p21WAF1/CIP1介導(dǎo)腫瘤化療耐藥機(jī)制的研究進(jìn)展

李秋華綜述 鐘 軍 鄭 智審校

p21WAF1/CIP1是由細(xì)胞增殖抑制基因WAF1(wide type p53 activated factor 1)編碼的21kDa的蛋白，屬于Cip/Kip家族。作為1種細(xì)胞周期蛋白激酶(Cds)抑制劑，p21WAF1/CIP1與腫瘤的分化、浸潤(rùn)深度、增生和轉(zhuǎn)移有關(guān)，具有判斷腫瘤分化程度和預(yù)后的價(jià)值[1-4]，與腫瘤化療耐藥機(jī)制的產(chǎn)生密切相關(guān)。

1 p21WAF1/CIP1介導(dǎo)化療耐藥的作用機(jī)制

1.1 p21WAF1/CIP1介導(dǎo)細(xì)胞周期G1停滯機(jī)制

p21WAF1/CIP1是細(xì)胞周期依賴性激酶抑制蛋白(CDKs)家族中重要的調(diào)節(jié)因子，其調(diào)節(jié)細(xì)胞周期的作用途徑分為p53依賴型途徑和p53非依賴型途徑[1-2]。

p53依賴型途徑導(dǎo)致細(xì)胞周期G1停滯：由于p53的活化使p21WAF1/CIP1表達(dá)水平升高，p21WAF1/CIP1能抑制CDK磷酸化或Cyclin-CDK的活性，從而阻止Rb的產(chǎn)物(pRb) 磷酸化,使得pRb和轉(zhuǎn)錄因子E2F-1結(jié)合，pRb無(wú)法適時(shí)分離，E2F-1不能發(fā)揮作用，從而使腫瘤細(xì)胞停滯在G1期，從而阻滯細(xì)胞周期從G1期向S期的進(jìn)展[3-7]；而原癌基因mdm2 的轉(zhuǎn)錄也在DNA 受損后由p53激活,但mdm2不是和細(xì)胞周期停滯直接相關(guān),而是與p53 結(jié)合，并起抑制p53的作用[8-9]。

p53非依賴型途徑介導(dǎo)G1停滯：一些生長(zhǎng)因子(PDGF、FGF、EGF、NGF、TGF等)通過(guò)促細(xì)胞分裂素激活蛋白激酶MAPK途徑活化p21WAF1/CIP1[10-11]。Lois Ramondetta 和Wu Lin等研究人卵巢癌細(xì)胞27749wl p53-/-和人子宮內(nèi)膜癌細(xì)胞CSPEC-2轉(zhuǎn)染p21WAF1/CIP1，結(jié)果發(fā)現(xiàn)p21WAF1/CIP1在介導(dǎo)細(xì)胞凋亡的過(guò)程中并無(wú)BAX、BCL-2的活化，而p53介導(dǎo)的細(xì)胞凋亡一般有BAX、BCL-2的活化，因此推測(cè)p21WAF1/CIP1介導(dǎo)細(xì)胞凋亡是通過(guò)非依賴p53途徑[12-13]。

1.2 p21WAF1/CIP1抑制腫瘤細(xì)胞增殖的機(jī)制

細(xì)胞G1期停滯是惡性腫瘤化療不敏感的重要原因,而p21WAF1/CIP1是調(diào)節(jié)細(xì)胞G1停滯的關(guān)鍵基因。研究證實(shí)p21WAF1/CIP1抑癌作用是通過(guò)CDK或增殖細(xì)胞核抗原PCNA結(jié)合，從而抑制它們的活性，最終導(dǎo)致腫瘤細(xì)胞周期停滯和抑制DNA的合成及細(xì)胞增殖。在小細(xì)胞肺癌、結(jié)直腸癌、宮頸癌及頭頸癌等惡性腫瘤中，很多研究證實(shí)p21WAF1/CIP1的缺失與腫瘤的發(fā)生及預(yù)后差有關(guān)[14-15]。相反，一些研究證實(shí)提高p21WAF1/CIP1的表達(dá)與卵巢癌、宮頸癌、乳腺癌及食管鱗癌進(jìn)展有關(guān)[16-17]。這可能與p21WAF1/CIP1本身的狀態(tài)或者腫瘤組織類(lèi)型有關(guān)[2,5,7]。

2 p21WAF1/CIP與常見(jiàn)腫瘤化療耐藥

2.1 p21WAF1/CIP與肺癌化療耐藥

Rintaro Noro等研究5-FU 對(duì)NSCLC化療敏感性，通過(guò)組蛋白的乙?；险{(diào)p21WAF1/CIP1的表達(dá)，從而提高NSCLC對(duì)5-FU的敏感性[18]。Marchetti等比較了43例非小細(xì)胞肺癌患者癌組織與肺正常組織p21WAF1/CIP1mRNA及其蛋白表達(dá)，運(yùn)用RT-PCR和免疫組化分別檢測(cè)p21WAF1/CIP1mRNA及其蛋白表達(dá)，發(fā)現(xiàn)肺正常組織p21WAF1/CIP1及其蛋白表達(dá)很低，散在表達(dá)于已分化的支氣管、肺泡及基質(zhì)細(xì)胞中，而腫瘤組織中p21WAF1/CIP1mRNA及其蛋白表達(dá)均過(guò)度表達(dá)，分別為65%和63%，分布在分化尚好的腫瘤組織中。因此推測(cè)p21WAF1/CIP1的表達(dá)與肺癌的發(fā)生和分化有關(guān)[19]。

2.2 p21WAF1/CIP與乳腺癌化療耐藥

L.Busia等在研究孕激素受體抑制劑Lonaprisan(洛那立生)對(duì)乳腺癌細(xì)胞T47D增殖抑制作用中發(fā)現(xiàn)通過(guò)基因敲除，降低T47D的p21WAF1/CIP表達(dá)，將削弱Lonaprisan對(duì)乳腺癌細(xì)胞的增殖抑制作用[20]。

2.3 p21WAF1/CIP與卵巢癌化療耐藥

X Xia等研究卵巢癌C13細(xì)胞株胞質(zhì)p21WAF1/CIP高表達(dá)與鉑類(lèi)耐藥有關(guān)，通過(guò)siRAN 干擾細(xì)胞質(zhì)p21WAF1/CIP的表達(dá)，可增加順鉑介導(dǎo)的細(xì)胞凋亡[16]。G.He等研究順鉑介導(dǎo)的DNA損傷激活p53/p21WAF1/CIP細(xì)胞毒性通路，非交叉耐藥Ⅳ鉑類(lèi)DAP處理p53、p21WAF1/CIP表達(dá)正常的人卵巢癌細(xì)胞A2780為對(duì)照組。結(jié)果發(fā)現(xiàn)：順鉑通過(guò)阻滯CDK2/CyclinA復(fù)合物的活性，使腫瘤細(xì)胞停滯在S期(12 h),隨后(12～18 h)永久停滯在G2/M期，其作用機(jī)制是：抑制Chk1和Chk2的活化及其磷酸化，順鉑還可以阻滯Cdk4/cyclin D1 及Cdk2/cyclin復(fù)合物活性化，使腫瘤細(xì)胞停滯在G1期[21]。Besson等報(bào)道不同部位的p21(胞質(zhì)還是核內(nèi))功能不同。通過(guò)2株人卵巢癌細(xì)胞順鉑耐藥株C13和平行對(duì)照細(xì)胞株OV2008細(xì)胞轉(zhuǎn)染p21WAF1/CIPsiRNA，AKt2 shRNA和Akt調(diào)節(jié)胞質(zhì)p21WAF1/CIP表達(dá)。結(jié)果發(fā)現(xiàn)：人卵巢癌細(xì)胞順鉑耐藥株C13與人卵巢癌細(xì)胞OV2008比較，胞質(zhì)p21WAF1/CIP表達(dá)增加；同時(shí)通過(guò)細(xì)胞轉(zhuǎn)染p21siRAN下調(diào)人卵巢癌細(xì)胞順鉑耐藥株C13胞質(zhì)p21WAF1/CIP的表達(dá)，凋亡增加，這與半胱天冬酶3活化有關(guān)。通過(guò)轉(zhuǎn)染AKt2 shRNA抑制人卵巢癌細(xì)胞順鉑耐藥株C13 核內(nèi)：p21WAF1/CIP向細(xì)胞質(zhì)轉(zhuǎn)移來(lái)降低細(xì)胞質(zhì)p21WAF1/CIP濃度，增加順鉑誘導(dǎo)的凋亡。而轉(zhuǎn)染活化AKt2的人卵巢癌細(xì)胞OV2008，增加p21WAF1/CIP向細(xì)胞質(zhì)轉(zhuǎn)移，從而導(dǎo)致順鉑耐藥。臨床免疫組化也證實(shí)細(xì)胞質(zhì)p21WAF1/CIP與順鉑為基礎(chǔ)化療療效呈負(fù)相關(guān)[4]。

2.4 p21WAF1/CIP與血液腫瘤化療耐藥

J.N.Winter等研究p21WAF1/CIP與DLBCL患者(彌漫性大B細(xì)胞淋巴瘤)預(yù)后關(guān)系示：p21WAF1/CIP高表達(dá)能延長(zhǎng)DLBCL患者DFS[22]。C.Gareau等研究硼替佐米(蛋白酶抑制)通過(guò)應(yīng)激顆粒連接蛋白CUGBP1上調(diào)p21WAF1/CIP1的表達(dá),從而增強(qiáng)細(xì)胞凋亡效應(yīng)。這種應(yīng)激顆粒連接蛋白CUGBP1介導(dǎo)的p21WAF1/CIP1在轉(zhuǎn)錄后和轉(zhuǎn)錄水平上的表達(dá)上調(diào)，主要通過(guò)蛋白酶抑制劑MG132來(lái)穩(wěn)定p21WAF1mRNA，使之不被降解，而蛋白酶抑制劑MG132與細(xì)胞質(zhì)RNA 應(yīng)激顆粒的形成有關(guān)。硼替佐米在治療骨髓瘤和其他血液腫瘤方面非常有效，但對(duì)一些實(shí)體腫瘤無(wú)效，這與CUGBP缺乏，繼而p21WAF1/CIP表達(dá)缺乏，細(xì)胞凋亡受阻有關(guān)。其他一些細(xì)胞株(hela、Calu-I and MCF-7)FISH聯(lián)合mRNA 穩(wěn)定性試驗(yàn)都證實(shí)缺乏(細(xì)胞質(zhì)RNA)應(yīng)激顆粒，p21WAF1/CIPmRNA不穩(wěn)定而被降解，p21WAF1/CIP表達(dá)下調(diào)，硼替佐米耐藥[23]。

2.5 p21WAF1/CIP與結(jié)腸癌化療耐藥

M.Kalimutho等研究四代鉑類(lèi)衍生物-satraplatin(沙特鉑)與p53-p21WAF1/CIP信號(hào)傳導(dǎo)通路誘導(dǎo)結(jié)腸癌細(xì)胞G2/M期停滯。上調(diào)p53野生型人結(jié)腸癌細(xì)胞HCT-116與人結(jié)腸癌細(xì)胞LOVO的p53的表達(dá)將增加p21WAF1/CIP1的表達(dá)；而satraplatin介導(dǎo)p53突變?nèi)私Y(jié)腸癌細(xì)胞HCT-15、HT-29及WiDr細(xì)胞周期停滯，p53的表達(dá)缺乏,p21WAF1/CIP1表達(dá)也下降，但14-3-3σ蛋白表達(dá)增加，這說(shuō)明satraplatin可以通過(guò)非依賴p53-p21WAF1/CIP1通路使結(jié)腸癌細(xì)胞停滯在G2/M。satraplatin介導(dǎo)細(xì)胞凋亡是通過(guò)下調(diào)BCL-2蛋白表達(dá)，體外細(xì)胞實(shí)驗(yàn)也發(fā)現(xiàn)人結(jié)腸癌細(xì)胞成瘤能力也下降[24]。Jave lanud和Besancon等研究發(fā)現(xiàn)降低p21WAF1/CIP1表達(dá)對(duì)人結(jié)腸癌細(xì)胞HCT-116凋亡起增敏作用-通過(guò)增加p53和P14ARF及BAX/Bcl-2比例倒置途徑。結(jié)果發(fā)現(xiàn)p53wt p21WAF1/CIP1-/-HCT-116細(xì)胞株對(duì)化療藥更敏感。可能機(jī)制是:敲除HCT-116細(xì)胞株p21WAF1/CIP1，將提高p53轉(zhuǎn)錄后水平，繼而P14ARF表達(dá)增加，P14ARF可抑制MDM2活性[25]。p21WAF1/CIP1介導(dǎo)的細(xì)胞凋亡與真核生物DNA保真性有關(guān)，當(dāng)DNA受到內(nèi)外因素刺激時(shí)，可以造成不同程度的損傷,當(dāng)DNA輕度損傷時(shí)，p21WAF1/CIP1一方面介導(dǎo)細(xì)胞周期停滯，另一方面負(fù)反饋機(jī)制抑制p53的活性，從而阻滯p53介導(dǎo)的細(xì)胞凋亡，結(jié)果DNA損傷得以修復(fù)，細(xì)胞周期恢復(fù)循環(huán)，細(xì)胞增殖，腫瘤進(jìn)展；當(dāng)DNA嚴(yán)重?fù)p傷時(shí)，p53介導(dǎo)的細(xì)胞凋亡可以清除損傷DNA細(xì)胞，減少DNA損傷的累積，這與DNA保真性有關(guān)[26-27]。Han等研究p21WAF1/CIP1在伊立替康(CPT-11)誘導(dǎo)人結(jié)腸癌細(xì)胞凋亡與衰老過(guò)程中的作用，發(fā)現(xiàn)p21WAF1/CIP1介導(dǎo)的細(xì)胞凋亡與CPT-11的劑量有關(guān)：低劑量CPT-11誘導(dǎo)人結(jié)腸癌細(xì)胞HCT-116 p53-/-p21WAF1/CIP-/-細(xì)胞凋亡，但高劑量CPT-11誘導(dǎo)人結(jié)腸癌細(xì)胞HCT-116 p53wt p21wt細(xì)胞凋亡。推測(cè)低劑量CPT-11引起DNA輕度損傷，真核生物DNA保真性，啟動(dòng)DNA修復(fù)機(jī)制，p21WAF1/CIP1介導(dǎo)的細(xì)胞周期停滯，DNA損傷得到修復(fù)[28]。

因此，p21WAF1/CIP1作為細(xì)胞周期素依賴蛋白激酶(cdks)抑制劑,在G1/S 期的轉(zhuǎn)化過(guò)程中起著重要抑制作用,使細(xì)胞停滯于G1期。p21WAF1/CIP1可在不同水平發(fā)揮功能,與p21WAF1/CIP本身的狀態(tài)或者腫瘤組織類(lèi)型有關(guān)[2,5,7]，盡管對(duì)該基因多種功能的了解在不斷增加,但它的許多功能仍有待探索[29]。

總之，大量體外細(xì)胞實(shí)驗(yàn)研究表明，p21WAF1/CIP高表達(dá)，促使細(xì)胞周期停滯、促進(jìn)細(xì)胞凋亡，抑制癌細(xì)胞的增殖、轉(zhuǎn)移和侵襲。臨床研究實(shí)驗(yàn)也證實(shí):p21WAF1/CIP高表達(dá)患者，對(duì)化療更敏感，無(wú)病生存期及總生存期延長(zhǎng)[30]。因此如何提高p21WAF1/CIP表達(dá)，將可能是今后抗腫瘤新藥作用靶點(diǎn)之一。

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