李澎瀛　張東陽(yáng)　吳美玲　董克磊　施冬云

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系　上海　200032)

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缺氧條件下肝癌細(xì)胞和正常肝細(xì)胞能量代謝通路的差異

李澎瀛張東陽(yáng)吳美玲董克磊施冬云△

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)系上海200032)

【摘要】目的比較缺氧條件下肝癌細(xì)胞與正常肝細(xì)胞能量代謝通路的差異,探索腫瘤細(xì)胞的耐缺氧機(jī)制。方法采用環(huán)境缺氧(0.2%O2缺氧培養(yǎng)箱)模擬腫瘤的體內(nèi)環(huán)境,比較肝癌細(xì)胞株HepG2與原代分離的小鼠肝臟細(xì)胞在不同缺氧時(shí)間下的能量代謝情況。流式細(xì)胞術(shù)檢測(cè)各組活性氧(reactive oxygen species,ROS)水平,MTT法檢測(cè)各組細(xì)胞缺氧后細(xì)胞活力及生存率,real-time PCR方法檢測(cè)己糖激酶2(hexokinase 2)、異檸檬酸脫氫酶(isocitrate dehydrogenase)等糖代謝關(guān)鍵酶及p53、TIGAR/Tigar、SCO2/Sco2基因的mRNA水平,NADH比色法檢測(cè)乳酸脫氫酶(lactate dehydrogenase)活性,氧電極法檢測(cè)各組細(xì)胞耗氧量,高效液相色譜法檢測(cè)細(xì)胞內(nèi)ATP含量。結(jié)果缺氧后,腫瘤細(xì)胞HepG2與正常小鼠肝臟細(xì)胞相比生存率更高,活性氧增幅更小。兩種細(xì)胞在缺氧下耗氧量都下降,但肝癌細(xì)胞的ATP產(chǎn)量代償性增加,而正常肝細(xì)胞的ATP顯著下降;有氧氧化相關(guān)酶基因在缺氧小鼠肝臟細(xì)胞中代償性上調(diào),而在缺氧HepG2細(xì)胞中下降;糖酵解相關(guān)酶基因在缺氧HepG2細(xì)胞中上調(diào)更迅速,幅度更大;進(jìn)一步研究表明,缺氧時(shí),小鼠肝臟細(xì)胞p53、Tigar、Sco2基因表達(dá)上調(diào),而HepG2細(xì)胞p53、TIGAR、SCO2基因表達(dá)下調(diào)。結(jié)論在缺氧應(yīng)激狀態(tài)下,正常細(xì)胞主要依賴有氧氧化補(bǔ)償能量,腫瘤細(xì)胞則主要依賴糖酵解。ROS或可通過(guò)調(diào)節(jié)腫瘤細(xì)胞p53/TIGAR、p53/SCO2通路及糖酵解關(guān)鍵酶活性促進(jìn)Warburg效應(yīng)與腫瘤耐缺氧能力。

【關(guān)鍵詞】腫瘤缺氧;能量代謝通路;活性氧;Warburg效應(yīng);小鼠

*This work was supported by the National Natural Science Foundation of China (31270901,30970684).

新生血管畸變導(dǎo)致血液供應(yīng)不足,腫瘤與周邊正常組織最大的差別就是腫瘤內(nèi)部的缺氧微環(huán)境。不同于正常組織,腫瘤在氧氣及養(yǎng)分不足的環(huán)境中仍得以存活生長(zhǎng),并獲得抗藥性。大量研究已證實(shí),腫瘤部分區(qū)域極低濃度的氧氣反而會(huì)增加腫瘤的侵襲性行為和顯示更差的預(yù)后[1]。腫瘤細(xì)胞對(duì)缺氧的強(qiáng)耐受力被認(rèn)為是腫瘤發(fā)展、侵襲、轉(zhuǎn)移的重要因素[2]。缺氧對(duì)組織細(xì)胞帶來(lái)的危害主要包括活性氧(reactive oxygen species,ROS)的大量增加以及氧氣不足造成的能量耗竭[3]。因此,缺氧腫瘤細(xì)胞維持自身代謝活動(dòng)的方式便是研究腫瘤缺氧耐受能力的關(guān)鍵。

Warburg效應(yīng)揭示了腫瘤細(xì)胞有氧糖酵解的獨(dú)特能量代謝方式。我們以前的研究發(fā)現(xiàn)[4-5],缺氧和活性氧可以上調(diào)細(xì)胞內(nèi)的糖酵解通路,Warburg效應(yīng)可能與氧化應(yīng)激微環(huán)境相關(guān)。然而,腫瘤細(xì)胞究竟如何利用低效率的糖酵解滿足其體內(nèi)快速生長(zhǎng)所需要的能量需求,其機(jī)制尚不清楚。

p53在代謝通路調(diào)控中起重要作用。p53可轉(zhuǎn)錄激活Tigar蛋白,后者通過(guò)降解細(xì)胞內(nèi)2,6-二磷酸果糖抑制糖酵解,使葡萄糖通量涌入PPP通路[4-6]。p53還可上調(diào)呼吸鏈復(fù)合物Ⅳ中的細(xì)胞色素C氧化酶組裝蛋白2(cytochrome C oxidase assembly protein,SCO2)[7],從而促進(jìn)氧化磷酸化。我們推測(cè)缺氧腫瘤細(xì)胞可能通過(guò)p53/TIGAR和p53/SCO2通路調(diào)控缺氧腫瘤能量代謝。

本文采用環(huán)境缺氧方式(0.2% O2缺氧培養(yǎng)箱)模擬腫瘤內(nèi)部的缺氧環(huán)境,比較腫瘤細(xì)胞與正常細(xì)胞在糖酵解、有氧氧化、能量生成、p53及其下游信號(hào)等方面的變化,探討腫瘤細(xì)胞缺氧耐受的產(chǎn)生機(jī)制,為腫瘤的靶向治療提供新的思路。

材 料 和 方 法

實(shí)驗(yàn)動(dòng)物、細(xì)胞株與儀器SPF級(jí)昆明小鼠購(gòu)自復(fù)旦大學(xué)實(shí)驗(yàn)動(dòng)物科學(xué)部[生產(chǎn)許可證號(hào)：SCXK(滬) 2014-0004];人肝癌細(xì)胞株HepG2購(gòu)自ATCC細(xì)胞庫(kù);三氣培養(yǎng)箱(德國(guó)Binder CB系列);CO2細(xì)胞培養(yǎng)箱(美國(guó)Thermo Forma公司);液相氧電極(Oxygraph,英國(guó)Hansatech公司);高效液相色譜HPLC系統(tǒng)(日本島津公司);高效液相色譜柱[ODS-SPC18(4.6 mm×250 mm,粒徑5 μm),日本島津公司];熒光定量PCR儀(7900HT,美國(guó)ABI公司);流式細(xì)胞儀(FC500,美國(guó)Beckman Coulter公司)。

小鼠肝臟細(xì)胞原代分離小鼠麻醉后剖開(kāi)腹腔,暴露門靜脈并插入塑料導(dǎo)管,灌注37 ℃的Hanks平衡鹽溶液5～10 min以除去肝內(nèi)血液。結(jié)扎下腔靜脈,并插入另一根塑料導(dǎo)管,同時(shí)結(jié)扎胸腔段靜脈。灌注37℃膠原酶消化液(Ⅳ型膠原酶,0.05%),待整肝鼓脹泛白后,撕去表面包膜并將肝細(xì)胞轉(zhuǎn)移至DMEM培養(yǎng)液(含10%FBS,105U/L青霉素與鏈霉素)。細(xì)胞懸液經(jīng)200目不銹鋼濾網(wǎng)除去細(xì)胞團(tuán)塊,并以上述DMEM培養(yǎng)液洗滌3次,低速離心(1 000 r/min,離心半徑為13 cm,5 min)收取純化肝細(xì)胞并接種于無(wú)菌細(xì)胞培養(yǎng)皿。接種4 h后更換細(xì)胞培養(yǎng)液。

細(xì)胞培養(yǎng)與缺氧HepG2細(xì)胞株在含有10% FBS DMEM培養(yǎng)液(含青霉素和鏈霉素各100 U/mL)中,于37 ℃、5% CO2條件下培養(yǎng)至貼壁面積達(dá)80%以上。使用含0.02% EDTA的0.25%胰酶消化后,按1∶2或1∶4比例傳代,貼壁4～6 h后備用。原代小鼠肝臟細(xì)胞自分離接種4 h后備用。將已貼壁細(xì)胞轉(zhuǎn)移放入常規(guī)細(xì)胞培養(yǎng)箱繼續(xù)培養(yǎng)24 h,作為缺氧0 h對(duì)照組;先于常規(guī)培養(yǎng)箱中繼續(xù)培養(yǎng)18 h,再轉(zhuǎn)入37℃、0.2% O2缺氧培養(yǎng)箱中培養(yǎng)6 h,作為缺氧6 h組;先于常規(guī)培養(yǎng)箱中繼續(xù)培養(yǎng)12 h,再轉(zhuǎn)入37℃、0.2% O2缺氧培養(yǎng)箱中培養(yǎng)12 h,作為缺氧12 h組;直接轉(zhuǎn)入37 ℃、0.2% O2缺氧培養(yǎng)箱中繼續(xù)培養(yǎng)24 h,作為缺氧24 h組。

MTT法檢測(cè)細(xì)胞活力取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于96孔培養(yǎng)板,每孔200 μL細(xì)胞懸液(5×103細(xì)胞)。培養(yǎng)4～6 h待細(xì)胞貼壁后,每組設(shè)置至少3個(gè)平行孔。于培養(yǎng)常氧培養(yǎng)箱與缺氧培養(yǎng)箱中各培養(yǎng)24、48、72 h。達(dá)到缺氧時(shí)間后,迅速向各孔加入20 μL (5 mg/mL)MTT溶液,37 ℃孵育4 h后吸去各孔內(nèi)溶液,加入150 μL DMSO,置搖床上低速震蕩10 min,待結(jié)晶充分溶解后,以DMSO調(diào)零,用多功能酶標(biāo)儀檢測(cè)儀在490 nm處測(cè)定各孔吸光度D,并計(jì)算細(xì)胞存活率(survival rate,SR)。SR =實(shí)驗(yàn)組D490/對(duì)照組D490×100%。

高效液相色譜(HPLC)檢測(cè)ATP含量細(xì)胞ATP樣品收集及HPLC檢測(cè)方法參考Zur等[8]的研究。

熒光定量PCR檢測(cè)胞內(nèi)目的基因mRNA表達(dá)水平用冰冷PBS清洗細(xì)胞,按105～106細(xì)胞/mL加入Trizol試劑充分吹打,靜置10 min后轉(zhuǎn)入 1.5 mL離心管;加入200 μL氯仿,渦旋混勻,室溫靜置10 min;12 000 ×g 4 ℃離心15 min,上清(約500 μL)轉(zhuǎn)移至另一離心管;加500 μL異丙醇,震蕩混勻,室溫靜置10 min;12 000×g 4 ℃離心15 min,可見(jiàn)RNA絮狀沉淀,將上清小心棄去,并加入500 μL預(yù)冷的75%乙醇洗滌RNA沉淀1次,棄去乙醇,適度揮發(fā)后,用DEPC處理水溶解RNA,檢測(cè)RNA濃度并調(diào)整為2 μg /10 μL,置冰上備用。逆轉(zhuǎn)錄PCR體系按照DBI公司RT-PCR試劑盒(DBI-2226)說(shuō)明書進(jìn)行,反轉(zhuǎn)錄cDNA儲(chǔ)備液稀釋適當(dāng)倍數(shù)后,按DBI公司熒光定量PCR試劑盒(DBI-2043)說(shuō)明書配置熒光定量PCR體系并進(jìn)行熒光定量PCR。

流式細(xì)胞術(shù)檢測(cè)細(xì)胞ROS水平細(xì)胞接種于6孔板,培養(yǎng)完成后,棄培養(yǎng)液,PBS清洗1次后棄盡,每孔加1 mL DCF工作液(10 μmol/L),鋪勻后避光37 ℃孵育20 min,后續(xù)步驟避光操作。洗棄DCF工作液,用PBS清洗細(xì)胞表面3次,胰酶消化收集細(xì)胞于500 μL PBS,冰浴送檢。設(shè)置流式細(xì)胞儀激發(fā)波長(zhǎng)為488 nm,發(fā)射波長(zhǎng)為525 nm,檢測(cè)各樣品的平均熒光強(qiáng)度,作為ROS水平的度量。

細(xì)胞耗氧量檢測(cè)設(shè)定氧電極溫度37 ℃,于檢測(cè)小室中加入2 mL純凈水并持續(xù)攪拌至溫度恒定且氧氣飽和。吸出檢測(cè)室內(nèi)純凈水,加入2 mL細(xì)胞培養(yǎng)液攪拌至信號(hào)平衡,記錄培養(yǎng)液起始氧濃度。向檢測(cè)小室內(nèi)注入100 μL樣品細(xì)胞懸液(含105細(xì)胞),并立即計(jì)時(shí)5 min,記錄溶解氧變化。

統(tǒng)計(jì)學(xué)分析所有數(shù)據(jù)使用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行分析,采用Student’st檢測(cè)或One-way Anova方法進(jìn)行檢驗(yàn),P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié)果

缺氧條件下肝癌細(xì)胞和正常肝細(xì)胞ROS水平及生存的差異隨缺氧時(shí)間延長(zhǎng),肝癌細(xì)胞與正常肝細(xì)胞的ROS水平均逐步提高,但正常細(xì)胞增加得更快,增幅更大。缺氧24 h組小鼠肝臟細(xì)胞的ROS水平是缺氧0 h組小鼠肝臟細(xì)胞的4倍,而缺氧24 h組HepG2細(xì)胞的ROS水平則僅為缺氧0 h組HepG2細(xì)胞的1.8倍。在此過(guò)程中,HepG2細(xì)胞各組的ROS水平始終高于同條件下的正常細(xì)胞組(圖1A)。缺氧條件下,兩種細(xì)胞的存活率都隨缺氧時(shí)間延長(zhǎng)而減少,但小鼠肝臟細(xì)胞下降幅度更大,缺氧24 h組小鼠肝臟細(xì)胞的生存率已降至40%以下,而缺氧24h組HepG2細(xì)胞的生存率仍維持在80%左右,說(shuō)明腫瘤細(xì)胞具有較強(qiáng)的缺氧耐受能力(圖1B)。

缺氧條件下肝癌細(xì)胞和正常肝細(xì)胞在糖酵解通路相關(guān)基因的差異如圖2A所示,兩種細(xì)胞己糖激酶基因的水平在缺氧6 h、12 h時(shí)都代償升高,而在24 h則呈下降趨勢(shì)。圖2B表示,缺氧后HepG2細(xì)胞丙酮酸脫氫酶基因PKM2的水平比較穩(wěn)定,缺氧24 h組HepG2細(xì)胞的PKM2表達(dá)與其缺氧0 h組差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),而缺氧24 h組小鼠肝臟細(xì)胞PKM2基因的表達(dá)與其缺氧0 h組相比則顯著降低(P<0.05)。如圖2C所示,缺氧后小鼠肝臟細(xì)胞及HepG2細(xì)胞LDH活力均隨缺氧時(shí)間延長(zhǎng)而增加,且HepG2細(xì)胞增加更為迅速,漲幅更大。缺氧24 h組小鼠肝臟細(xì)胞的LDH活性是0 h組的10倍,而缺氧24 h組HepG2細(xì)胞的LDH活性則為0 h組的22倍以上。這些結(jié)果都說(shuō)明在缺氧的情況下,相對(duì)于正常細(xì)胞,腫瘤細(xì)胞更傾向于通過(guò)糖酵解通路相關(guān)酶的表達(dá)或活性代償升高來(lái)補(bǔ)償能量供應(yīng)的不足。

The introcellular ROS level(A) and survival curve (B) of mice hepatocellular and HepG2 under hypoxia. Cell viability (%control) represents the ratio of hypoxic cell viability to nomoxic cell viability being cultured for the same time.(1)vs. hepatocytes 0 h group,P<0.05;(2)vs. HepG2 0 h group,P<0.05 (n≥3).

圖1缺氧小鼠肝臟細(xì)胞與HepG2細(xì)胞的生存曲線與胞內(nèi)ROS水平

Fig 1Survival rate and ROS level of mice hepatocytes and HepG2 cells under hypoxia

(1)vs. hepatocytes 0 h group,P<0.05;(2)vs. HepG2 0 h group,P<0.05 (n≥3).

圖2缺氧小鼠肝臟細(xì)胞與HepG2細(xì)胞的糖酵解水平

Fig 2The glycolysis level of mice hepatocytes and HepG2 cells under hypoxia

缺氧條件下肝癌細(xì)胞和正常肝細(xì)胞在有氧氧化通路相關(guān)基因的差異如圖3A所示,兩種細(xì)胞異檸檬酸脫氫酶的基因水平在缺氧6 h時(shí)都呈現(xiàn)代償升高然后下降的趨勢(shì)。在缺氧12 h時(shí),小鼠肝臟細(xì)胞Idh表達(dá)雖有回落,但仍與缺氧0 h對(duì)照組持平(P>0.05),而相同缺氧時(shí)長(zhǎng)的HepG2細(xì)胞已經(jīng)顯著低于其0 h對(duì)照組(P<0.05)。如圖3B所示,與IDH的基因表達(dá)相似,缺氧后HepG2琥珀酸脫氫酶SDH基因表達(dá)水平隨缺氧時(shí)間迅速下降,缺氧6 h組差異已出現(xiàn)統(tǒng)計(jì)學(xué)意義(P<0.05,vs. HepG2缺氧0 h組),而小鼠肝臟細(xì)胞Sdh表達(dá)水平直到缺氧12 h依然處于代償性升高,與其缺氧0 h組差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。進(jìn)一步說(shuō)明在缺氧的情況下,正常細(xì)胞比腫瘤細(xì)胞更依賴于有氧氧化通路。此結(jié)果說(shuō)明與腫瘤細(xì)胞不同,正常細(xì)胞在缺氧的情況下主要依靠有氧氧化通路代償性升高來(lái)補(bǔ)償能量供應(yīng)的不足。

缺氧條件下肝癌細(xì)胞和正常肝細(xì)胞在耗氧率與ATP生成的差異如圖4A所示,隨缺氧時(shí)間的延長(zhǎng),兩種細(xì)胞的耗氧率都顯著降低(P<0.05),但HepG2細(xì)胞的耗氧率始終顯著高于小鼠肝臟細(xì)胞,說(shuō)明在缺氧的情況下,腫瘤細(xì)胞消耗的氧氣仍比正常細(xì)胞多。 如圖4B所示,兩種細(xì)胞在缺氧時(shí)ATP生成量的變化截然相反。缺氧后,HepG2細(xì)胞的ATP的生成代償性升高,直至24 h依然不低于其缺氧0 h組(P>0.05)。而小鼠肝臟細(xì)胞ATP含量則隨缺氧時(shí)間迅速下降。說(shuō)明在缺氧的情況下,腫瘤細(xì)胞依然能產(chǎn)生足夠的ATP,而正常細(xì)胞則可能已經(jīng)處于ATP匱乏的狀態(tài)。缺氧24 h內(nèi),腫瘤細(xì)胞耗氧率的下降與ATP的增加,說(shuō)明這部分增加的ATP可能主要來(lái)自于糖酵解。

(1)vs. hepatocytes 0 h group,P<0.05;(2)vs. HepG2 0 h group,P<0.05 (n≥3).

圖3缺氧小鼠肝臟細(xì)胞與缺氧HepG2細(xì)胞的有氧氧化關(guān)鍵酶的基因表達(dá)

Fig 3The expression of key enzymes involved in aerobic oxidation in mice hepatocytes and HepG2 cells under hypoxia

The oxygen consumption rate (A) and ATP production of mice hepatocytes and HepG2 cells under hypoxia (B). All values were mean±SD.(1)vs. hepatocytes 0 h group,P<0.05;(2)vs. HepG2 0 h group,P<0.05 (n≥3);(3)vs. hepatocytes 6 h group,P<0.05;(4)vs. HepG2 6 h group,P<0.05 (n≥3).

圖4缺氧小鼠肝臟細(xì)胞與缺氧HepG2細(xì)胞的能量代謝狀態(tài)

Fig 4The energy metabolism state of mice hepatocytes and HepG2 cells under hypoxia

腫瘤細(xì)胞對(duì)缺氧耐受與p53、tigar及sco2基因表達(dá)相關(guān)如圖5,小鼠肝臟細(xì)胞p53、tigar、sco2的基因mRNA水平在缺氧12 h后分別增長(zhǎng)為缺氧0 h對(duì)照組的1.7倍、1.22倍與4.29倍,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。這3種基因在缺氧12 h組HepG2細(xì)胞內(nèi)表達(dá)變化則截然相反。相比于缺氧0 h組,缺氧12 h組HepG2細(xì)胞p53、TIGAR、SCO2的基因mRNA水平分別降低了36%、63%和46%,差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。

討論

腫瘤細(xì)胞生長(zhǎng)增殖迅速,而血管生成相對(duì)滯后,且新生血管普遍存在異常,這種情況導(dǎo)致了腫瘤細(xì)胞大多數(shù)情況下處于缺氧及養(yǎng)分供應(yīng)不足的微環(huán)境中。細(xì)胞在缺氧環(huán)境中會(huì)經(jīng)歷一系列生理變化,缺氧嚴(yán)重時(shí)則會(huì)引發(fā)凋亡。腫瘤細(xì)胞在衍變過(guò)程中產(chǎn)生了對(duì)缺氧微環(huán)境較強(qiáng)的耐受能力,使其不僅可以存活而且能維持一定程度的增殖,同時(shí)還伴隨著惡化轉(zhuǎn)移[1]及放化療抗性,我們的實(shí)驗(yàn)結(jié)果也證實(shí)了腫瘤細(xì)胞具有更高的缺氧生存率(圖1B)。因此,腫瘤細(xì)胞對(duì)于缺氧應(yīng)激所做出的各種適應(yīng)性應(yīng)答將為有效的抗癌療法提供靶點(diǎn)。本研究采用0.2%環(huán)境氧濃度對(duì)細(xì)胞進(jìn)行缺氧處理。在該濃度下平衡的無(wú)細(xì)胞培養(yǎng)液溶解氧濃度為32 mmHg(1 mmHg=0.133 kPa,下同),介于人體內(nèi)正常組織氧分壓60 mmHg與腫瘤內(nèi)部氧分壓15 mmHg之間[9-10]。

(1)vs. hepatocytes 0 h group,P<0.05;(2)vs. HepG2 0 h group,P<0.05 (n≥3).

圖5缺氧小鼠肝臟細(xì)胞與缺氧HepG2細(xì)胞p53,TIGAR/Tigar及SCO2/Sco2基因表達(dá)

Fig 5The expression ofp53,TIGAR/TigarandSCO2/Sco2 in mice hepatocytes or HepG2 cells under hypoxia

缺氧對(duì)組織細(xì)胞帶來(lái)的危害之一是氧氣不足造成的能量耗竭[11]。ATP含量是細(xì)胞能量產(chǎn)出的直接指標(biāo),本研究通過(guò)對(duì)比腫瘤細(xì)胞與正常細(xì)胞在缺氧后ATP產(chǎn)量的變化,探究?jī)烧吣芰慨a(chǎn)出受缺氧環(huán)境的影響程度。研究結(jié)果表明(圖4B),缺氧腫瘤細(xì)胞的能量耗竭速率要遠(yuǎn)低于正常細(xì)胞。相比于正常細(xì)胞迅速降低的ATP含量,腫瘤細(xì)胞ATP含量在短時(shí)間缺氧時(shí)甚至還出現(xiàn)一定程度的代償性增加。細(xì)胞ATP產(chǎn)出來(lái)源于糖酵解通路及線粒體有氧氧化,1分子葡萄糖經(jīng)由糖酵解通路分解為丙酮酸,產(chǎn)出2個(gè)ATP,丙酮酸在氧氣充足的條件下進(jìn)入線粒體繼續(xù)氧化,此過(guò)程中可直接或間接地生成36個(gè)ATP。因此,線粒體有氧氧化是需氧生物能量產(chǎn)出的主要來(lái)源。然而,本研究發(fā)現(xiàn)正常細(xì)胞與腫瘤細(xì)胞在缺氧后耗氧量均受到抑制(圖4A),而腫瘤細(xì)胞在耗氧量降低的同時(shí)ATP產(chǎn)出卻是大幅增加的,說(shuō)明腫瘤細(xì)胞通過(guò)線粒體有氧氧化以外的其他方式有效地補(bǔ)償了線粒體能量產(chǎn)出的不足,即糖酵解通路,以此維持缺氧條件下自身的能量供應(yīng)。

IDH與SDH是細(xì)胞參與有氧氧化的重要成員,前者參與三羧酸循環(huán)(TCA cycle),將NAD還原為NADH,SDH既是TCA循環(huán)成員,同時(shí)也是線粒體傳遞鏈的復(fù)合體2。本實(shí)驗(yàn)對(duì)比了缺氧后正常細(xì)胞與腫瘤細(xì)胞IDH/Idh與SDH/Sdh基因的表達(dá),用以說(shuō)明細(xì)胞內(nèi)有氧氧化水平。結(jié)果表明(圖3)缺氧后的正常細(xì)胞會(huì)通過(guò)提高有氧氧化相關(guān)基因的表達(dá)水平,來(lái)彌補(bǔ)氧氣缺失所造成的能量產(chǎn)出不足,說(shuō)明正常細(xì)胞在缺氧環(huán)境中仍然主要依靠有氧氧化功能。然而,這顯然并不能長(zhǎng)久維持,隨著缺氧時(shí)間的延長(zhǎng),以氧氣作為最終電子受體的呼吸鏈?zhǔn)茏?導(dǎo)致細(xì)胞能量耗竭而死亡。Warburg效應(yīng)的提出揭示了腫瘤細(xì)胞不同于正常細(xì)胞的代謝方式,該假說(shuō)認(rèn)為,腫瘤細(xì)胞即便在氧氣充足的條件下也依賴糖酵解供能的原因在于線粒體缺陷,說(shuō)明腫瘤細(xì)胞已不以線粒體呼吸作為主要能量產(chǎn)出方式。本研究中,與正常小鼠肝臟細(xì)胞不同,腫瘤細(xì)胞HepG2在缺氧后IDH與SDH基因表達(dá)水平迅速下調(diào)。我們推測(cè),缺氧腫瘤細(xì)胞有氧氧化相關(guān)酶的下調(diào),使得TCA循環(huán)減慢,減少了TCA循環(huán)產(chǎn)物NADH對(duì)呼吸鏈的遞氫量,不但降低了缺氧狀態(tài)下細(xì)胞在有氧氧化通路上不必要的物料浪費(fèi),將葡萄糖更多地送入無(wú)氧糖酵解與磷酸戊糖通路(pentose phosphate pathway,PPP),同時(shí)也阻止了由于呼吸鏈?zhǔn)茏?、電子溢出而?dǎo)致的ROS大量增加,降低細(xì)胞缺氧損傷。本研究的結(jié)果也驗(yàn)證了這一觀點(diǎn),腫瘤細(xì)胞缺氧后ROS增幅小于正常細(xì)胞(圖1A),兩種細(xì)胞糖酵解相關(guān)酶HK2/Hk2、PKM2/Pkm2基因表達(dá)水平均上調(diào),但腫瘤細(xì)胞上調(diào)幅度更大(圖2A、2B),其無(wú)氧糖酵解末端酶LDH活性也大幅上調(diào),增幅遠(yuǎn)高于正常細(xì)胞(圖2C)。這些結(jié)果說(shuō)明,缺氧條件下正常細(xì)胞仍依賴有氧氧化代償增加補(bǔ)充能量,而腫瘤細(xì)胞則依賴迅速高度活化的無(wú)氧糖酵解。

我們以前的研究表明[12],缺氧可誘導(dǎo)ROS釋放,并可通過(guò)缺氧誘導(dǎo)因子(hypoxia-inducible factor-1)上調(diào)細(xì)胞內(nèi)的糖酵解通路,說(shuō)明腫瘤Warburg效應(yīng)可能與其缺氧氧化應(yīng)激微環(huán)境相關(guān)。此外,ROS也被發(fā)現(xiàn)可通過(guò)調(diào)節(jié)p53基因影響細(xì)胞生長(zhǎng)。p53是一種重要的腫瘤抑制基因,且有超過(guò)半數(shù)以上的惡性腫瘤被發(fā)現(xiàn)存在p53突變,失去了對(duì)細(xì)胞生長(zhǎng)凋亡的調(diào)控作用[13-14]。 但近年來(lái)發(fā)現(xiàn)p53可以通過(guò)誘導(dǎo)TIGAR降解2,6-二磷酸果糖從而抑制糖酵解,并可活化SCO2提升有氧氧化,在腫瘤能量代謝中起重要調(diào)控作用[15-16]。本研究對(duì)比了缺氧后腫瘤細(xì)胞與正常細(xì)胞P53/p53、TIGAR/Tigar及SCO2/Sco2的mRNA表達(dá)量(圖5),結(jié)果與設(shè)想一致,正常細(xì)胞缺氧12 h后p53基因表達(dá)上調(diào),Tigar基因表達(dá)也隨之上調(diào),提示正常細(xì)胞缺氧后糖酵解通路可能因2,6-二磷酸果糖的降解而受到抑制。由于2,6-二磷酸果糖處于己糖激酶的下游,故Tigar表達(dá)上調(diào)并不會(huì)影響Hk2的基因表達(dá),且Tigar與Hk2表達(dá)共同上調(diào)可進(jìn)一步促進(jìn)葡萄糖通量涌入PPP通路。因此,正常細(xì)胞缺氧己糖激酶基因表達(dá)代償性增加并非主要用于補(bǔ)充能量,而是用于促進(jìn)PPP通路產(chǎn)生更多的NADPH以對(duì)抗缺氧產(chǎn)生的ROS。但此代償作用并不持久,缺氧24 h后,正常細(xì)胞Hh2與其Pkm2基因表達(dá)一樣,受到了顯著抑制。此外,缺氧12 h后正常細(xì)胞Sco2的mRNA水平大幅提升,這與正常細(xì)胞缺氧后Idh、Sdh基因表達(dá)代償性增加的結(jié)果一致,說(shuō)明正常細(xì)胞缺氧后通過(guò)有氧氧化代償補(bǔ)充能量。與正常細(xì)胞不同,本研究中腫瘤細(xì)胞p53、TIGAR、SCO2基因均降低。Tigar基因的降低穩(wěn)定了2,6-二磷酸果糖對(duì)糖酵解通路的促進(jìn)作用,處于下游的PKM2基因表達(dá)穩(wěn)定(圖2B),LDH活性大幅增加(圖2C),說(shuō)明缺氧腫瘤細(xì)胞極大地增加提高了無(wú)氧糖酵解水平,原本進(jìn)入線粒體繼續(xù)氧化的丙酮酸轉(zhuǎn)而大量生成乳酸。此過(guò)程雖然產(chǎn)能低效,卻很大程度上通過(guò)降低有氧氧化及NADH生成,阻止NADH在缺氧的環(huán)境下向線粒體持續(xù)遞氫,防止電子傳遞鏈由于電子終受體氧分子不足而大量溢出,保護(hù)線粒體不受電子溢出所產(chǎn)生的ROS損傷,抑制線粒體介導(dǎo)的細(xì)胞凋亡。本研究中,相對(duì)于正常細(xì)胞,腫瘤細(xì)胞的ROS水平在缺氧后更為穩(wěn)定也進(jìn)一步證實(shí)了此觀點(diǎn)(圖1A)。即便如此,腫瘤細(xì)胞的ROS水平始終是高于正常細(xì)胞的,暗示了ROS在腫瘤細(xì)胞中的重要作用。結(jié)合ROS對(duì)p53、HIF-1等基因的多重調(diào)控作用,以及我們?cè)缙趯?duì)ROS調(diào)控p53基因表達(dá)的研究[17],仍可認(rèn)為腫瘤細(xì)胞所維持的一定量ROS參與調(diào)控了腫瘤細(xì)胞缺氧能量代謝及缺氧耐受。

本研究發(fā)現(xiàn)在缺氧應(yīng)激狀態(tài)下,細(xì)胞可通過(guò)代償性提高能量代謝通路來(lái)補(bǔ)償能量供應(yīng),正常細(xì)胞主要依賴有氧氧化補(bǔ)償能量,而腫瘤細(xì)胞則主要依賴糖酵解。缺氧腫瘤細(xì)胞獨(dú)特的能量代謝方式可能歸因于ROS參與調(diào)控的p53/TIGAR及p53/SCO2通路,這不僅可維持其能量供應(yīng),還降低了缺氧應(yīng)激對(duì)線粒體的損傷,抑制線粒體介導(dǎo)的細(xì)胞凋亡(圖6)。這一發(fā)現(xiàn)為腫瘤細(xì)胞缺氧耐受能力提供了理論基礎(chǔ),并一定程度上解釋了腫瘤Warburg效應(yīng),為腫瘤的靶向治療提供了思路,但ROS參與腫瘤細(xì)胞缺氧能量調(diào)節(jié)及缺氧耐受能力形成的具體方式、作用節(jié)點(diǎn)等問(wèn)題,仍需進(jìn)一步研究和探討。

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The difference of energy metabolism pathways between normalhepatocytes and hepatoma cells under hypoxia

LI Peng-ying, ZHANG Dong-yang, WU Mei-ling, DONG Ke-lei, SHI Dong-yun△

(DepartmentofBiochemistryandMolecularBiology,SchoolofBasicMedicalSciences,FudanUniversity,Shanghai200032,China)

【Abstract】ObjectiveTo compare the difference between energy metabolism pathways of hypoxic normal hepatocytes cells and tumor cells, and try to understand the underlying mechanism of tumor hypoxia resistance.MethodsWe adopted environmental hypoxia (Hypoxia incubator with 0.2%O2) to mimic the tumor environment in vitro,and compared the difference of energy metabolism response to hypoxic stress between HepG2 cells and mice hepatocytes.Flow cytometry was used to detect the level of different kinds of ROS.MTT was used to compare the viability of cells before and afterhypoxia.The mRNA level of p53,Tigar,SCO2, and key enzymes in the metabolism including hexokinase 2 and isocitrate dehydrogenase,were detected by real-time PCR.NADH colorimetric assay was used to detect lactate dehydrogenase activity.Oxygen electrode was used to detect the oxygen consumption rate of cells.HPLC was used to detect the ATP levels in cells.ResultsUnder moderate hypoxia, HepG2 cells had a higher survival rate and underwent a more steady reactive oxygen species (ROS) fluctuation. The oxygen consumption rate declined in both types of cells, ATP production in hepatoma cells increased with cellular proliferation being maintained,but decreased significantly in normal liver cells. The results also showed that key enzymes involved in aerobic oxidation were compensatively increased in normal liver cells but decreased in hepatoma cells under hypoxia. The key enzymes involved in glycolysis were both upregulated in response to hypoxia, but the hepatoma cells increased more significantly. Further studies showed that in response to hypoxia, the mRNA expression of p53,Tigar and Sco2 were all upregulated in normal liver cells but p53,TIGAR and SCO2 downregulated in hepatoma cells.ConclusionsUnder hypoxia stress, normal cells mainly depend on aerobic oxidation to compensate the energy, but hepatoma cells mainly rely on glycolysis. ROS may enhance glycolysis through p53/TIGAR and p53/SCO2 pathways, so as to promote the Warburg effect and hypoxia tolerance.

【Key words】tumor hypoxia;energy metabolism pathway;reactive oxygen species;Warburg effect;mouse

(收稿日期:2015-11-24;編輯:王蔚)

【中圖分類號(hào)】R730

【文獻(xiàn)標(biāo)識(shí)碼】A

doi:10.3969/j.issn.1672-8467.2016.02.001

國(guó)家自然科學(xué)基金(31270901, 30970684)

△Corresponding authorE-mail:dyshi@fudan.edu.cn