失巢凋亡與中醫(yī)藥抗腫瘤研究進(jìn)展

1、失巢凋亡與中醫(yī)藥抗腫瘤研究進(jìn)展*通訊作者簡介：沈克平，男，主任醫(yī)師，碩士生導(dǎo)師，主要從事消化道腫瘤研究。地址：上海市宛平南路725號(hào)，龍華醫(yī)院腫瘤五科 郵編200032 電話153 E-mail：沈克平，劉威，胡兵（上海中醫(yī)藥大學(xué)附屬龍華醫(yī)院腫瘤五科， 200032)摘要：失巢凋亡是細(xì)胞脫離胞外基質(zhì)或與胞外基質(zhì)不結(jié)合而發(fā)生的一種凋亡。失巢凋亡耐受是轉(zhuǎn)移細(xì)胞的基本特性，可使腫瘤細(xì)胞在脫離與基質(zhì)粘附狀態(tài)時(shí)仍能存活。細(xì)胞失巢凋亡主要有死亡受體途徑及線粒體途徑。參與失巢凋亡的信號(hào)轉(zhuǎn)導(dǎo)通路主要有磷脂酰肌醇3激酶AKT（PI3-k/AKT）通路和絲裂原活化蛋白激酶（MPAK）通

2、路。近期發(fā)現(xiàn)癌基因 TrkB、抑癌基因PTEN也參與失巢凋亡調(diào)控。目前研究發(fā)現(xiàn)部分中藥抗腫瘤的作用機(jī)制之一是抑制腫瘤細(xì)胞錨著不依賴性增殖，促進(jìn)腫瘤細(xì)胞失巢凋亡。關(guān)鍵詞： 失巢凋亡；腫瘤轉(zhuǎn)移；信號(hào)轉(zhuǎn)導(dǎo)；中醫(yī)藥基金資助：國家自然科學(xué)基金（30873258），上海市浦東新區(qū)中醫(yī)領(lǐng)軍型人才項(xiàng)目（PWZ2008-20-104）Anoikis and anti-cancer effects of Chinese herbsShen-keping, Liu-wei, Hu-bing5th Department of Oncology, Longhua Hospital, Shanghai Universit

3、y of Traditional Chinese Medicine,Abstract: Anoikis is a form of apoptosis induced by detachment of adherent cells from the extracellular matrix or inappropriate cell-matrix interactions. Resistance to anoikis is a hallmark of metastatic cancer cells which leads to cancer cells evading anoikis and t

4、hereby survive after detachment from their primary site, and metastasizing to distant organs. Both death receptor-mediated extrinsic pathway and mitochondrial-mediated intrinsic pathway contribute to anoikis. Phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway and mitogen-activated protein kinas

5、e (MAPK) signal are major regulator of anoikis. In addition, oncogene TrkB and tumor suppressor gene PTEN may also contribute to anoikis regulation. Some studies suggested some anti-cancer Chinese herbs or components may inhibit anchoring-independent cancer cell growth and induces anoikis. Key words

6、: Anoikis, metastasis, signal transduction, Chinese herbProject supported by the National Natural Science Foundation of China (30873258) andTCM Leading Talents Project of Shanghai Pu-dong New Area(PWZ2008-20-104)細(xì)胞凋亡（Apoptosis）是機(jī)體生長、分化、發(fā)育及病理過程中，由細(xì)胞基因調(diào)控的自發(fā)死亡過程，又稱程序性細(xì)胞死亡（programmed cell death）。正常上皮或內(nèi)皮

7、細(xì)胞具有粘附依賴性，其存活依賴于細(xì)胞間和細(xì)胞與基質(zhì)間的信號(hào)傳遞，即錨定依賴，一旦脫離細(xì)胞基質(zhì)粘附，失去胞間聯(lián)系即發(fā)生凋亡，這種形式的細(xì)胞死亡被命名為“anoikis”，中文譯為“失巢凋亡”1。失巢凋亡具有重要的生理意義，細(xì)胞脫離基質(zhì)粘附狀態(tài)發(fā)生失巢凋亡，對(duì)機(jī)體發(fā)育和組織自身平衡起重要作用。失巢凋亡耐受或抑制與惡性腫瘤細(xì)胞轉(zhuǎn)移和存活密切相關(guān)2。本文就失巢凋亡的分子機(jī)制，涉及的主要細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路及中藥對(duì)腫瘤細(xì)胞失巢凋亡的影響闡述如下。1 .失巢凋亡分子機(jī)制1.1死亡受體途徑失巢凋亡一部分是通過細(xì)胞死亡受體途徑激活半胱氨酸蛋白酶調(diào)節(jié)的3-5。在死亡受體途徑當(dāng)中，細(xì)胞外配體（如Fas-L、TRAIL

8、等）結(jié)合導(dǎo)致死亡受體（如FAS、TRAIL-R等）寡聚化，活化FAS相關(guān)死亡域蛋白（FAS-Associated Death Domain，F(xiàn)ADD），進(jìn)而募集caspase-8形成死亡信號(hào)誘導(dǎo)復(fù)合物（death inducing signalling complex，DISC），活化caspase-8可激活caspase-3、caspase-7等凋亡執(zhí)行蛋白，導(dǎo)致細(xì)胞凋亡6。死亡受體途徑也可為非受體依賴型，Stupack等7研究表明非結(jié)合的整合素可以介導(dǎo)caspase-8外膜活化而非受體依賴。與正常細(xì)胞相比，惡性腫瘤細(xì)胞在脫離細(xì)胞質(zhì)基質(zhì)后盡管能夠上調(diào)FAS及配體，但卻不能活化caspase激

9、活死亡受體途徑，并可能與上調(diào)內(nèi)源性的caspase-8抑制劑FLIP(FADD-like interleukin-1 beta-converting enzyme-inhibitory protein)相關(guān)。1.2 線粒體途徑線粒體途徑激活caspase也是另一個(gè)主要的細(xì)胞死亡路徑，它在失巢凋亡/抵抗過程中也起著重要作用。該途徑是由線粒體損傷開始的，接著會(huì)釋放細(xì)胞色素C，與促使凋亡蛋白活化因子（Apaf-1）和caspase-9形成凋亡復(fù)合體(apoptosome) 活化caspase-9，活化的caspase-9進(jìn)而激活caspase-3等凋亡執(zhí)行蛋白引發(fā)細(xì)胞凋亡8。細(xì)胞凋亡受Bcl-2家族

10、的促凋亡蛋白和抗凋亡蛋白調(diào)控，Bcl-2家族蛋白分為三個(gè)亞家族，第一個(gè)亞家族包括Bcl-2，Bcl-XL,和Bcl-w，可以介導(dǎo)抗凋亡；剩余兩個(gè)亞家族在自然狀態(tài)下則是促細(xì)胞凋亡的，主要包括Bax、Bak、Box及Bim、Bid。Bak或Bax的活化都是細(xì)胞凋亡內(nèi)源性通路的起始因素9；Bax和Bak以單體形式存在于胞漿，當(dāng)接受死亡信號(hào)時(shí)Bax發(fā)生構(gòu)象上的變化，插入線粒體外膜形成小孔，透明化的線粒體外膜則會(huì)允許細(xì)胞色素C等因子的釋放。在凋亡過程當(dāng)中，促凋亡蛋白和抗凋亡蛋白能異二聚體化，影響相互的功能，二者之間的平衡狀態(tài)決定細(xì)胞對(duì)凋亡的敏感性10。研究發(fā)現(xiàn)腸內(nèi)皮細(xì)胞發(fā)生失巢凋亡與Bcl-XL下調(diào)有關(guān)

11、，Ras轉(zhuǎn)染可使Bax降低和Bcl-XL下調(diào)失敗，使細(xì)胞不能釋放Omi/HtrA2，從而耐受失巢凋亡11。2. 失巢凋亡信號(hào)轉(zhuǎn)導(dǎo) 腫瘤細(xì)胞失巢凋亡受磷脂酰肌醇3激酶- AKT (phosphatidylinositol 3-kinases，PI3K/AKT)、絲裂原活化蛋白激酶(mitogen -activated protein kinase，MAPK) 信號(hào)轉(zhuǎn)導(dǎo)通路的調(diào)控。2.1 PI3-KAKT信號(hào)通路PI3K是一個(gè)復(fù)雜的大家族，根據(jù)其結(jié)構(gòu)可分為3類：I、型，型PI3K又分為IA和IB兩個(gè)亞型，PI3K上游受受體酪氨酸激酶和Ras蛋白等調(diào)控12， PI3K可將PI(4)P和PI(4,5)

12、P磷酸化生成PI(3,4)P2和PI(3,4,5)P3，并依賴它們向下游傳遞信號(hào)，PKB是PI3K通路下游主要信號(hào)轉(zhuǎn)導(dǎo)分子；PKB是一種絲/蘇氨酸蛋白激酶，與PKA、PKC同源，由病毒癌基因v-akt對(duì)應(yīng)的原癌基因c-akt編碼，亦稱Akt；激活的PKB能使促凋亡蛋白BAD和caspase-9磷酸化而失活，有抗失巢凋亡作用13。胰島素和胰島素樣生長因子1（insulin-like growth factor1，IGF1）是AKt的主要激活劑，IGFs能夠保護(hù)血清饑餓鼠成纖維肝細(xì)胞和人乳腺癌細(xì)胞MDA-436A免于失巢凋亡 14。2.2 MAPK信號(hào)通路絲裂原活化的蛋白激酶(MAPK)是一組可被

13、多種信號(hào)激活的絲蘇氨酸激酶，MAPK家族包括p38 MAPK、ERK5、ERK 以及JNK4個(gè)亞族，可參與細(xì)胞的多種生物活性，如調(diào)節(jié)基因轉(zhuǎn)錄、誘導(dǎo)細(xì)胞凋亡、調(diào)節(jié)細(xì)胞周期等。p38 MAPK、JNK、ERK三條通路與腫瘤細(xì)胞凋亡關(guān)系密切。在腫瘤細(xì)胞中，活化的p38可增強(qiáng)c- myc表達(dá)、磷酸化p53、參與Fas Fasl及線粒體途徑介導(dǎo)的凋亡15。JNK信號(hào)轉(zhuǎn)導(dǎo)通路參與多種凋亡反應(yīng)，研究表明JNK通路介導(dǎo)腫瘤細(xì)胞凋亡的機(jī)制是通過磷酸化Bcl-2和Bcl - xL，促進(jìn)線粒體釋放細(xì)胞色素 C，激活Caspase 級(jí)聯(lián)反應(yīng)，導(dǎo)致細(xì)胞凋亡16。ERK通路是目前研究的最為透徹的MAPK通路，其激酶可被依

14、賴于高濃度鈣離子的Ras激活，而其靶基因就包括c-myc 等， ERK誘導(dǎo)腫瘤細(xì)胞凋亡的機(jī)制還包括：下調(diào)Bc1-2 、上調(diào)Bax，作用于Caspase家族等17。 3 .腫瘤相關(guān)基因與失巢凋亡抑癌基因PTEN是最近發(fā)現(xiàn)的一個(gè)具有雙重特異性磷酸酶活性的抑癌基因，其編碼蛋白可參與整合蛋白介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路，表達(dá)增加可使PIP3脫磷酸失活，從而抑制PIP3-AKTPKB細(xì)胞生存信號(hào)途徑，也可依賴其磷酸酶活性抑制FAK的磷酸化使細(xì)胞凋亡。研究了多種腫瘤細(xì)胞如乳腺癌、膀胱癌等發(fā)現(xiàn)18-19，PTEN表達(dá)下降或缺失可使細(xì)胞AKTPKB磷酸化水平保持在較高水平且能改變癌細(xì)胞對(duì)化療藥物的敏感性，磷酸化的PKB

15、能使促凋亡蛋白(BAD)和胱冬肽酶-9(easpase-9)磷酸化而失活，具有抗凋亡作用。TrkB是由酪氨酸激酶原癌基因編碼的一種NTs神經(jīng)生長因子家族(neurotrophins，NTs)成員之一，與BDNF具有特異性的高親和力。BDNF和TrkB結(jié)合后,可以引起其磷酸化并激活胞內(nèi)的酪氨酸激酶信號(hào)通路，從而誘導(dǎo)細(xì)胞逃逸凋亡20。研究人員發(fā)現(xiàn)，Trkb的下游可以通過改變細(xì)胞內(nèi)部和其他的形式來抗失巢凋亡和其他形式的細(xì)胞死亡，如Trkb激活PI3Ks，活化AKT/PKB，最終抑制caspases，阻止失巢凋亡的發(fā)生。4. 中藥對(duì)腫瘤細(xì)胞失巢凋亡的影響徐則豐等人21-22用不同濃度的姜黃素分別處理結(jié)

16、腸癌SW-480細(xì)胞、SW-620細(xì)胞、采用末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP切口末端標(biāo)記法檢測結(jié)腸癌細(xì)胞失巢凋亡，結(jié)果發(fā)現(xiàn)姜黃素能有效地抑制結(jié)腸癌細(xì)胞錨著不依賴性增殖，促進(jìn)結(jié)腸癌細(xì)胞失巢凋亡。亦有研究報(bào)道23青蒿琥酯對(duì)乳腺癌MCF-7細(xì)胞有同樣的增值抑制，促進(jìn)凋亡的作用。李瑩等研究證實(shí)白藜蘆醇及葡萄籽提取物均有誘導(dǎo)胃癌細(xì)胞HGC-27、BGC-823失巢凋亡的作用24。此外有報(bào)道稱掌葉半夏提取物有效部位對(duì)體外培養(yǎng)的宮頸癌細(xì)胞有明顯促凋亡作用25。廣泛臨床與實(shí)驗(yàn)研究證實(shí)中醫(yī)藥具有一定程度的抗腫瘤作用，其機(jī)制可能涉及調(diào)節(jié)機(jī)體免疫，抑制腫瘤細(xì)胞增殖、促腫瘤細(xì)胞凋亡等多個(gè)方面；中藥干預(yù)腫瘤細(xì)胞失巢凋

17、亡，抑制腫瘤轉(zhuǎn)移將可能是一個(gè)新的突破口。雖然近年進(jìn)行了一些研究，但由于失巢凋亡主要是體外研究，尚存在一些問題，如中藥復(fù)方有效成份的確定、提取，以及對(duì)細(xì)胞的作用方式等；雖然有中藥血清的作用方式，但由于中藥血清中的中藥含量偏低，加之血清本身對(duì)腫瘤細(xì)胞生長有一定作用，因此建立一系列有效的方法模式，是中醫(yī)藥干預(yù)腫瘤細(xì)胞失巢凋亡研究，深化分子機(jī)制研究亟待解決的問題。REFERENCES：1 Frisch SM, Screaton RA. Anoikis mechanisms. Curr Opin cell Biol, 2001,13(5):555-562.2 Chiarugi P, Giannoni E

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