p21WAF1/CIP1介導腫瘤化療耐藥機制的研究進展

李秋華綜述 鐘 軍 鄭 智審校

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

1 p21WAF1/CIP1介導化療耐藥的作用機制

1.1 p21WAF1/CIP1介導細胞周期G1停滯機制

p21WAF1/CIP1是細胞周期依賴性激酶抑制蛋白(CDKs)家族中重要的調節因子，其調節細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依賴型途徑導致細胞周期G1停滯：由于p53的活化使p21WAF1/CIP1表達水平升高，p21WAF1/CIP1能抑制CDK磷酸化或Cyclin-CDK的活性，從而阻止Rb的產物(pRb) 磷酸化,使得pRb和轉錄因子E2F-1結合，pRb無法適時分離，E2F-1不能發揮作用，從而使腫瘤細胞停滯在G1期，從而阻滯細胞周期從G1期向S期的進展[3-7]；而原癌基因mdm2 的轉錄也在DNA 受損后由p53激活,但mdm2不是和細胞周期停滯直接相關,而是與p53 結合，并起抑制p53的作用[8-9]。

p53非依賴型途徑介導G1停滯：一些生長因子(PDGF、FGF、EGF、NGF、TGF等)通過促細胞分裂素激活蛋白激酶MAPK途徑活化p21WAF1/CIP1[10-11]。Lois Ramondetta 和Wu Lin等研究人卵巢癌細胞27749wl p53-/-和人子宮內膜癌細胞CSPEC-2轉染p21WAF1/CIP1，結果發現p21WAF1/CIP1在介導細胞凋亡的過程中并無BAX、BCL-2的活化，而p53介導的細胞凋亡一般有BAX、BCL-2的活化，因此推測p21WAF1/CIP1介導細胞凋亡是通過非依賴p53途徑[12-13]。

1.2 p21WAF1/CIP1抑制腫瘤細胞增殖的機制

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

2 p21WAF1/CIP與常見腫瘤化療耐藥

2.1 p21WAF1/CIP與肺癌化療耐藥

Rintaro Noro等研究5-FU 對NSCLC化療敏感性，通過組蛋白的乙酰化上調p21WAF1/CIP1的表達，從而提高NSCLC對5-FU的敏感性[18]。Marchetti等比較了43例非小細胞肺癌患者癌組織與肺正常組織p21WAF1/CIP1mRNA及其蛋白表達，運用RT-PCR和免疫組化分別檢測p21WAF1/CIP1mRNA及其蛋白表達，發現肺正常組織p21WAF1/CIP1及其蛋白表達很低，散在表達于已分化的支氣管、肺泡及基質細胞中，而腫瘤組織中p21WAF1/CIP1mRNA及其蛋白表達均過度表達，分別為65%和63%，分布在分化尚好的腫瘤組織中。因此推測p21WAF1/CIP1的表達與肺癌的發生和分化有關[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)對乳腺癌細胞T47D增殖抑制作用中發現通過基因敲除，降低T47D的p21WAF1/CIP表達，將削弱Lonaprisan對乳腺癌細胞的增殖抑制作用[20]。

2.3 p21WAF1/CIP與卵巢癌化療耐藥

X Xia等研究卵巢癌C13細胞株胞質p21WAF1/CIP高表達與鉑類耐藥有關，通過siRAN 干擾細胞質p21WAF1/CIP的表達，可增加順鉑介導的細胞凋亡[16]。G.He等研究順鉑介導的DNA損傷激活p53/p21WAF1/CIP細胞毒性通路，非交叉耐藥Ⅳ鉑類DAP處理p53、p21WAF1/CIP表達正常的人卵巢癌細胞A2780為對照組。結果發現：順鉑通過阻滯CDK2/CyclinA復合物的活性，使腫瘤細胞停滯在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復合物活性化，使腫瘤細胞停滯在G1期[21]。Besson等報道不同部位的p21(胞質還是核內)功能不同。通過2株人卵巢癌細胞順鉑耐藥株C13和平行對照細胞株OV2008細胞轉染p21WAF1/CIPsiRNA，AKt2 shRNA和Akt調節胞質p21WAF1/CIP表達。結果發現：人卵巢癌細胞順鉑耐藥株C13與人卵巢癌細胞OV2008比較，胞質p21WAF1/CIP表達增加；同時通過細胞轉染p21siRAN下調人卵巢癌細胞順鉑耐藥株C13胞質p21WAF1/CIP的表達，凋亡增加，這與半胱天冬酶3活化有關。通過轉染AKt2 shRNA抑制人卵巢癌細胞順鉑耐藥株C13 核內：p21WAF1/CIP向細胞質轉移來降低細胞質p21WAF1/CIP濃度，增加順鉑誘導的凋亡。而轉染活化AKt2的人卵巢癌細胞OV2008，增加p21WAF1/CIP向細胞質轉移，從而導致順鉑耐藥。臨床免疫組化也證實細胞質p21WAF1/CIP與順鉑為基礎化療療效呈負相關[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細胞淋巴瘤)預后關系示：p21WAF1/CIP高表達能延長DLBCL患者DFS[22]。C.Gareau等研究硼替佐米(蛋白酶抑制)通過應激顆粒連接蛋白CUGBP1上調p21WAF1/CIP1的表達,從而增強細胞凋亡效應。這種應激顆粒連接蛋白CUGBP1介導的p21WAF1/CIP1在轉錄后和轉錄水平上的表達上調，主要通過蛋白酶抑制劑MG132來穩定p21WAF1mRNA，使之不被降解，而蛋白酶抑制劑MG132與細胞質RNA 應激顆粒的形成有關。硼替佐米在治療骨髓瘤和其他血液腫瘤方面非常有效，但對一些實體腫瘤無效，這與CUGBP缺乏，繼而p21WAF1/CIP表達缺乏，細胞凋亡受阻有關。其他一些細胞株(hela、Calu-I and MCF-7)FISH聯合mRNA 穩定性試驗都證實缺乏(細胞質RNA)應激顆粒，p21WAF1/CIPmRNA不穩定而被降解，p21WAF1/CIP表達下調，硼替佐米耐藥[23]。

2.5 p21WAF1/CIP與結腸癌化療耐藥

M.Kalimutho等研究四代鉑類衍生物-satraplatin(沙特鉑)與p53-p21WAF1/CIP信號傳導通路誘導結腸癌細胞G2/M期停滯。上調p53野生型人結腸癌細胞HCT-116與人結腸癌細胞LOVO的p53的表達將增加p21WAF1/CIP1的表達；而satraplatin介導p53突變人結腸癌細胞HCT-15、HT-29及WiDr細胞周期停滯，p53的表達缺乏,p21WAF1/CIP1表達也下降，但14-3-3σ蛋白表達增加，這說明satraplatin可以通過非依賴p53-p21WAF1/CIP1通路使結腸癌細胞停滯在G2/M。satraplatin介導細胞凋亡是通過下調BCL-2蛋白表達，體外細胞實驗也發現人結腸癌細胞成瘤能力也下降[24]。Jave lanud和Besancon等研究發現降低p21WAF1/CIP1表達對人結腸癌細胞HCT-116凋亡起增敏作用-通過增加p53和P14ARF及BAX/Bcl-2比例倒置途徑。結果發現p53wt p21WAF1/CIP1-/-HCT-116細胞株對化療藥更敏感。可能機制是:敲除HCT-116細胞株p21WAF1/CIP1，將提高p53轉錄后水平，繼而P14ARF表達增加，P14ARF可抑制MDM2活性[25]。p21WAF1/CIP1介導的細胞凋亡與真核生物DNA保真性有關，當DNA受到內外因素刺激時，可以造成不同程度的損傷,當DNA輕度損傷時，p21WAF1/CIP1一方面介導細胞周期停滯，另一方面負反饋機制抑制p53的活性，從而阻滯p53介導的細胞凋亡，結果DNA損傷得以修復，細胞周期恢復循環，細胞增殖，腫瘤進展；當DNA嚴重損傷時，p53介導的細胞凋亡可以清除損傷DNA細胞，減少DNA損傷的累積，這與DNA保真性有關[26-27]。Han等研究p21WAF1/CIP1在伊立替康(CPT-11)誘導人結腸癌細胞凋亡與衰老過程中的作用，發現p21WAF1/CIP1介導的細胞凋亡與CPT-11的劑量有關：低劑量CPT-11誘導人結腸癌細胞HCT-116 p53-/-p21WAF1/CIP-/-細胞凋亡，但高劑量CPT-11誘導人結腸癌細胞HCT-116 p53wt p21wt細胞凋亡。推測低劑量CPT-11引起DNA輕度損傷，真核生物DNA保真性，啟動DNA修復機制，p21WAF1/CIP1介導的細胞周期停滯，DNA損傷得到修復[28]。

因此，p21WAF1/CIP1作為細胞周期素依賴蛋白激酶(cdks)抑制劑,在G1/S 期的轉化過程中起著重要抑制作用,使細胞停滯于G1期。p21WAF1/CIP1可在不同水平發揮功能,與p21WAF1/CIP本身的狀態或者腫瘤組織類型有關[2,5,7]，盡管對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，大量體外細胞實驗研究表明，p21WAF1/CIP高表達，促使細胞周期停滯、促進細胞凋亡，抑制癌細胞的增殖、轉移和侵襲。臨床研究實驗也證實:p21WAF1/CIP高表達患者，對化療更敏感，無病生存期及總生存期延長[30]。因此如何提高p21WAF1/CIP表達，將可能是今后抗腫瘤新藥作用靶點之一。

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