p21WAF1/CIP1介导肿瘤化疗耐药机制的研究进展

李秋华综述 钟 军 郑 智审校

p21WAF1/CIP1是由细胞增殖抑制基因WAF1(wide type p53 activated factor 1)编码的21kDa的蛋白，属于Cip/Kip家族。作为1种细胞周期蛋白激酶(Cds)抑制剂，p21WAF1/CIP1与肿瘤的分化、浸润深度、增生和转移有关，具有判断肿瘤分化程度和预后的价值[1-4]，与肿瘤化疗耐药机制的产生密切相关。

1 p21WAF1/CIP1介导化疗耐药的作用机制

1.1 p21WAF1/CIP1介导细胞周期G1停滞机制

p21WAF1/CIP1是细胞周期依赖性激酶抑制蛋白(CDKs)家族中重要的调节因子，其调节细胞周期的作用途径分为p53依赖型途径和p53非依赖型途径[1-2]。

p53依赖型途径导致细胞周期G1停滞：由于p53的活化使p21WAF1/CIP1表达水平升高，p21WAF1/CIP1能抑制CDK磷酸化或Cyclin-CDK的活性，从而阻止Rb的产物(pRb) 磷酸化,使得pRb和转录因子E2F-1结合，pRb无法适时分离，E2F-1不能发挥作用，从而使肿瘤细胞停滞在G1期，从而阻滞细胞周期从G1期向S期的进展[3-7]；而原癌基因mdm2 的转录也在DNA 受损后由p53激活,但mdm2不是和细胞周期停滞直接相关,而是与p53 结合，并起抑制p53的作用[8-9]。

p53非依赖型途径介导G1停滞：一些生长因子(PDGF、FGF、EGF、NGF、TGF等)通过促细胞分裂素激活蛋白激酶MAPK途径活化p21WAF1/CIP1[10-11]。Lois Ramondetta 和Wu Lin等研究人卵巢癌细胞27749wl p53-/-和人子宫内膜癌细胞CSPEC-2转染p21WAF1/CIP1，结果发现p21WAF1/CIP1在介导细胞凋亡的过程中并无BAX、BCL-2的活化，而p53介导的细胞凋亡一般有BAX、BCL-2的活化，因此推测p21WAF1/CIP1介导细胞凋亡是通过非依赖p53途径[12-13]。

1.2 p21WAF1/CIP1抑制肿瘤细胞增殖的机制

细胞G1期停滞是恶性肿瘤化疗不敏感的重要原因,而p21WAF1/CIP1是调节细胞G1停滞的关键基因。研究证实p21WAF1/CIP1抑癌作用是通过CDK或增殖细胞核抗原PCNA结合，从而抑制它们的活性，最终导致肿瘤细胞周期停滞和抑制DNA的合成及细胞增殖。在小细胞肺癌、结直肠癌、宫颈癌及头颈癌等恶性肿瘤中，很多研究证实p21WAF1/CIP1的缺失与肿瘤的发生及预后差有关[14-15]。相反，一些研究证实提高p21WAF1/CIP1的表达与卵巢癌、宫颈癌、乳腺癌及食管鳞癌进展有关[16-17]。这可能与p21WAF1/CIP1本身的状态或者肿瘤组织类型有关[2,5,7]。

2 p21WAF1/CIP与常见肿瘤化疗耐药

2.1 p21WAF1/CIP与肺癌化疗耐药

Rintaro Noro等研究5-FU 对NSCLC化疗敏感性，通过组蛋白的乙酰化上调p21WAF1/CIP1的表达，从而提高NSCLC对5-FU的敏感性[18]。Marchetti等比较了43例非小细胞肺癌患者癌组织与肺正常组织p21WAF1/CIP1mRNA及其蛋白表达，运用RT-PCR和免疫组化分别检测p21WAF1/CIP1mRNA及其蛋白表达，发现肺正常组织p21WAF1/CIP1及其蛋白表达很低，散在表达于已分化的支气管、肺泡及基质细胞中，而肿瘤组织中p21WAF1/CIP1mRNA及其蛋白表达均过度表达，分别为65%和63%，分布在分化尚好的肿瘤组织中。因此推测p21WAF1/CIP1的表达与肺癌的发生和分化有关[19]。

2.2 p21WAF1/CIP与乳腺癌化疗耐药

L.Busia等在研究孕激素受体抑制剂Lonaprisan(洛那立生)对乳腺癌细胞T47D增殖抑制作用中发现通过基因敲除，降低T47D的p21WAF1/CIP表达，将削弱Lonaprisan对乳腺癌细胞的增殖抑制作用[20]。

2.3 p21WAF1/CIP与卵巢癌化疗耐药

X Xia等研究卵巢癌C13细胞株胞质p21WAF1/CIP高表达与铂类耐药有关，通过siRAN 干扰细胞质p21WAF1/CIP的表达，可增加顺铂介导的细胞凋亡[16]。G.He等研究顺铂介导的DNA损伤激活p53/p21WAF1/CIP细胞毒性通路，非交叉耐药Ⅳ铂类DAP处理p53、p21WAF1/CIP表达正常的人卵巢癌细胞A2780为对照组。结果发现：顺铂通过阻滞CDK2/CyclinA复合物的活性，使肿瘤细胞停滞在S期(12 h),随后(12～18 h)永久停滞在G2/M期，其作用机制是：抑制Chk1和Chk2的活化及其磷酸化，顺铂还可以阻滞Cdk4/cyclin D1 及Cdk2/cyclin复合物活性化，使肿瘤细胞停滞在G1期[21]。Besson等报道不同部位的p21(胞质还是核内)功能不同。通过2株人卵巢癌细胞顺铂耐药株C13和平行对照细胞株OV2008细胞转染p21WAF1/CIPsiRNA，AKt2 shRNA和Akt调节胞质p21WAF1/CIP表达。结果发现：人卵巢癌细胞顺铂耐药株C13与人卵巢癌细胞OV2008比较，胞质p21WAF1/CIP表达增加；同时通过细胞转染p21siRAN下调人卵巢癌细胞顺铂耐药株C13胞质p21WAF1/CIP的表达，凋亡增加，这与半胱天冬酶3活化有关。通过转染AKt2 shRNA抑制人卵巢癌细胞顺铂耐药株C13 核内：p21WAF1/CIP向细胞质转移来降低细胞质p21WAF1/CIP浓度，增加顺铂诱导的凋亡。而转染活化AKt2的人卵巢癌细胞OV2008，增加p21WAF1/CIP向细胞质转移，从而导致顺铂耐药。临床免疫组化也证实细胞质p21WAF1/CIP与顺铂为基础化疗疗效呈负相关[4]。

2.4 p21WAF1/CIP与血液肿瘤化疗耐药

J.N.Winter等研究p21WAF1/CIP与DLBCL患者(弥漫性大B细胞淋巴瘤)预后关系示：p21WAF1/CIP高表达能延长DLBCL患者DFS[22]。C.Gareau等研究硼替佐米(蛋白酶抑制)通过应激颗粒连接蛋白CUGBP1上调p21WAF1/CIP1的表达,从而增强细胞凋亡效应。这种应激颗粒连接蛋白CUGBP1介导的p21WAF1/CIP1在转录后和转录水平上的表达上调，主要通过蛋白酶抑制剂MG132来稳定p21WAF1mRNA，使之不被降解，而蛋白酶抑制剂MG132与细胞质RNA 应激颗粒的形成有关。硼替佐米在治疗骨髓瘤和其他血液肿瘤方面非常有效，但对一些实体肿瘤无效，这与CUGBP缺乏，继而p21WAF1/CIP表达缺乏，细胞凋亡受阻有关。其他一些细胞株(hela、Calu-I and MCF-7)FISH联合mRNA 稳定性试验都证实缺乏(细胞质RNA)应激颗粒，p21WAF1/CIPmRNA不稳定而被降解，p21WAF1/CIP表达下调，硼替佐米耐药[23]。

2.5 p21WAF1/CIP与结肠癌化疗耐药

M.Kalimutho等研究四代铂类衍生物-satraplatin(沙特铂)与p53-p21WAF1/CIP信号传导通路诱导结肠癌细胞G2/M期停滞。上调p53野生型人结肠癌细胞HCT-116与人结肠癌细胞LOVO的p53的表达将增加p21WAF1/CIP1的表达；而satraplatin介导p53突变人结肠癌细胞HCT-15、HT-29及WiDr细胞周期停滞，p53的表达缺乏,p21WAF1/CIP1表达也下降，但14-3-3σ蛋白表达增加，这说明satraplatin可以通过非依赖p53-p21WAF1/CIP1通路使结肠癌细胞停滞在G2/M。satraplatin介导细胞凋亡是通过下调BCL-2蛋白表达，体外细胞实验也发现人结肠癌细胞成瘤能力也下降[24]。Jave lanud和Besancon等研究发现降低p21WAF1/CIP1表达对人结肠癌细胞HCT-116凋亡起增敏作用-通过增加p53和P14ARF及BAX/Bcl-2比例倒置途径。结果发现p53wt p21WAF1/CIP1-/-HCT-116细胞株对化疗药更敏感。可能机制是:敲除HCT-116细胞株p21WAF1/CIP1，将提高p53转录后水平，继而P14ARF表达增加，P14ARF可抑制MDM2活性[25]。p21WAF1/CIP1介导的细胞凋亡与真核生物DNA保真性有关，当DNA受到内外因素刺激时，可以造成不同程度的损伤,当DNA轻度损伤时，p21WAF1/CIP1一方面介导细胞周期停滞，另一方面负反馈机制抑制p53的活性，从而阻滞p53介导的细胞凋亡，结果DNA损伤得以修复，细胞周期恢复循环，细胞增殖，肿瘤进展；当DNA严重损伤时，p53介导的细胞凋亡可以清除损伤DNA细胞，减少DNA损伤的累积，这与DNA保真性有关[26-27]。Han等研究p21WAF1/CIP1在伊立替康(CPT-11)诱导人结肠癌细胞凋亡与衰老过程中的作用，发现p21WAF1/CIP1介导的细胞凋亡与CPT-11的剂量有关：低剂量CPT-11诱导人结肠癌细胞HCT-116 p53-/-p21WAF1/CIP-/-细胞凋亡，但高剂量CPT-11诱导人结肠癌细胞HCT-116 p53wt p21wt细胞凋亡。推测低剂量CPT-11引起DNA轻度损伤，真核生物DNA保真性，启动DNA修复机制，p21WAF1/CIP1介导的细胞周期停滞，DNA损伤得到修复[28]。

因此，p21WAF1/CIP1作为细胞周期素依赖蛋白激酶(cdks)抑制剂,在G1/S 期的转化过程中起着重要抑制作用,使细胞停滞于G1期。p21WAF1/CIP1可在不同水平发挥功能,与p21WAF1/CIP本身的状态或者肿瘤组织类型有关[2,5,7]，尽管对该基因多种功能的了解在不断增加,但它的许多功能仍有待探索[29]。

总之，大量体外细胞实验研究表明，p21WAF1/CIP高表达，促使细胞周期停滞、促进细胞凋亡，抑制癌细胞的增殖、转移和侵袭。临床研究实验也证实:p21WAF1/CIP高表达患者，对化疗更敏感，无病生存期及总生存期延长[30]。因此如何提高p21WAF1/CIP表达，将可能是今后抗肿瘤新药作用靶点之一。

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