:相差顯微鏡觀察MGC803細(xì)胞形態(tài)的影響。采用反轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription polymerase chain reaction,RT-PCR)、蛋白[質(zhì)]印跡法(Western blot)、免疫熒光與免疫組織化學(xué)檢測EMT相關(guān)分子表達(dá)。裸鼠實(shí)驗(yàn)檢測對移植瘤生長的影響。結(jié)果:相差顯微鏡顯示,DADS組與RORα高表達(dá)組細(xì)胞大小較MGC803細(xì)胞一致,呈圓形或橢圓形,梭形細(xì)胞少見,異型性降低。RORα高表達(dá)加DADS后,上述改變更為明顯。Western blot檢測結(jié)果顯示,DADS組與RORα高表達(dá)組Snail蛋白表達(dá)明顯下調(diào),DADS+RORα高表達(dá)組更明顯(P<0.05)。RT-PCR與Western blot檢測顯示,DADS與RORα高表達(dá)可明顯下調(diào)Vimentin和上調(diào)E-cadherin mRNA與蛋白表達(dá),DADS+RORα高表達(dá)組更為顯著(P<0.05)。免疫熒光顯示,DADS組與RORα高表達(dá)組細(xì)胞Snail與Vimentin表達(dá)較對照組明顯減弱和E-cadherin表達(dá)顯著增強(qiáng),DADS+RORα高表達(dá)組更為顯著,與Western blot結(jié)果一致。裸鼠實(shí)驗(yàn)顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS組移植瘤較對照組生長減慢(P<0.05),并且,移植瘤體重較對照組明顯降低,抑瘤率分別為15.07%、26.55%與49.27%(P<0.05)。免疫組織化學(xué)檢測結(jié)果顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS組較對照組的Ki-67、CD34與Vimentin表達(dá)均明顯降低,E-cadherin表達(dá)明顯增強(qiáng)。結(jié)論:RORα高表達(dá)可增強(qiáng)DADS體內(nèi)外抑制人胃癌細(xì)胞EMT的作用。
二烯丙基二硫;RORα;人胃癌MGC803細(xì)胞;上皮-間質(zhì)轉(zhuǎn)化;Snail;Vimentin;E-cadherin
胃癌是最常見的惡性腫瘤之一,發(fā)生率與死亡率分別為全球第四位與第三位。據(jù)2015年最新統(tǒng)計(jì),胃癌在我國的發(fā)生率與死亡率位于第二位,每年約新發(fā)67.9萬和死亡49.8萬人。由于患者就診時大多已發(fā)生侵襲轉(zhuǎn)移,5年生存率低于10%[1-3]。因此,研究胃癌侵襲轉(zhuǎn)移機(jī)制,尋找靶點(diǎn)具有重要的意義。
最近,我們運(yùn)用蛋白質(zhì)組學(xué)技術(shù)鑒定二烯丙基二硫(diallyl disulfide,DADS)處理人胃癌MGC803細(xì)胞的差異蛋白,結(jié)果發(fā)現(xiàn)維甲酸相關(guān)孤核受體α(retinoid acid receptor related orphan receptor α,RORα)蛋白表達(dá)明顯上調(diào)[4]。我們發(fā)現(xiàn),RORα在胃癌組織中低表達(dá),與胃癌發(fā)生和分化程度有關(guān)[5]。RORα是核受體超家族成員之一,可調(diào)節(jié)多種正常組織細(xì)胞的發(fā)育和分化、生物代謝、機(jī)體穩(wěn)態(tài)維持及高級神經(jīng)功能等,并且,RORα在腫瘤組織中表達(dá)下調(diào)與腫瘤發(fā)生密切相關(guān),可能是腫瘤治療的靶點(diǎn)[6-7]。DADS是大蒜中的一種脂溶性的有效成分,對多種腫瘤均有明顯的抑制作用,是一種很有開發(fā)潛力的抗腫瘤藥物[8]。我們前期工作表明,DADS可通過Rac1-Pak1/Rock1通路下調(diào)LIMK1、MMP-9和上調(diào)TIMP-3,抑制人胃癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transformation,EMT)與遷移侵襲[9]。本研究進(jìn)一步探討DADS與RORα高表達(dá)對胃癌細(xì)胞EMT的影響及其相關(guān)機(jī)制。
1.1 細(xì)胞培養(yǎng)
人胃癌MGC803細(xì)胞由本實(shí)驗(yàn)室保存,高表達(dá)RORα人胃癌MGC803細(xì)胞由本實(shí)驗(yàn)室構(gòu)建[10],置于含10%小牛血清的RPMI-1640培養(yǎng)基中,于37 ℃,CO2體積分?jǐn)?shù)為5%、飽和濕度的培養(yǎng)箱內(nèi)傳代培養(yǎng)。取對數(shù)生長期的細(xì)胞用于實(shí)驗(yàn)。
1.2 主要試劑
DADS購自美國Fluka公司,RNA提取試劑盒購自美國OMEGA公司,逆轉(zhuǎn)錄試劑盒與BCA蛋白定量試劑盒購自美國Promega公司,RORα、E-cadherin、Vimentin與β-actin抗體購自美國Abcam公司,Snail、Ki-67與CD34抗體與ECL發(fā)光試劑盒購自美國Santa Cruz公司,新生牛血清購自杭州四季青生物工程材料有限公司。引物用Primer Premier 5.0軟件設(shè)計(jì),由生工生物工程(上海)股份有限公司合成。羊抗兔IgGHR和羊抗小鼠IgG-HRP購自江蘇凱基生物技術(shù)股份有限公司,羊抗小鼠IgG(H+L)購自美國Protech公司,DAPI和正常山羊血清購自武漢博士德生物工程有限公司,MaxVisionTM試劑盒購自福州邁新生物技術(shù)開發(fā)有限公司。
1.3 相差顯微鏡觀察
人胃癌MGC803細(xì)胞與RORα高表達(dá)MGC803細(xì)胞以及經(jīng)DADS處理的兩種細(xì)胞培養(yǎng)24 h后,置于倒置相差顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化。
1.4 采用反轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription polymerase chain reaction,RTPCR)
采用總RNA試劑盒提取細(xì)胞總RNA,在AMV酶作用下逆轉(zhuǎn)錄合成cDNA。設(shè)計(jì)并合成PCR引物序列。Vimentin順義鏈為5’-ACACCCTGCAATCTTTCAGACA-3’,反義鏈為5’-AGAAATCCTGCTCTCCTCGCCT-3’,產(chǎn)物長度635 bp;E-cadherin順義鏈為5’-CTCCCAATACATCTCCCTTCA C-3’,反義鏈為5’-CGCCTCCTTCTTCATCATAGTAA-3’,產(chǎn)物長度423 bp;β-actin順義鏈為5’-TCTACA AT G A G C T G C G T G T G G-3’,反義鏈:5’-GGAACCGCTCATTGCCAATG-3’,產(chǎn)物長度498 bp。PCR反應(yīng)條件:94 ℃,5 min;94 ℃,40 s,各基因Tm,45 s,72 ℃ 80 s,28個循環(huán);72 ℃ 10 min。5 μL的PCR產(chǎn)物經(jīng)1%的瓊脂糖電泳,嗅化乙啶染色,通過IS1000圖像分析軟件讀取條帶灰度值,相對值以目的基因與β-actin灰度值之比表示。
1.5 蛋白[質(zhì)]印跡法(Western blot)檢測
收集細(xì)胞,提取細(xì)胞總蛋白,BCA法測定蛋白濃度,每組取等量樣本進(jìn)行SDS-PAGE凝膠電泳,電泳后轉(zhuǎn)膜,封閉1 h,加一抗,4 ℃過夜,TBST洗膜,加二抗溫育1 h,洗膜,ECL發(fā)光,X片曝光、顯影、定影。
1.6 細(xì)胞免疫熒光實(shí)驗(yàn)
在6孔板中滴加培養(yǎng)基,將消毒的蓋玻片放入6孔板。將對數(shù)生長期的MGC803細(xì)胞制成懸液,每孔接種5×105個細(xì)胞,細(xì)胞貼壁融合至80%時,取出蓋玻片,PBS洗3次,每次5 min,采用4%多聚甲醛固定,常溫靜置15 min,PBS洗3次,每次5 min;將0.5%Triton覆蓋細(xì)胞后,常溫靜置30 min,PBS洗3次,每次5 min,吸干PBS液;山羊血清封閉,于37 ℃溫育1 h;吸凈封閉液,加入一抗,濕盒內(nèi)4℃溫育過夜;第2天移置37 ℃下復(fù)溫1 h,PBS洗3次,每次5 min;暗室中加FITC的二抗,37 ℃下避光濕盒中溫育1 h,PBS洗3次,每次5 min。染核:0.4 μg/μL DAPI覆蓋細(xì)胞,避光靜置2~5 min,PBS洗3次,每次5 min,甘油封片在熒光顯微鏡下觀察。
1.7 裸鼠成瘤實(shí)驗(yàn)
裸鼠購自北京維通利華實(shí)驗(yàn)動物技術(shù)有限公司,為4周齡雄性,分為MGC803細(xì)胞組、MGC803+DADS組、RORα高表達(dá)組和RORα高表達(dá)+DADS組,每組5只。將處于對數(shù)生長期的細(xì)胞1×107個/mL,分別取0.2 mL細(xì)胞懸液接種于各組裸鼠的腋下。觀察裸鼠進(jìn)食、飲水、精神及活動等情況。每周測量移植瘤大小,腫瘤體積:V=ab2/2,以裸鼠移植瘤體積的平均值,繪制生長曲線。第10周麻醉處死裸鼠,完整剝離腫瘤,稱移植瘤重量,瘤重抑瘤率=(1-實(shí)驗(yàn)組瘤重/對照組瘤重)×100%。移植瘤組織固定于4%的中性甲醛溶液中。
1.8 免疫組織化學(xué)檢測
采用MaxVisionTM法,分別滴加一抗室溫60 min,4 ℃過夜,用PBS洗3次,每次3 min,滴加即用型MaxVisionTM試劑,室溫下15 min,PBS洗3次,每次5 min。DAB顯色,自來水沖洗,蘇木素復(fù)染,脫水、透明、封固、鏡檢。
1.9 統(tǒng)計(jì)學(xué)處理
2.1 相差顯微鏡觀察
采用相差顯微鏡對細(xì)胞進(jìn)行觀察。鏡下可見MGC803細(xì)胞大小不一,大部分呈長梭形,纖維母細(xì)胞樣,細(xì)胞膜可見突起,核質(zhì)比例增大,異型性明顯。30 mg/L DADS組細(xì)胞大小較一致,大部分呈圓形或橢圓形,梭形細(xì)胞少見,細(xì)胞膜突起減少,核質(zhì)比值下降,異型性降低。RORα高表達(dá)細(xì)胞大小較一致,呈圓形或橢圓形,細(xì)胞膜突起少見,核質(zhì)比例減少,異型性顯著降低。RORα高表達(dá)加DADS后,上述改變更為明顯。表明DADS與RORα高表達(dá)可從形態(tài)上抑制MGC803細(xì)胞EMT,RORα高表達(dá)可增強(qiáng)DADS的抑制作用(圖1)。
圖1 RORα高表達(dá)對MGC803細(xì)胞形態(tài)的影響Fig. 1 The morphological effect of overexpression of RORα in MGC803 cells
2.2 DADS與RORα高表達(dá)抑制MGC803細(xì)胞EMT
Western blot檢測結(jié)果顯示,DADS與RORα高表達(dá)可明顯下調(diào)MGC803細(xì)胞Snail蛋白表達(dá)(P<0.05)。并且,RT-PCR與Western blot檢測結(jié)果顯示,DADS與RORα高表達(dá)可下調(diào)Vimentin和上調(diào)E-cadherin mRNA與蛋白表達(dá)(P<0.05)。上述改變RORα/MGC803+DADS作用更為明顯(P<0.05)。表明DADS與RORα高表達(dá)可通過下調(diào)Snail與Vimentin和上調(diào)E-cadherin抑制MGC803細(xì)胞EMT,RORα高表達(dá)可增強(qiáng)DADS的抑制作用(圖2~4)。
2.3 細(xì)胞免疫熒光檢測DADS與RORα高表達(dá)對EMT相關(guān)蛋白表達(dá)的影響
免疫熒光檢測結(jié)果顯示,Snail蛋白定位細(xì)胞核,Vimentin與E-cadherin蛋白主要定位于細(xì)胞質(zhì)。DADS與RORα高表達(dá)組細(xì)胞Snail與Vimentin陽性表達(dá)較對照組明顯減弱,而E-cadherin陽性表達(dá)顯著增強(qiáng),與Western blot檢測結(jié)果一致(圖5)。
2.4 DADS與RORα高表達(dá)對裸鼠移植瘤生長的影響
生長曲線圖顯示,隨著時間的延長,移植瘤體積逐漸增大,但是,DADS較對照組移植瘤生長明顯減慢;而RORα高表達(dá)組生長速度減慢程度更加顯著,特別是DADS處理后,生長速度減慢最為顯著(P<0.05,圖6)。圖7顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS移植瘤平均體重分別為4.51±0.29、3.90±0.33和2.80±0.27,較對照組的5.31±0.37明顯降低,抑瘤率分別為15.07%、26.55%和49.27%,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。該結(jié)果表明DADS與RORα可在體內(nèi)抑制MGC803細(xì)胞移植瘤形成,RORα高表達(dá)具有增強(qiáng)DADS的作用。
圖2 DADS與RORα高表達(dá)對Snail蛋白表達(dá)的影響Fig. 2 The effect of Snail expression by DADS and overexpression of RORα
圖3 DADS與RORα高表達(dá)對Vimentin表達(dá)的影響Fig. 3 The e ff ect of Vimentin expression by DADS and overexpression of RORα
圖4 DADS與RORα高表達(dá)對E-cadherin表達(dá)的影響Fig. 4 The e ff ect of E-cadherin expression by DADS and overexpression of RORα
圖5 DADS與RORα高表達(dá)對EMT相關(guān)蛋白表達(dá)的影響Fig. 5 The e ff ect of EMT associated proteins expression by DADS and overexpression of RORα
圖6 DADS與RORα高表達(dá)對裸鼠移植瘤生長的影響Fig. 6 The effect of growth in mouse xenograft models by DADS and overexpression of RORα
圖7 DADS與RORα高表達(dá)抑制裸鼠移植瘤形成的作用Fig. 7 DADS and overexpression of RORα inhibit the formation in transplantation tumor
2.5 RORα高表達(dá)對裸鼠移植瘤組織EMT相關(guān)蛋白表達(dá)的影響
免疫組織化學(xué)檢測結(jié)果顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS組移植瘤組織的Ki-67、CD34與Vimentin陽性表達(dá)分別較對照組均明顯減弱,而E-cadherin陽性表達(dá)明顯增強(qiáng)(圖8)。
圖8 裸鼠移植瘤組織EMT相關(guān)蛋白的表達(dá)Fig. 8 The expression of EMT associated proteins in transplantation tumor tissue
研究表明,RORα在胃癌、結(jié)腸癌、食管癌、胰腺癌、肝癌、乳腺癌、子宮頸癌、卵巢癌、前列腺癌、膀胱癌、頭頸部癌及白血病等多種腫瘤中表達(dá)下調(diào)[5-7]?;謴?fù)RORα表達(dá)可體內(nèi)外抑制乳腺癌細(xì)胞增殖與侵襲,提示RORα可能是腫瘤治療靶點(diǎn)[7]。研究發(fā)現(xiàn),RORα在肝癌組織表達(dá)明顯下調(diào),與血清AFP、病理分級、腫瘤復(fù)發(fā)、血管侵襲和預(yù)后密切相關(guān)。并且,RORα高表達(dá)可減少肝癌細(xì)胞需氧糖酵解和下調(diào)生物合成途徑,上調(diào)p21,抑制PDK2表達(dá)和磷酸化[11-12]。但是,RORα抑制腫瘤細(xì)胞遷移侵襲與轉(zhuǎn)移是否與EMT有關(guān)尚不清楚。
腫瘤細(xì)胞獲得遷移和侵襲能力是腫瘤轉(zhuǎn)移起始的重要步驟,而EMT是腫瘤細(xì)胞獲得遷移和侵襲能力的關(guān)鍵。上皮源性腫瘤細(xì)胞發(fā)生EMT后,除形態(tài)發(fā)生改變外,具有較高的遷移與侵襲、抗凋亡和降解細(xì)胞外基質(zhì)的能力,上皮標(biāo)志物E-cadherin、ZO-1等表達(dá)下調(diào),間質(zhì)標(biāo)志物Vimentin、α-SMA、N-cadherin等上調(diào)以及Snail、Slug和Twist等轉(zhuǎn)錄因子活性增強(qiáng)。此時,腫瘤細(xì)胞間的連接變得疏松,細(xì)胞骨架蛋白發(fā)生重組,黏附能力下降,遷移能力增強(qiáng),更易于離開原有位置發(fā)生侵襲與轉(zhuǎn)移。因此,腫瘤細(xì)胞出現(xiàn)EMT改變是啟動腫瘤侵襲與轉(zhuǎn)移的關(guān)鍵所在,阻止EMT發(fā)生已經(jīng)成為抑制惡性腫瘤轉(zhuǎn)移的新的治療策略[13-14]。
本研究在在DADS上調(diào)人胃癌MGC803細(xì)胞RORα的基礎(chǔ)上,探討DADS與RORα高表達(dá)對胃癌細(xì)胞EMT的影響。相差顯微鏡顯示,DADS組與RORα高表達(dá)組細(xì)胞大小較MGC803細(xì)胞一致,呈圓形或橢圓形,梭形細(xì)胞少見,異型性降低,DADS處理RORα高表達(dá)細(xì)胞后,上述改變更為明顯。基于Snail、E-cadherin與Vimentin是腫瘤EMT與進(jìn)展的關(guān)鍵因子[15-16],Ki-67是檢測腫瘤增殖能力的重要指標(biāo)[17],CD34是血管形成的標(biāo)志[18]。RT-PCR與Western blot檢測結(jié)果顯示,DADS與RORα高表達(dá)可明顯下調(diào)Snail與Vimentin和上調(diào)E-cadherin表達(dá),DADS+RORα高表達(dá)組更為顯著。免疫熒光檢測結(jié)果與Western blot檢測結(jié)果一致。裸鼠實(shí)驗(yàn)顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS組移植瘤較對照組生長減慢,移植瘤體重明顯降低,抑瘤率分別為15.07%、26.55%與49.27%。免疫組織化學(xué)檢測結(jié)果顯示,DADS組、RORα高表達(dá)組與RORα高表達(dá)+DADS組較對照組的Ki-67、CD34與Vimentin陽性表達(dá)均明顯降低和E-cadherin陽性表達(dá)明顯增強(qiáng)。上述證明DADS與RORα高表達(dá)通過下調(diào)Snail與Vimentin和上調(diào)E-cadherin表達(dá)可體內(nèi)外抑制人胃癌細(xì)胞EMT,RORα高表達(dá)可增強(qiáng)DADS的作用。
研究表明,RORα可通過Wnt/β-catenin、Wnt5a/PKC、Hypoxia/Angiogenesis、NF-κB和p53等多種途徑調(diào)控腫瘤細(xì)胞增殖與遷移侵襲[7]。Lee等[19]的研究結(jié)果顯示,RORα可通過絲氨酸35殘基磷酸化,競爭結(jié)合β-catenin,抑制Wnt/β-catenin靶基因cyclin D1、c-myc和Axin,從而調(diào)控細(xì)胞增殖與腫瘤進(jìn)展。此外,RORα可依賴PGE2/PKCα途徑磷酸化減弱結(jié)腸癌細(xì)胞Wnt靶基因表達(dá)[20]。近來,我們發(fā)現(xiàn),RORα高表達(dá)可抑制人胃癌細(xì)胞Wnt/β-catenin通路靶基因表達(dá)和增殖與遷移侵襲[7,21]。然而,DADS上調(diào)RORα抑制人胃癌細(xì)胞EMT是否通過Wnt/β-catenin通路調(diào)控的分子機(jī)制尚待深入研究。
[1] TORRE L A, BRAY F, SIEGEL R L, et al. Global cancer statistics, 2012[J]. CA Cancer J Clin, 2015, 65(2): 87-108.
[2] ORDITURA M, GALIZIA G, SFORZA V, et al. Treatment of gastric cancer[J]. World J Gastroenterol, 2014, 20(7): 1635-1649.
[3] CHEN W, ZHENG R, BAADE P D, et al. Cancer statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 66(2): 115-132.
[4] SU B, SU J, HE H, et al. Identification of potential targets for diallyl disulfide in human gastric cancer MGC-803 cells using proteomics approaches[J]. Oncol Rep, 2015, 33(5): 2484-2494.
[5] 石 鶯, 黃建軍, 蘇 堅(jiān), 等. RORα蛋白在胃癌中的表達(dá)及臨床病理意義[J]. 臨床與實(shí)驗(yàn)病理學(xué)雜志, 2012, 28(3): 270-273.
[6] 趙曉紅, 蘇 琦. 維甲酸相關(guān)孤核受體α與Wnt信號途徑及腫瘤的關(guān)系[J]. 國際病理科學(xué)與臨床雜志, 2011, 31(3): 234-237.
[7] DU J, XU R. RORα, a potential tumor suppressor and therapeutic target of breast cancer[J]. Int J Mol Sci, 2012, 13(12):15755-17566.
[8] YI L, SU Q. Molecular mechanisms for the anti-cancer effects of diallyl disulfide[J]. Food Chem Toxicol, 2013, 57:362-370.
[9] SU B, SU J, ZENG Y, et al. Diallyl disulfide suppresses epithelial-mesenchymal transition, invasion and proliferation by downregulation of LIMK1 in gastric cancer[J]. Oncotarget, 2016, 7(9): 10498-10512.
[10] 趙曉紅, 向姝霖, 劉 芳, 等. RORα高表達(dá)對人胃癌MGC803細(xì)胞增殖與遷移侵襲的影響[J]. 腫瘤防治研究, 2016, 43(11): 926-932.
[11] FU R D, QIU C H, CHEN H A, et al. Retinoic acid receptorrelated receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma[J]. Tumour Biol, 2014, 35(8): 7603-7610.
[12] BYUN J K, CHOI Y K, KANG Y N, et al. Retinoic acidrelated orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells[J]. Hepatology, 2015, 61(3): 953-964.
[13] GOMES L R, TERRA L F, SOGAYAR M C, et al. Epithelialmesenchymal transition: implications in cancer progression and metastasis[J]. Curr Pharm Biotechnol, 2011, 12(11): 1881-1890.
[14] MENG F, WU G. The rejuvenated scenario of epithelial-mesenchymal transition (EMT) and cancer metastasis[J]. Cancer Metastasis Rev, 2012, 31(3-4): 455-467.
[15] ABOUHASHEM N S, IBRAHIM D A, MOHAMED A M. Prognostic implications of epithelial to mesenchymal transitionrelated proteins (E-cadherin, Snail) and hypoxia inducible factor 1α in endometrioid endometrial carcinoma[J]. Ann Diagn Pathol, 2016, 22: 1-11.
[16] LAZAROVA D L, BORDONARO M. Vimentin, colon cancer progression and resistance to butyrate and other HDACis[J]. J Cell Mol Med, 2016, 20(6): 989-993.
[17] LEE W S, PARK Y L, KIM N, et al. Myeloid cell leukemia-1 regulates the cell growth and predicts prognosis in gastric cancer[J]. Int J Oncol, 2015, 46(5): 2154-2162.
[18] LEE O, CHOI M R, CHRISTOV K, et al. Progesterone receptor antagonism inhibits progestogen-related carcinogenesis and suppresses tumor cell proliferation[J]. Cancer Lett, 2016, 376(2): 310-317.
[19] LEE J M, KIM I S, KIM H, et al. RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer[J]. Mol Cell, 2010, 37(2): 183-195.
[20] SHIN D, KIM I S, LEE J M, et al. The hidden switches underlying RORα-mediated circuits that critically regulate uncontrolled cell proliferation[J]. J Mol Cell Biol, 2014, 6(4): 338-348.
[21] 蘇 堅(jiān), 趙曉紅, 劉 芳, 等. RORα高表達(dá)抑制人胃癌細(xì)胞Wnt/β-catenin通路靶基因[J]. 中國細(xì)胞生物學(xué)學(xué)報(bào), 2016, 38(11): 1358-1365.
Effect of RORα overexpression on inhibition of epithelial-mesenchymal transformation by DADS in human gastric MGC803 cells
XIA Hong1, SU Jian1,2, ZHAO Xiaohong1,3, LIU Fang1, SU Bo1, LING Hui1, ZHEN Xi1, SU Qi1
(1. Center for Gastric Cancer Research of Hunan Province, Key Laboratory of Cancer Cellular and Molecular Pathology of Hunan Provincial University, Cancer Research Institute, University of South China, Hengyang 421001, Hunan Province, China; 2. Department of Pathology, the Second Affiliated Hospital, University of South China, Hengyang 421001, Hunan Province, China; 3. Department of Gynaecology and Obstetrics, Hainan Maternal and Child Health Hospital, Haikou 570206, Hainan Province, China)
SU Qi E-mail: suqi1945@163.com
Background and purpose: Diallyl disulfide (DADS) could inhibit the growth of cancer cells. This study aimed to investigate the effect of DADS and overexpression of RORα on EMT in human gastric cancerMGC803 cells with upregulation of RORα by DADS. Methods: The morphological effect on MGC803 cells was observed by phase-contrast microscope. The correlative molecules with EMT were detected by RT-PCR, Western blot, immunof l uorescence and immunohistochemistry. The inf l uence on xenograft tumor growth in nude mice was observed in MGC803 cells. Results: Phase-contrast microscope showed that MGC803 cells were of identical size, round or oval with decreased spindle cells and lower level of heteromorphism in DADS group and RORα/MGC803 group. The above-mentioned alterations were more obvious in RORα/MGC803+DADS group. Western blot exhibited obviously the downregulation of Snail protein in DADS group and RORα/MGC803 group (P<0.05). RT-PCR and Western blot disclosed that the expression of Vimentin was downregulated notably and E-cadherin was upregulated in DADS group, RORα/MGC803 group, and RORα/MGC803+DADS group more obviously (P<0.05). Immunofluorescence revealed that the positive expression of Snail and Vimentin protein was attenuate, while E-cadherin was strengthened in DADS group, RORα/MGC803 group and RORα/MGC803+DADS group compared with MGC803 cells. Moreover, the xenograft tumor growth was markedly decreased, and body weight of transplanted tumor was visibly reduced with the inhibition ratio of 15.07%, 26.55 % and 49.27%, respectively (P<0.05). The positive expression of Ki-67, CD34 and Vimentin were obviously decreased, while the positive expression of E-cadherin was increased. Conclusion: Overexpression of RORα can remarkably enhance inhibition of EMT in MGC803 cells by DADS in vivo and in vivo.
Diallyl disulfide; RORα; Human gastric cancer MGC803 cells; Epithelial-mesenchymal transformation; Snail; Vimentin; E-cadherin
10.19401/j.cnki.1007-3639.2017.05.007
R735.2
A
1007-3639(2017)05-0359-09
2016-12-30
2017-02-10)
國家自然科學(xué)基金(81374013)。
蘇 琦 E-mail:suqi1945@163.com