抑癌基因PDCD4和PDCD5在卵巢癌中的作用及其机制研究
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摘要
目的
卵巢癌是妇科常见的恶性肿瘤,种类繁多,其中原发性卵巢上皮癌(最常见的是浆液性囊腺癌)占卵巢恶性肿瘤的85-90%。卵巢癌早期不易发现,就诊率低,病死率居女性生殖系统恶性肿瘤首位。临床上对于卵巢癌的治疗主要是采用手术及以铂类为主的联合化疗,尽管起初化疗效应约50-80%,但由于原发性与获得性耐药,患者的5年生存率仍然很低。因此,提高化疗敏感性,逆转化疗耐药(尤其是最常用的铂类化疗药物,如顺铂)是改善卵巢癌预后的关键。近些年来,基因治疗在肿瘤领域的成功应用为卵巢癌基因治疗奠定了基础,对恶性卵巢癌患者化疗药增敏基因和耐药基因的研究,成为卵巢癌治疗领域的热门课题。
程序性细胞死亡-4(programmed cell death 4, PDCD4)是一种新的抑癌基因,可以通过抑制蛋白转录和翻译过程抑制肿瘤细胞的生长。我们和其他学者已经发现PDCD4在肺癌和神经胶质瘤等肿瘤中表达下调并且在肿瘤中发挥着非常重要的作用,但在卵巢癌中的作用尚未见报道。那么,PDCD4在卵巢癌中的表达状态如何?其对卵巢癌的发生发展到底有什么作用?能否影响卵巢癌的化疗敏感性并成为基因治疗的靶点之一?PDCD4的作用机制是什么,能否通过调控自噬的发生来发挥其在疾病中的作用?这些都是非常值得探索的问题。
为了明确PDCD4在卵巢癌发生发展中的关系,阐明其发挥作用的机制,为PDCD4的临床应用提供理论依据和实验基础,本论文拟从以下三个方面进行研究：
(一) PDCD4在人卵巢癌中的表达状态及其作用：通过多种方法检测正常卵巢、浆液性囊腺瘤和浆液性囊腺癌中PDCD4的表达发现,PDCD4在囊腺癌的表达出现明显的下调与缺失,并且与囊腺癌的病理级别密切相关。进一步研究发现,PDCD4在体外能够明显抑制卵巢癌细胞增殖和克隆形成能力,在裸鼠体内能降低卵巢癌细胞的成瘤能力,延长肿瘤发生的潜伏期。
(二) PDCD4对卵巢癌化疗敏感性的影响及其机制研究：我们通过过表达和沉默表达PDCD4,从正反两方面证实PDCD4能显著增强卵巢癌细胞对铂类化疗药物的敏感性,在裸鼠体内PDCD4和顺铂联合治疗能有效地抑制卵巢癌的生长。进一步阐明其机制：PDCD4能够明显增强顺铂诱导的细胞凋亡,并且主要通过死亡受体通路介导的。
(三) PDCD4在细胞发生自噬过程中的作用及其机制研究：PDCD4在卵巢癌细胞中过表达可以抑制自噬的发生,PDCD4-/-小鼠的巨噬细胞自噬水平明显高于C57小鼠。进一步的研究证明PDCD4是通过调控与自噬发生有关的p38MAPK通路来发挥作用。
方法
一、PDCD4在卵巢癌中的作用及其临床意义
1.分别用RT-PCR. western blot和免疫组化方法检测人卵巢癌细胞系以及人正常卵巢、浆液性囊腺瘤和浆液性囊腺癌组织标本中PDCD4 mRNA和蛋白的表达情况。x2检验分析PDCD4在浆液性囊腺癌病例中的表达与其临床病理特点间的关系。
2.利用脂质体转染PDCD4重组表达载体和G418筛选的方法建立PDCD4稳定表达的细胞系SKOV3-PDCD4和对照细胞系SKOV3-MOCK,用活细胞计数法检测PDCD4对细胞增殖能力的影响,平板集落形成实验检测PDCD4对细胞克隆形成能力的影响。
3.将SKOV3,SKOV3-MOCK, SKOV3-PDCD4细胞分别注射到裸鼠腋窝皮下荷瘤,观察PDCD4表达对卵巢癌细胞SKOV3在裸鼠体内成瘤能力的影响。
二、PDCD4对卵巢癌化疗敏感性的影响及其机制研究
1.用MTT法检测四种卵巢癌细胞系,SKOV3、CAOV3、OVCAR3和3AO对顺铂的化疗敏感性,分析PDCD4表达水平与卵巢癌细胞对顺铂化疗敏感性的关系。
2.在卵巢癌PDCD4相对低表达的卵巢癌细胞系SKOV3和CAOV3中,稳定或瞬时表达PDCD4,显微镜下观察细胞的状态,用MTT法检测PDCD4对卵巢癌细胞化疗敏感性的影响。
3.设计合成两条PDCD4特异的siRNA,用siRNA沉默PDCD4高表达的卵巢癌细胞系OVCAR3中PDCD4的表达,干扰48小时后用RT-PCR和western blot检测PDCD4的表达,确定干扰效率。用MTT实验检测PDCD4沉默表达对卵巢癌化疗敏感性的影响。
4.用卵巢癌细胞SKOV3注射到裸鼠腋窝皮下荷瘤后,随机分为6组,分别采用生理盐水,空载体,PDCD4载体,顺铂,空载体+顺铂,PDCD4+顺铂6种方法进行治疗,探讨PDCD4和顺铂联合治疗对肿瘤生长的影响。
5.用PI染色,流式细胞仪检测PDCD4过表达对顺铂诱导的细胞周期变化的影响。进一步通过Hoechst染色从细胞形态学上观察PDCD4过表达对顺铂诱导的凋亡的影响,采用Tunnel染色对细胞的晚期凋亡率进行检测。
6.应用western blot检测PDCD4过表达对顺铂诱导的凋亡相关蛋白caspase-3、caspase-8、caspase-9的活性变化及Bcl-2、Bax, c-FLIP蛋白表达变化。
7.用caspase-8抑制剂抑致caspase-8的活性,检测PDCD4过表达对顺铂的化疗增敏作用的影响。
8.利用流式细胞术检测PDCD4 j过表达对顺铂诱导的死亡受体DR5的表达状态的变化。
三、PDCD4在细胞发生自噬过程中的作用及其机制研究
1.用空载体或PDCD4重组表达载体转染卵巢癌细胞系SKOV3,转染48小时之后,采用饥饿的方法诱导细胞产生自噬,检测自噬标志性蛋白LC3的表达情况。
2.分别获取C57和PDCD4基因敲除小鼠的腹腔巨噬细胞和骨髓来源的巨噬细胞,采用饥饿,LPS或雷帕霉素刺激诱导自噬的产生,western blot检测LC3表达情况。分别在C57和PDCD4基因敲除小鼠的腹腔巨噬细胞转染GFP-LC3,诱导自噬发生后,荧光显微镜观察GFP-LC3的表达分布情况。
3.分别获取C57和PDCD4基因敲除小鼠的腹腔巨噬细胞,诱导自噬发生后,用ROS活性检测试剂盒,流式细胞术检测PDCD4对ROS活性的影响。Western blot检测PDCD4对自噬相关蛋白Beclin-1和自噬发生有关的p38MAPK通路中p38MAPK表达的影响。
结果
一、PDCD4在卵巢癌中的表达、临床意义及对肿瘤恶性行为的影响
1. PDCD4在人卵巢癌细胞系和卵巢癌组织中的表达状况及临床意义
为了研究PDCD4在卵巢癌中的作用,首先应用RT-PCR和western blot的方法检测了四种卵巢癌细胞系SKOV3、CAOV3、OVCAR3和3AO中PDCD4的表达情况,结果显示PDCD4在SKOV3和3AO中表达较低,CAOV3中度表达,OVCAR3表达相对较高。进而我们采用RT-PCR和免疫组化的方法检测了20例正常卵巢、26例浆液性囊腺瘤、43例浆液性囊腺癌组织中PDCD4的表达,发现与正常卵巢和浆液性囊腺瘤相比,PDCD4在浆液性囊腺癌中的表达明显下调甚至缺失。进一步分析发现,PDCD4的表达下调和缺失与浆液性囊腺癌患者高的病理级别密切相关。
2.外源性PDCD4的表达能够明显抑制卵巢癌细胞的生长及克隆形成能力
为了进一步研究PDCD4在卵巢癌中的作用,我们用脂质体介导将PDCD4重组表达载体和空载体转染到卵巢癌细胞系SKOV3中,并用G418进一步筛选,通过RT-PCR和western blot鉴定,得到稳定表达PDCD4的细胞系SKOV3-PDCD4和对照细胞系SKOV3-MOCK。通过活细胞计数的方法检测细胞增殖能力发现,SKOV3-PDCD4的增殖能力明显减弱(p<0.001),克隆形成实验显示SKOV3-PDCD4的克隆形成数明显降低(p<0.05),提示PDCD4在体外能明显抑制卵巢癌的恶性增殖能力。
3.外源性PDCD4表达降低了卵巢癌细胞在裸鼠体内的成瘤能力
为了研究PDCD4在体内是否同样具有抑制卵巢癌恶性行为的能力,我们用SKOV3、SKOV3-MOCK、SKOV3-PDCD4细胞分别在裸鼠皮下进行荷瘤,观察肿瘤的生长。结果显示,SKOV3-PDCD4组的成瘤率明显降低,成瘤时间明显延长,而且形成的肿瘤大小和重量也明显低于其他两组(p<0.05)。这些结果显示,PDCD4过表达在体内同样可以抑制卵巢癌细胞的恶性行为。
二、PDCD4对卵巢癌化疗敏感性的影响及其机制研究
1. PDCD4表达水平与卵巢癌敏感性的关系
上一部分研究提出PDCD4不仅在卵巢癌中表达降低,而且PDCD4在体内外都可以抑制卵巢癌的恶性增殖能力。然而,PDCD4能否增强卵巢癌的化疗敏感性,从而增强其疗效仍有待于进一步研究。我们通过MTT法检测上述四种卵巢癌细胞系SKOV3、CAOV3、OVCAR3及3AO对顺铂的化疗敏感性,分析PDCD4表达水平和其敏感程度的关系。结果显示,相对高表达PDCD4的CAOV3和OVCAR3细胞对顺铂更加敏感,而低表达细胞SKOV3和3AO则较不敏感,提示PDCD4表达水平可能与卵巢癌的化疗敏感性相关。
2.外源性PDCD4表达可以增强卵巢癌细胞的化疗敏感性
为了确定PDCD4对化疗敏感性的影响,我们在第一部分得到的稳定表达细胞系SKOV3-PDCD4和SKOV3-MOCK中分别加入不同浓度的顺铂,MTT检测细胞的生存率。结果显示,在12.5μg/ml浓度的顺铂作用下,SKOV3-PDCD4的细胞形态出现变圆或死亡,而SKOV3-MOCK没有明显的改变。MTT结果显示SKOV3-PDCD4的细胞生存率明显低于SKOV3-MOCK。我们通过瞬时转染的方法在CAOV3细胞中过表达PDCD4也发现了相似的结果,表明外源性的PDCD4表达可以增强卵巢癌细胞对顺铂的化疗敏感性。进一步检测了PDCD4过表达对其他化疗药物的影响,结果显示PDCD4同样也可以增强卵巢癌细胞对卡铂的化疗敏感性,但是对环磷酰胺,VP-16及紫杉醇的化疗敏感性没有明显的影响。
3.沉默表达PDCD4减低了卵巢癌对顺铂的化疗敏感性
为了进一步确定PDCD4对卵巢癌化疗敏感性的影响,我们设计合成了两条PDCD4特异性siRNA片段,将其转染PDCD4高表达的卵巢癌细胞系OVCAR3, RT-PCR和,western blot检测PDCD4表达发现,两条siRNA片段都能够有效的沉默PDCD4的表达(mRNA水平降低77%和76%,蛋白表达降低80%和88%)。进一步,我们采用这两条siRNA沉默OVCAR3中的PDCD4表达,发现其对顺铂的化疗敏感性明显降低(p<0.05),从反方面验证了PDCD4能够增强卵巢癌对顺铂的化疗敏感性。
4. PDCD4和顺铂体内联合治疗能明显抑制卵巢癌的生长
为了验证PDCD4在体内能否增强卵巢癌对顺铂的化疗敏感性,我们用SKOV3细胞在裸鼠皮下荷瘤,随即分为6组进行治疗：生理盐水对照组,空载体组,PDCD4组,顺铂组,空载体+顺铂组,PDCD4+顺铂组。结果显示：PDCD4+顺铂组肿瘤的大小和重量(0.0375士0.0069 mm3,0.0300+0.0044 g)都明显的低于顺铂组(0.1386±0.0185 mm3,0.0860±0.0204 g), PDCD4组(0.0779±0.0115 mm3,0.0533±0.0042 g),和空载体+顺铂组(0.1621±0.0090 mm3,0.07667±0.0126 g)。通过免疫组化检测以上各组肿瘤中PDCD4的表达显示,有PDCD4载体治疗组的PDCD4表达明显增高,说明体内表达成功。体内实验结果显示PDCD4和顺铂联合治疗能明显抑制卵巢癌的生长,提示PDCD4在体内仍具有增强卵巢癌对顺铂的化疗敏感性的作用。
5. PDCD4增强顺铂的化疗敏感性是通过诱导细胞凋亡,而不是改变细胞周期
为了进一步检测PDCD4增强卵巢癌对顺铂化疗敏感性的机制,我们首先采用流式细胞术检测PDCD4对顺铂作用后细胞周期的影响,结果显示顺铂能明显诱导SKOV3细胞S期阻滞,这和其他有关顺铂的作用机制的研究报道是相一致的,但是PDCD4过表达对顺铂诱导的细胞周期改变没有明显的影响。进一步,我们通过Hoechst染色和Tunnel染色检测了细胞凋亡,结果显示在相同浓度顺铂作用下,与SKOV3-MOCK对照组(凋亡率为1.5%)相比,PDCD4过表达组SKOV3-PDCD4细胞的核固缩、碎裂明显增多,Tunnel染色流式细胞术检测显示PDCD4过表达组细胞凋亡率(30.1%)明显增高。这些结果说明PDCD4增强卵巢癌对顺铂化疗敏感性的机制主要是通过增强顺铂诱导的细胞凋亡,而对细胞周期的变化没有什么影响。
6. PDCD4增强顺铂诱导的凋亡通路主要通过死亡受体通路
介导细胞凋亡主要的信号通路分为线粒体通路和死亡受体通路。为了确定PDCD4增强顺铂诱导的凋亡通路,我们通过western blot检测了凋亡相关蛋白caspase-3,8,9以及Bcl-2,Bax的变化。结果显示,在相同浓度顺铂作用下,与SKOV3-MOCK对照组相比,PDCD4过表达组SKOV3-PDCD4细胞的caspase-3和caspase-8活性蛋白表达明显升高,而caspase-9和Bax具有轻微的升高,Bcl-2的表达没有明显变化,免疫组化检测肿瘤切片中的表达也得到了相似结果,提示死亡受体通路可能是PDCD4的主要作用通路。采用caspase-8抑制剂能够逆转PDCD4对顺铂的化疗增敏作用,进一步验证了这个结论。另外,在顺铂作用下,PDCD4过表达能明显上调死亡受体DR5的表达,内源性caspase-8的抑制蛋白c-FLIP-L的表达明显下调,可能是casapse-8活性蛋白升高的原因之一。根据这些结果,我们认为PDCD4主要通过死亡受体介导的凋亡通路促进了顺铂诱导的凋亡,从而增强了卵巢癌细胞对顺铂的化疗敏感性。
三、PDCD4在细胞发生自噬过程中的作用及其机制研究
尽管我们和其它学者的研究显示PDCD4可能通过诱导细胞凋亡或干扰细胞周期发挥作用,但其作用并不明显,不能完全解释PDCD4对肿瘤的有效抑制。自噬是近几年发现的细胞死亡的另一种形式,故进一步探讨了PDCD4对自噬的影响。
1.外源性PDCD4表达抑制卵巢癌细胞发生自噬
为了研究PDCD4对细胞自噬的作用,我们在SKOV3中过表达PDCD4之后,用饥饿诱导因素EBSS诱导自噬的发生,检测自噬的标记分子LC3蛋白的表达。结果发现,PDCD4过表达组LC3-Ⅱ明显降低,说明其自噬水平降低。进一步检测体内实验显示空载体和PDCD4表达载体治疗的肿瘤组织中LC3蛋白的表达也得到了相似的结果,说明PDCD4
确实具有抑制卵巢癌细胞自噬发生的作用。
2. PDCD4敲除后巨噬细胞自噬明显增强
为了进一步确定PDCD4在自噬发生中的作用,我们获取了C57和PDCD4-/-小鼠的腹腔巨噬细胞和骨髓来源的巨噬细胞,分别用EBSS, LPS和雷帕霉素诱导自噬的发生,结果都显示PDCD4-/-小鼠的两种来源的巨噬细胞中LC3-Ⅱ表达明显升高。进一步在C57和PDCD4-/-两种小鼠的腹腔巨噬细胞中转染GFP-LC3质粒,LPS诱导自噬之后发现,PDCD4-/-小鼠的巨噬细胞中绿色荧光斑点明显多于C57小鼠,说明自噬小体形成增多,自噬水平升高。通过上述实验,我们得出结论：PDCD4敲除之后,自噬水平明显升高。
3. PDCD4抑制LPS诱导的自噬可能是通过p38MAPK通路
PDCD4本身可以抑制自噬的发生,PDCD4-/-小鼠的自噬也明显升高,其作用机制是什么呢?我们主要研究PDCD4对LPS诱导的自噬通路的影响,对LPS诱导自噬的几个通路进行了分析。我们通过流式细胞术检测ROS活性,结果发现PDCD4过表达对氧化应激通路没有明显的影响。进一步检测LPS诱导自噬作用下,自噬相关蛋白Beclin-1的表达,结果发现虽然LPS确实可以诱导Beclin-1的表达,但是PDCD4对这种诱导表达没有什么明显的影响,说明PDCD4不是通过ROS和Beclin-1介导的通路影响自噬。进一步探索发现LPS可以诱导磷酸化p38MAPK的表达,并且在PDCD4-/-小鼠的巨噬细胞中,这种诱导作用更加明显,磷酸化p38MAPK的表达明显高于C57小鼠,因此p38MAPK通路可能是PDCD4调控自噬发生的主要通路。
结论
1. PDCD4在人卵巢癌中表达明显降低甚至缺失,PDCD4的这种表达状态和卵巢癌高的病理级别密切相关。
2. PDCD4过表达在体内外均可以抑制卵巢癌的恶性增殖能力。
3. PDCD4在体内外都可以增强卵巢癌对顺铂的化疗敏感性。
4. PDCD4增强顺铂化疗敏感性的机制主要是通过促进顺铂诱导的细胞凋亡,其
通路主要是死亡受体介导的凋亡通路。
5. PDCD4具有抑制自噬发生的作用,其作用可能是通过调控与自噬发生相关的p38MAPK通路来实现的。
创新性和意义
1.发现PDCD4在卵巢癌中表达降低甚至缺失,并且与肿瘤的病理级别密切相关。进一步通过体内外实验证明PDCD4过表达确实具有抑制肿瘤生长和恶性行为的能力,为PDCD4的临床治疗提供了理论和实验依据。
2.国内外首次系统地研究了PDCD4与卵巢癌化疗敏感性的关系,并深入阐明了其机制。PDCD4在体内外均能够明显的增强卵巢癌对顺铂的化疗敏感性,其作用机制主要是通过促进顺铂诱导的细胞凋亡,其作用通路主要是死亡受体介导的凋亡通路。此研究不仅深入阐明了PDCD4的作用机制,而且为PDCD4应用与临床化疗药物联合应用提供了更有力的依据。
3.国内外首次证明了PDCD4对细胞自噬的影响。细胞自噬是存在于所有细胞的一个广泛现象,生理水平对维持细胞的稳定有重要的作用,自噬失调将会参与很多疾病的发生。我们证明了PDCD4具有抑制自噬发生的作用,这不仅是对PDCD4的功能进行了补充,而且对深入研究很多疾病的发生机制具有重要的意义。
研究的局限性
1. PDCD4诱导死亡受体表达及降低c-FLIP-L的表达机制还有待于进一步研究。
2. PDCD4抑制自噬发生的机制还有待于深入的研究。
第二部分PDCD5在卵巢癌中的表达及其临床意义目的
程序化死亡因子5基因(PDCD5)是北京大学人类疾病基因研究中心从白血病细胞株TF—1细胞中克隆的新基因。PDCD5蛋白在凋亡过程中不仅表达明显增加,而且在凋亡早期出现明显核转位现象,可能是一种细胞凋亡的早期信号。PDCD5单独不能对细胞产生明显的影响,只有在诱导凋亡的因素的存在下可明显促进凋亡,表明PDCD5是凋亡促进剂,而不是凋亡诱导剂。PDCD5作为促凋亡因子,已经证实在人类某些肿瘤如胃癌、胶质瘤等中表达降低,并且与病人的病理和预后相关,提示PDCD5可能和肿瘤的发生发展相关。但PDCD5在卵巢癌中表达和意义尚未见报道。
本研究的目的主要是通过检测PDCD5在卵巢癌细胞系,浆液性囊腺癌、浆液性囊腺瘤以及正常卵巢中的表达水平,分析PDCD5表达与临床病理特征及预后之间的关系,为下一步研究其机制及与PDCD5的关系提供实验基础。
方法
一. PDCD5在人卵巢癌细胞和组织中的表达
1.病例收集：收集山东大学齐鲁医院妇产科2001-2007年间41例浆液性囊腺癌,26例浆液性囊腺瘤,和20例正常对照(取自手术切除卵巢的非卵巢疾病患者)。所有患者手术前未接受任何放、化疗治疗。
2.分别用RT-PCR、western blot及免疫组化的方法检测卵巢癌细胞系(SKOV3、CAOV3和OVCAR3)以及上述组织中PDCD5mRNA及蛋白质的表达。
二、PDCD5在人卵巢癌组织中表达的临床意义
1.利用x2检验及Fisher's精确概率分析PDCD5表达和浆液性囊腺癌患者的年龄,发病部位,转移情况,病理分级和FIGO分期之间的关系。
2.用Kaplan-Meier生存曲线分析PDCD5的表达与卵巢癌患者生存率之间的关系。
结果
一、PDCD5mRNA和蛋白在人卵巢癌细胞系中的表达
为了探讨PDCD5在卵巢癌发生发展中的作用,我们首先用RT-PCR及western blot的方法检测了PDCD5在来源于浆液性囊腺癌的细胞系SKOV3、CAOV3和OVCAR3中的表达。结果显示,OVCAR3中表达较高的PDCD5mRNA和蛋白,SKOV3中表达较低,在CAOV3中虽然mRNA水平相对较高,但是蛋白水平仍然较低,表明在人浆液性囊腺癌细胞系中PDCD5的表达是降低的,提示PDCD5可能在卵巢癌中表达下调。
二、PDCD5mRNA和蛋白在人浆液性囊腺癌组织中的表达
进一步我们检测了正常卵巢,浆液性囊腺瘤(良性卵巢瘤)和浆液性囊腺癌组织中PDCD5的表达情况,结果显示在mRNA和蛋白水平,浆液性囊腺癌组织中的PDCD5表达下调甚至缺失,而在正常卵巢和浆液性囊腺瘤中均存在中等或高水平的PDCD5表达。进一步我们采用免疫组化的方法检测了20例正常卵巢,26例浆液性囊腺瘤和41例浆液性囊腺癌组织,结果显示所有的正常卵巢和浆液性囊腺瘤中PDCD5的表达都是阳性的,80%(16/20)的正常卵巢组织和76.9%(20/26)的浆液性囊腺瘤组织中PDCD5呈现中度或高度表达,然而,46.3%(19/41)的浆液性囊腺癌组织中呈现PDCD5低表达状态,甚至有22%(9/41)的浆液性囊腺癌组织中没有检测到PDCD5蛋白的表达。这些结果表明,PDCD5在卵巢癌组织中表达下调甚至缺失。
三、PDCD5在浆液性囊腺癌的低表达状态和卵巢癌的FIGO分期相关
为了进一步确定PDCD5在浆液性囊腺癌中低表达或缺失的临床意义,我们用χ2和Fisher's精确检验法分析了PDCD5表达水平和患者的年龄,发病部位,转移情况,病理分级和FIGO分期之间的相关性。结果显示,PDCD5的表达水平和其他临床指标都没有明显的相关性,但是和患者的FIGO分期有显著的相关性,PDCD5高表达的比率在FIGO分期Ⅲ期和Ⅳ期病人中为19.2%(5/26),明显低于其在FIGO分期Ⅰ期和Ⅱ期病人中的比率53.3%(8/15)。这些结果提示PDCD5在卵巢癌中的低表达状态和卵巢癌的进展相关。
四、PDCD5在浆液性囊腺癌的低表达或缺失和病人的预后密切相关
进一步,我们分析了PDCD5的表达和病人预后之间的关系。根据PDCD5的表达情况将病人分为两组：PDCD5高表达组(3-6分,n=16),PDCD5低表达组(0-3分,n=14)。将两组的生存时间应用Kaplan-Meier生存曲线及生存率log-rank检验进行分析之后发现,PDCD5高表达患者的生存时间明显长于低表达组(p<0.05),提示PDCD5的表达水平和患者的预后密切相关。
结论
1. PDCD5在人卵巢浆液性囊腺癌中表达显著降低甚至缺失。
2. PDCD5在卵巢癌中的降低表达和卵巢癌病人的FIGO分期密切相关。
3. PDCD5在卵巢癌中的表达水平和病人的预后有显著相关性。
创新性和意义
1.本研究发现PDCD5在卵巢浆液性囊腺癌中表达降低甚至缺失,并且和患者的FIGO分期密切相关,提出PDCD5可能在卵巢癌的发病中发挥重要作用。
2.发现了PDCD5和卵巢癌病人的预后密切相关,为判断病人预后的依据提供了理论基础。并且初步提出PDCD5在卵巢癌发生发展的作用,为进一步研究PDCD5的作用和将来在临床治疗中的应用提供了依据和线索。
局限性
1. PDCD5对卵巢癌的生长及其恶性行为的影响有待于进一步研究。
2. PDCD5在肿瘤中的作用机制至今还不清楚,有待于深入的研究。
Part 1 The effect and mechanism of PDCD4 in ovarian cancer
Objective
Ovarian cancer is the leading cause of death from gynecological malignancy worldwide and serous cystadenocarcinomas is the most common histologic type of epithelial ovarian carcinomas. Because early symptoms of patients with serous cystadenocarcinomas are frequently nonspecific and early detection is lack of reliable methods, the majority of ovarian cancer patients were diagnosed at late stage, and the prognosis is very poor. Platinum-based chemotherapy is the most commonly used method for treatment of advanced ovarian cancer. However, although the initial efficacy is 50-80%, its final efficacy is often limited to less than 30% because the existence or development of chemoresistance. Factors that enhance sensitivity of ovarian cancer cells to chemotherapeutic drugs could provide predictive biomarkers or targets for therapy. Therefore, it is necessary to develop drugs to target specific molecular pathways and improve the chemosensitivity of ovarian cancer.
PDCD4 (programmed cell death 4) is new tumor suppressor, which can inhibit tumor growth through suppressing protein translation and transcriptional activity of AP-1. It has been reported that PDCD4 expression was decreased in some cancers and PDCD4 played important roles in the development of cancer. Recently, some studies indicated that PDCD4 was also involved in inflammatory diseases. However, whether PDCD4 expression was decreased in ovarian cancer? What roles did PDCD4 play in the formation and development of ovarian cancer? Whether PDCD4 can change the chemosensitivity of ovarian cancer? What is the mechanism of PDCD4, and whether PDCD4 played roles in diseases by regulating autophagy? All these problems remain to be investigated.
In this study, we detected expression of PDCD4 in ovarian cancer, study the effect of PDCD4 on the development and chemosensitivity of ovarian cancer, and further explore the mechanisms of PDCD4, such as whether PDCD4 can promote apoptosis and regulate autophagy.
Methods
1. The expression status and role of PDCD4 in ovarian cancer
(1) We detected PDCD4 mRNA and protein expression in normal ovarian tissue, serous cystadenomas and serous cystadenocarcinomas by RT-PCR, western blot and immunohistochemistry. The Chi-square test was used to compare the expression of PDCD4 with clinico-pathological parameters.
(2) The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established by Lipofectamine 2000 transfection and G418 selection. Cell proliferation and colony formation assay were used to detected the effect of PDCD4 on ovarian cancer cells.
(3) SKOV3, SKOV3-MOCK, SKOV3-PDCD4 cells were injected subcutaneously in the left flank of nude mice. The effect of PDCD4 expression on tumorigenesis of ovarian cancer cells was detected.
2. The effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer
(1) The chemosensitivity of four kinds of ovarian cancer cells (SKOV3,CAOV3, OVCAR3 and 3AO) to cisplatin were detected by MTT assay. The relationship of PDCD4 expression and chemosensitivity was analyzed.
(2) PDCD4 was overexpressed in SKOV3 and CAOV3 cells, and whether PDCD4 overexpression can enhance chemosensitivity to cisplatin and other drugs in ovarian cancer cell lines was investigated by microscope and MTT assay.
(3) Two siRNAs targeted PDCD4 and non-silencing control siRNA were synthesized, then siRNAs were transfected into OVCAR-3 cells, after 48h, cells were subjected to RT-PCR and immunoblotting analysis. The effect of knockdown of PDCD4 with PDCD4-specific siRNAs on the chemosensitivity was detected by MTT assay.
(4) SKOV3 cells were inoculated subcutaneously into the left flank of nude mice. When tumors reached to about 100 mm3 one week later, these mice were randomly divided into six groups as follows:Normal saline; MOCK; PDCD4; cisplatin; MOCK+cisplatin; PDCD4+cisplatin. The tumor volume and weight was detected to investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo.
(5) The effect of PDCD4 expression on cisplatin-induced alterations in cell cycle was analyzed by PI staining. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays.
(6) To further examine the particular apoptotic pathways by which PDCD4 promotes cisplatin-induced apoptosis, we detected expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, c-FLIP by western blot. The expression of DR4 and DR5 was analyzed by flow cytometer. A specific caspase-8 inhibitor Z-ITED-FMK was used to inhibit the activity of csapase-8, then cisplatin-induced apoptosis in PDCD4-overexpressing ovarian cancer cells was determined.
3. The role and mechanism of PDCD4 in autophagy
(1) PDCD4 was overexpressed in ovarian cancer cell line SKOV3,48h later, autophagy was induced by starvation, and then marker protein of autophagy (LC3) was detected by western blot.
(2) The abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice were obtained, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/-mice, and expression of GFP-LC3 was observed by fluorescence microscope after autophagy induction.
(3) The activity of ROS was analyzed by flow cytometer, and expression of Beclin-1 and p38MAPK were detected by western blot after autophagy were induced in abdominal macrophages of C57 and PDCD4-/- mice.
Results
1. The expression status and role of PDCD4 in ovarian cancer
(1) Expression and significance of PDCD4 in human ovarian carcinomas
We first examined the expression of PDCD4 in ovarian cancer cell lines by semi-quantitative RT-PCR and western blot. The result showed that PDCD4 expression was low in SKOV3 and 3AO, moderate in CAOV3 and high in OVCAR3 cells.
To explore the expression level of PDCD4 in ovarian carcinoma, we detected PDCD4 expression in normal ovarian tissues, serous cystadenomas and serous cystadenocarcinomas by RT-PCR and immunohistochemistry (IHC). The results showed that 57.1%(8/14) of serous cystadenocarcinomas samples showed loss or reduction of PDCD4 mRNA expression. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD4 expression. However, 39.5%(17/43) of serous cystadenocarcinomas had no detectable PDCD4 protein expression. PDCD4 protein expression was found to be lost or significantly lower in serous cystadenocarcinomas compared with normal ovaries and serous cystadenomas (P<0.05). Lost or decreased PDCD4 expression in serous cystadenocarcinomas was associated significantly with higher pathological grade (P=0.0118).
(2) Overexpression of PDCD4 in ovarian cancer cells inhibited their proliferation and colony formation capacity.
The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established, and the effect of PDCD4 on ovarian cancer cells was further investigated. Our results showed that SKOV3-PDCD4 cells grew significantly slower than control cells (SKOV3 and SKOV3-MOCK) (P<0.001) detected by live cell counting. In addition, the colony number of SKOV3-PDCD4 cells was significantly decreased compared with that of SKOV3-MOCK cells. All these results show that PDCD4 overexpression significantly prevents the proliferation and colony formation capacity of ovarian cancer cells.
(3) PDCD4 overexpression in ovarian cancer cells decreased their tumorigenic capacity in nude mice.
To determine the effect of PDCD4 on proliferation of SKOV3 in vivo, we inoculated SKOV3, SKOV3-MOCK and SKOV3-PDCD4 cells into nude mice. One week after inoculation, all mice in SKOV3 and SKOV3-MOCK group developed palpable tumors and grew rapidly, however, none of the mice in SKOV3-PDCD4 group showed any obvious sign of tumor formation. Three weeks later, only two of eight mice developed small tumors. The overall mean tumor volume and weight in SKOV3-PDCD4 group were much smaller than those in SKOV3-MOCK or SKOV3 group. Moreover, there was obvious PDCD4 expression in tumors of SKOV3-PDCD4 group compared with those of SKOV3 or SKOV3-MOCK group detected by IHC. These results indicate that overexpression of PDCD4 decreases tumorigenic capacity and delays tumor growth in vivo.
2. PDCD4 Enhances Chemosensitivity of Ovarian Cancer Cells by Activating Death Receptor Pathway in Vitro and Vivo
(1) PDCD4 expression correlates with the cytotoxic activity of cisplatin in different ovarian cancer cell lines.
In the above part, we indicated that PDCD4 expression was significantly decreased in ovarian cancer, and PDCD4 expression could inhibit proliferation of ovarian cancer cells in vitro and in vivo. However, the effect of PDCD4 expression on the sensitivity of ovarian cancer cells to cisplatin needs to be further investigated. We detected the sensitivity of three kinds of ovarian cancer cell lines to cisplatin by MTT assay, the results showed that OVCAR3 and CAOV3 cells with relatively high or moderate levels of PDCD4 expression were more sensitive to cisplatin. However, SKOV3 and 3AO cells with relatively low levels of PDCD4 expression were resistant to cisplatin. These results suggest that PDCD4 expression may be associated with high sensitivity to cisplatin in ovarian cancer cell lines.
(2) PDCD4 overexpression enhances chemosensitivity of ovarian cancer cells.
To investigate the direct impact of PDCD4 on chemosensitivity, we overexpressed PDCD4 in SKOV3 and CAOV3 cells. The results showed that in the presence of cisplatin (25μg/ml), SKOV3-PDCD4 cells appeared marked cell death while there was no observable cell death in SKOV3-MOCK cells. This result was confirmed by MTT assay which showed that cell viability of SKOV3-PDCD4 cells was significantly decreased (p<0.05).Similarly, CAOV3 cells transfected with PDCD4 were more sensitive to cisplatin than control cells. Moreover, our results showed that the cell viability of SKOV3-PDCD4 cells was more decreased than that of SKOV3-MOCK cells at the same concentration of carboplatin (p<0.05). However, there was no significant difference between the SKOV3-PDCD4 cells and SKOV3-MOCK cells when the cells were treated with CTX, VP-16 or paclitaxcl. These results indicate that PDCD4 expression selectively enhances platinum-based chemotherapy in ovarian cancer cells.
(3) Knockdown of PDCD4 confers resistance to cisplatin in ovarian cancer cells.
To further confirm the effect of PDCD4 on the chemosensitivity of ovarian cancer cells, we knocked down PDCD4 expression by PDCD4-specific siRNAs in OVCAR3 cells. Two PDCD4-specific siRNAs markedly inhibit expression of PDCD4 mRNA by 77% and 76% and PDCD4 protein by 80% and 88%, whereas nonspecific siRNA had no significant effect on PDCD4 expression. Knockdown of PDCD4 with PDCD4-specific siRNAs significantly increased cell viability when compared with controls treated with nonspecific siRNA (p<0.05). This indicates that downregulation of PDCD4 enhances resistance to cisplatin.
(4) Combination of PDCD4 and cisplatin significantly inhibits xenograft growth in vivo.
To investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo, we established a xenograft animal model and mice were randomly divided into six groups and treated as follows:Normal saline (NS); MOCK; PDCD4; cisplatin; MOCK+cisplatin; and PDCD4+cisplatin. After treatment, mice were sacrificed, and tumor size and weight were measured. Tumor volume and weight in PDCD4+cisplatin group (0.0375±0.0069 mm3 and 0.0300±0.0044 g) were markedly lower when compared with that in cisplatin group (0.1386±0.0185 mm3 and 0.0860±0.0204 g) or PDCD4 (0.0779±0.0115 mm3 and 0.0533±0.0042 g) or MOCK+cisplatin (0.1621±0.0090 mm3 and 0.07667±0.0126 g). These data indicate that PDCD4 can also enhance the sensitivity of ovarian cancer to cisplatin in vivo.
(5) PDCD4 expression promotes cisplatin-induced apoptosis, but not alterations in cell cycle.
To determine the mechanism by which PDCD4 enhances sensitivity of ovarian cancer cells to cisplatin, we first analyzed the effect of PDCD4 expression on cisplatin-induced alterations in cell cycle. Cisplatin treatment markedly increased cell number of S phase, however, PDCD4 overexpression had no significant impact on platinum-induced alterations in cell cycle. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays. Staining with Hoechst showed that PDCD4 overexpression increased the number of apoptotic cells, characterized by chromatin condensation and nuclear fragmentation after treatment with cisplatin. Likewise, results with the Tunnel assay revealed that the combination of PDCD4 expression with cisplatin resulted in a significant increase in apoptosis (30.1%), whereas control cells treated with cisplatin resulted in minimal apoptosis(1.5%). Therefore, we can conclude that PDCD4 expression promotes cisplatin-induced apoptosis.
(6) PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathways.
To further explore the apoptotic pathway by which PDCD4 promotes cisplatin- induced apoptosis, we next detected the expression of several apoptosis-related proteins by western blot. Our results showed that the expression of cleaved caspase-3,-8 in SKOV3-PDCD4 cells treated with cisplatin obviously elevated compared with those in SKOV3 and SKOV3-MOCK cells. However, a slight increase of cleaved caspase-9 and Bax was observed in SKOV3-PDCD4 cells compared with the control cells while there was nearly no difference about expression of Bcl-2. When Z-ITED-FMK was used to inhibit the activity of caspase-8, cisplatin-induced apoptosis was attenuated in SKOV3-PDCD4 cells. Furthermore, we detected the expression of these apoptosis-related proteins in the tumor tissues from tumor-bearing nude mice, similar results were found. In addition, we detected altered expression of caspase-8 inhibitory protein c-FLIP-L and death receptors DR5 in PDCD4-overexpressing cells. PDCD4 decreased the expression of c-FLIP-L and increased the expression of DR5. Taken together, these results suggest that PDCD4 promotes cisplatin-induced apoptosis mainly by the activation of death receptor pathway.
3. The role and mechanism of PDCD4 in autophagy
(1) Overexpression of PDCD4 inhibit autophagy of ovarian cancer cells
To investigate the effect of PDCD4 on autophagy, we overexpressed PDCD4 in SKOV3 cells,48h later, autophagy was induced by starvation, and LC3 (autophagy marker) expression was detected by western blot. Our results showed that overexpression of PDCD4 could inhibit the expression of LC3-Ⅱ, which indicated that PDCD4 could inhibit autophagy of SKOV3 cells. We further determined the LC3 expression of tumor tissue from tumor-bearing nude mice treated with MOCK and PDCD4 vector, similar results were found.
(2) Autophagy was significantly increased in macrophage of PDCD4-/- mice
To further confirm the effect of PDCD4 on autophagy, we obtained the abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. Our results showed that LC3-Ⅱexpression of macrophagy from PDCD4-/- was significantly higher than that of C57 mice. In addition, GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/- mice, and the expression of GFP-LC3 was observed by fluorescence microscope after autophagy. The formation of GFP-LC3-labeled structures (GFP-LC3 "dots"), which represents autophagosomes, was extensively induced in PDCD4-/-macrophage exposed to LPS, which indicated that knockout of PDCD4 could enhance autophagy.
(3) PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.
PDCD4 could inhibit autophagy, and knockout of PDCD4 could enhance autophagy. However, the mechanism of PDCD4 regulating autophagy is unknown. We mainly study the mechanism of PDCD4 regulating LPS-induced autophagy. We found that there were no difference about the activity of ROS between C57 and PDCD4-/- mice induced by LPS. Next, we detected the expression of Beclin-1 and p38MAPK by western blot, the results showed that although LPS could induce the expression of Beclin-1, there was no difference of Beclin-1 expression between C57 and PDCD4-/- mice induced by LPS. Interestingly, we found that LPS induced the expression of pho-p38MAPK, and its expression was significantly increased in PDCD4-/- mice compared with that of C57 mice, which indicated that PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.
Conclusions
1. PDCD4 expression was significantly decreased or lost in ovarian cancer, which correlated with the high pathological grade ovarian cancer.
2. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo.
3. PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo.
4. PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathway.
5. PDCD4 may inhibit autophagy through p38MAPK pathway.
Originality
1. We demonstrate, for the first time, that loss of PDCD4 expression is a frequent event in human ovarian cancer and significantly correlated with the pathological grade of ovarian cancer. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo, which provide first hand data for the gene therapy targeted PDCD4 for ovarian cancer.
2. We studied the effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer for the first time. We found that PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo, the mechanism was that PDCD4 promoted cisplatin-induced apoptosis by mainly activating death receptor pathway.
3. We demonstrate, for the first time, PDCD4 may inhibit autophagy through p38MAPK pathway. It was not only provides the new research ideas for investigating the function of PDCD4, but also reveals the mechanism of many diseases, seeking new methods to prevent and treat the disease.
Limitations of this study
1. The mechanism of PDCD4 inducing DR5 expression and PDCD4 inhibiting c-FLIP-L need to be further investigated.
2. The mechanism of PDCD4 inhibiting autophagy needs to be further investigated.
Part 2 Clinical and prognostic significance of lost or decreased PDCD5 expression in human epithelial ovarian carcinomas
Objective
Programmed cell death 5 (PDCD5), also designated as TF-1 cell apoptosis-related gene-19 (TFAR19), is a novel gene cloned from TF-1 cells undergoing apoptosis, and it is up-regulated in the process of cell apoptosis and translocation from the cytoplasm to the nucleus. PDCD5 is a strong candidate of apoptosis-regulated protein and its overexpression promotes apoptosis triggered by certain stimuli, while blocking PDCD5 action suppresses apoptosis. Recent studies have demonstrated that PDCD5 is down-regulated in some human tumor tissues such as gastric cancer, hepatocellular carcinoma, breast cancer, acute and chronic myeloid leukemia and glioma. However, PDCD5 expression and its association with prognosis in gynecological cancer have not been evaluated.
In this study, we detected the status of PDCD5 expression in three kinds of human serous cystadenocarcinoma cell lines, normal ovarian tissues and serous ovarian tumors using RT-PCR, western blot and immuohistochemistry. Furthermore, we analyzed the association of PDCD5 expression with clinico-pathological features and survival of patients.
Methods
1. Expression of PDCD5 in human ovarian cancer cell lines and ovarian carcinomas.
(1) Case collection:Sixty-seven serous ovarian tumor samples (26 serous cystadenomas and 41 serous cystadenocarcinomas) were obtained from patients who underwent surgical operations at the Department of Gynecology, Qilu Hospital from 2001 to 2007. None of the patients studied had received adjuvant immunosuppressive treatments such as radiotherapy or chemotherapy prior to surgery. Twenty normal ovarian tissues were obtained from the normal ovaries of donors during surgery for other gynecological diseases in Qilu Hospital, Shandong University.
(2) Expression of PDCD5 mRNA and protein in ovarian cancer cell lines(SKOV3,CAOV3,OVCAR3) and above tissues were detected by RT-PCR, western blot and immunohistochemistry.
2. Clinical significance of PDCD5 expression in human ovarian carcinomas.
(1) The Chi-square and Fisher's exact test was used to compare the expression of PDCD5 with clinico-pathological parameters.
(2) Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.
Results
1. Expression of PDCD5 mRNA and protein in human ovarian cancer cell lines.
To explore the potential roles of PDCD5 in ovarian cancer, we firstly detected the status of PDCD5 expression in three ovarian cancer cell lines which derived from serous cystadenocarcinomas by RT-PCR and western blot. Our results showed that the OVCAR3 cell line expressed high levels of PDCD5 mRNA and protein, whereas the SKOV3 cell line showed low levels of PDCD5 expression. Although CAOV3 cells showed relative high PDCD5 mRNA expression, their protein expression was relative low. These results suggested that the expression of PDCD5 may be decreased in human serous cystadenocarcinomas.
2. Expression of PDCD5 mRNA and protein in human normal ovarian tissues and ovarian carcinomas.
To explore the expression of PDCD5 in ovarian carcinoma, we firstly detected PDCD5 expression in normal ovarian tissues and serous ovarian tumors by RT-PCR and western blot. We found that PDCD5 expression was obviously reduced or even lost in serous cystadenocarcinomas compared with normal ovarian tissues and serous cystadenomas. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD5 expression. Among them,80%(16/20) of normal ovarian tissues and 76.9%(20/26) of serous cystadenomas showed moderate or strong PDCD5 protein expression. In contrast,46.3%(19/41) of serous cystadenocarcinomas exhibited weak PDCD5 expression and 22%(9/41) had no detectable PDCD5 protein expression. The overall expression of PDCD5 in serous cystadenocarcinoma was significantly lower compared with normal ovarian tissues or serous cystadenomas (p<0.01).
3. Correlation of the expression level of PDCD5 with the FIGO stage of serous cystadenocarcinoma.
To determine the clinical significance of lost or reduced PDCD5 expression in serous cystadenocarcinoma, we examined the correlation of PDCD5 expression with the clinico-pathological parameters of serous cystadenocarcinoma by Chi-square and Fisher's exact test. The results showed that there was no significant correlation among PDCD5 expression and age, site of origin, metastasis and pathological grade. However, the expression of PDCD5 correlated significantly with clinical FIGO stage. The percentage of cases with PDCD5 high expression is lower in FIGO stageⅢand IV patients (19.2%,5 of 26) than that in FIGO stageⅠandⅡpatients (53.3%,8 of 15) (p<0.05).
4. Loss or reduction of PDCD5 expression was significantly associated with survival of patients with serous cystadenocarcinoma.
To assess the association of PDCD5 expression with patient survival, the survival data from 30 patients with serous cystadenocarcinomas were generated by follow-up. According to the final PDCD5 staining score in the results of IHC, these patients were divided into a high expression group (score 3-6) and a low expression group (score 0-2). The difference in survival time between patients with high PDCD5 expression tumors (n=16) and those with low PDCD5 expression tumors (n=14) was evaluated by Kaplan-Meier method and log-rank test. The result demonstrated that patients with a low level of PDCD5 expression had a significantly poorer disease-specific survival than those with a high level of PDCD5 expression (p<0.05). This indicates that the level of PDCD5 expression significantly correlated with prognosis of patients with serous cystadenocarcinoma.
Conclusions:
1. PDCD5 expression is clearly reduced or lost in serous cystadenocarcinoma compared with normal ovaries and serous cystadenomas.
2. lost or decreased PDCD5 expression in serous cystadenocarcinomas was associated significantly with FIGO stage and poorer disease-specific survival of patients.
Originality
1. We found that PDCD5 expression was lost or decreased in serous cystadenocarcinomas, and was associated significantly with FIGO stage patients, which suggest that PDCD5 expression may contribute to the pathogenesis of human serous cystadenocarcinomas.
2. We demonstrate that PDCD5 expression in serous cystadenocarcinomas was associated significantly with disease-specific survival of patients, which provide new method for diagnosis and prognosis for evaluation of serous cystadenocarcinomas.
Limitations of this study
1. The effect of PDCD5 on malignant proliferation of ovarian cancer cells needs to be further investigated.
2. The effector mechanism of PDCD5 in tumor remains unknown.
卵巢癌是妇科常见的恶性肿瘤,种类繁多,其中原发性卵巢上皮癌(最常见的是浆液性囊腺癌)占卵巢恶性肿瘤的85-90%。卵巢癌早期不易发现,就诊率低,病死率居女性生殖系统恶性肿瘤首位。临床上对于卵巢癌的治疗主要是采用手术及以铂类为主的联合化疗,尽管起初化疗效应约50-80%,但由于原发性与获得性耐药,患者的5年生存率仍然很低。因此,提高化疗敏感性,逆转化疗耐药(尤其是最常用的铂类化疗药物,如顺铂)是改善卵巢癌预后的关键。近些年来,基因治疗在肿瘤领域的成功应用为卵巢癌基因治疗奠定了基础,对恶性卵巢癌患者化疗药增敏基因和耐药基因的研究,成为卵巢癌治疗领域的热门课题。
程序性细胞死亡-4(programmed cell death 4, PDCD4)是一种新的抑癌基因,可以通过抑制蛋白转录和翻译过程抑制肿瘤细胞的生长。我们和其他学者已经发现PDCD4在肺癌和神经胶质瘤等肿瘤中表达下调并且在肿瘤中发挥着非常重要的作用,但在卵巢癌中的作用尚未见报道。那么,PDCD4在卵巢癌中的表达状态如何?其对卵巢癌的发生发展到底有什么作用?能否影响卵巢癌的化疗敏感性并成为基因治疗的靶点之一?PDCD4的作用机制是什么,能否通过调控自噬的发生来发挥其在疾病中的作用?这些都是非常值得探索的问题。
为了明确PDCD4在卵巢癌发生发展中的关系,阐明其发挥作用的机制,为PDCD4的临床应用提供理论依据和实验基础,本论文拟从以下三个方面进行研究：
(一) PDCD4在人卵巢癌中的表达状态及其作用：通过多种方法检测正常卵巢、浆液性囊腺瘤和浆液性囊腺癌中PDCD4的表达发现,PDCD4在囊腺癌的表达出现明显的下调与缺失,并且与囊腺癌的病理级别密切相关。进一步研究发现,PDCD4在体外能够明显抑制卵巢癌细胞增殖和克隆形成能力,在裸鼠体内能降低卵巢癌细胞的成瘤能力,延长肿瘤发生的潜伏期。
(二) PDCD4对卵巢癌化疗敏感性的影响及其机制研究：我们通过过表达和沉默表达PDCD4,从正反两方面证实PDCD4能显著增强卵巢癌细胞对铂类化疗药物的敏感性,在裸鼠体内PDCD4和顺铂联合治疗能有效地抑制卵巢癌的生长。进一步阐明其机制：PDCD4能够明显增强顺铂诱导的细胞凋亡,并且主要通过死亡受体通路介导的。
(三) PDCD4在细胞发生自噬过程中的作用及其机制研究：PDCD4在卵巢癌细胞中过表达可以抑制自噬的发生,PDCD4-/-小鼠的巨噬细胞自噬水平明显高于C57小鼠。进一步的研究证明PDCD4是通过调控与自噬发生有关的p38MAPK通路来发挥作用。
方法
一、PDCD4在卵巢癌中的作用及其临床意义
1.分别用RT-PCR. western blot和免疫组化方法检测人卵巢癌细胞系以及人正常卵巢、浆液性囊腺瘤和浆液性囊腺癌组织标本中PDCD4 mRNA和蛋白的表达情况。x2检验分析PDCD4在浆液性囊腺癌病例中的表达与其临床病理特点间的关系。
2.利用脂质体转染PDCD4重组表达载体和G418筛选的方法建立PDCD4稳定表达的细胞系SKOV3-PDCD4和对照细胞系SKOV3-MOCK,用活细胞计数法检测PDCD4对细胞增殖能力的影响,平板集落形成实验检测PDCD4对细胞克隆形成能力的影响。
3.将SKOV3,SKOV3-MOCK, SKOV3-PDCD4细胞分别注射到裸鼠腋窝皮下荷瘤,观察PDCD4表达对卵巢癌细胞SKOV3在裸鼠体内成瘤能力的影响。
二、PDCD4对卵巢癌化疗敏感性的影响及其机制研究
1.用MTT法检测四种卵巢癌细胞系,SKOV3、CAOV3、OVCAR3和3AO对顺铂的化疗敏感性,分析PDCD4表达水平与卵巢癌细胞对顺铂化疗敏感性的关系。
2.在卵巢癌PDCD4相对低表达的卵巢癌细胞系SKOV3和CAOV3中,稳定或瞬时表达PDCD4,显微镜下观察细胞的状态,用MTT法检测PDCD4对卵巢癌细胞化疗敏感性的影响。
3.设计合成两条PDCD4特异的siRNA,用siRNA沉默PDCD4高表达的卵巢癌细胞系OVCAR3中PDCD4的表达,干扰48小时后用RT-PCR和western blot检测PDCD4的表达,确定干扰效率。用MTT实验检测PDCD4沉默表达对卵巢癌化疗敏感性的影响。
4.用卵巢癌细胞SKOV3注射到裸鼠腋窝皮下荷瘤后,随机分为6组,分别采用生理盐水,空载体,PDCD4载体,顺铂,空载体+顺铂,PDCD4+顺铂6种方法进行治疗,探讨PDCD4和顺铂联合治疗对肿瘤生长的影响。
5.用PI染色,流式细胞仪检测PDCD4过表达对顺铂诱导的细胞周期变化的影响。进一步通过Hoechst染色从细胞形态学上观察PDCD4过表达对顺铂诱导的凋亡的影响,采用Tunnel染色对细胞的晚期凋亡率进行检测。
6.应用western blot检测PDCD4过表达对顺铂诱导的凋亡相关蛋白caspase-3、caspase-8、caspase-9的活性变化及Bcl-2、Bax, c-FLIP蛋白表达变化。
7.用caspase-8抑制剂抑致caspase-8的活性,检测PDCD4过表达对顺铂的化疗增敏作用的影响。
8.利用流式细胞术检测PDCD4 j过表达对顺铂诱导的死亡受体DR5的表达状态的变化。
三、PDCD4在细胞发生自噬过程中的作用及其机制研究
1.用空载体或PDCD4重组表达载体转染卵巢癌细胞系SKOV3,转染48小时之后,采用饥饿的方法诱导细胞产生自噬,检测自噬标志性蛋白LC3的表达情况。
2.分别获取C57和PDCD4基因敲除小鼠的腹腔巨噬细胞和骨髓来源的巨噬细胞,采用饥饿,LPS或雷帕霉素刺激诱导自噬的产生,western blot检测LC3表达情况。分别在C57和PDCD4基因敲除小鼠的腹腔巨噬细胞转染GFP-LC3,诱导自噬发生后,荧光显微镜观察GFP-LC3的表达分布情况。
3.分别获取C57和PDCD4基因敲除小鼠的腹腔巨噬细胞,诱导自噬发生后,用ROS活性检测试剂盒,流式细胞术检测PDCD4对ROS活性的影响。Western blot检测PDCD4对自噬相关蛋白Beclin-1和自噬发生有关的p38MAPK通路中p38MAPK表达的影响。
结果
一、PDCD4在卵巢癌中的表达、临床意义及对肿瘤恶性行为的影响
1. PDCD4在人卵巢癌细胞系和卵巢癌组织中的表达状况及临床意义
为了研究PDCD4在卵巢癌中的作用,首先应用RT-PCR和western blot的方法检测了四种卵巢癌细胞系SKOV3、CAOV3、OVCAR3和3AO中PDCD4的表达情况,结果显示PDCD4在SKOV3和3AO中表达较低,CAOV3中度表达,OVCAR3表达相对较高。进而我们采用RT-PCR和免疫组化的方法检测了20例正常卵巢、26例浆液性囊腺瘤、43例浆液性囊腺癌组织中PDCD4的表达,发现与正常卵巢和浆液性囊腺瘤相比,PDCD4在浆液性囊腺癌中的表达明显下调甚至缺失。进一步分析发现,PDCD4的表达下调和缺失与浆液性囊腺癌患者高的病理级别密切相关。
2.外源性PDCD4的表达能够明显抑制卵巢癌细胞的生长及克隆形成能力
为了进一步研究PDCD4在卵巢癌中的作用,我们用脂质体介导将PDCD4重组表达载体和空载体转染到卵巢癌细胞系SKOV3中,并用G418进一步筛选,通过RT-PCR和western blot鉴定,得到稳定表达PDCD4的细胞系SKOV3-PDCD4和对照细胞系SKOV3-MOCK。通过活细胞计数的方法检测细胞增殖能力发现,SKOV3-PDCD4的增殖能力明显减弱(p<0.001),克隆形成实验显示SKOV3-PDCD4的克隆形成数明显降低(p<0.05),提示PDCD4在体外能明显抑制卵巢癌的恶性增殖能力。
3.外源性PDCD4表达降低了卵巢癌细胞在裸鼠体内的成瘤能力
为了研究PDCD4在体内是否同样具有抑制卵巢癌恶性行为的能力,我们用SKOV3、SKOV3-MOCK、SKOV3-PDCD4细胞分别在裸鼠皮下进行荷瘤,观察肿瘤的生长。结果显示,SKOV3-PDCD4组的成瘤率明显降低,成瘤时间明显延长,而且形成的肿瘤大小和重量也明显低于其他两组(p<0.05)。这些结果显示,PDCD4过表达在体内同样可以抑制卵巢癌细胞的恶性行为。
二、PDCD4对卵巢癌化疗敏感性的影响及其机制研究
1. PDCD4表达水平与卵巢癌敏感性的关系
上一部分研究提出PDCD4不仅在卵巢癌中表达降低,而且PDCD4在体内外都可以抑制卵巢癌的恶性增殖能力。然而,PDCD4能否增强卵巢癌的化疗敏感性,从而增强其疗效仍有待于进一步研究。我们通过MTT法检测上述四种卵巢癌细胞系SKOV3、CAOV3、OVCAR3及3AO对顺铂的化疗敏感性,分析PDCD4表达水平和其敏感程度的关系。结果显示,相对高表达PDCD4的CAOV3和OVCAR3细胞对顺铂更加敏感,而低表达细胞SKOV3和3AO则较不敏感,提示PDCD4表达水平可能与卵巢癌的化疗敏感性相关。
2.外源性PDCD4表达可以增强卵巢癌细胞的化疗敏感性
为了确定PDCD4对化疗敏感性的影响,我们在第一部分得到的稳定表达细胞系SKOV3-PDCD4和SKOV3-MOCK中分别加入不同浓度的顺铂,MTT检测细胞的生存率。结果显示,在12.5μg/ml浓度的顺铂作用下,SKOV3-PDCD4的细胞形态出现变圆或死亡,而SKOV3-MOCK没有明显的改变。MTT结果显示SKOV3-PDCD4的细胞生存率明显低于SKOV3-MOCK。我们通过瞬时转染的方法在CAOV3细胞中过表达PDCD4也发现了相似的结果,表明外源性的PDCD4表达可以增强卵巢癌细胞对顺铂的化疗敏感性。进一步检测了PDCD4过表达对其他化疗药物的影响,结果显示PDCD4同样也可以增强卵巢癌细胞对卡铂的化疗敏感性,但是对环磷酰胺,VP-16及紫杉醇的化疗敏感性没有明显的影响。
3.沉默表达PDCD4减低了卵巢癌对顺铂的化疗敏感性
为了进一步确定PDCD4对卵巢癌化疗敏感性的影响,我们设计合成了两条PDCD4特异性siRNA片段,将其转染PDCD4高表达的卵巢癌细胞系OVCAR3, RT-PCR和,western blot检测PDCD4表达发现,两条siRNA片段都能够有效的沉默PDCD4的表达(mRNA水平降低77%和76%,蛋白表达降低80%和88%)。进一步,我们采用这两条siRNA沉默OVCAR3中的PDCD4表达,发现其对顺铂的化疗敏感性明显降低(p<0.05),从反方面验证了PDCD4能够增强卵巢癌对顺铂的化疗敏感性。
4. PDCD4和顺铂体内联合治疗能明显抑制卵巢癌的生长
为了验证PDCD4在体内能否增强卵巢癌对顺铂的化疗敏感性,我们用SKOV3细胞在裸鼠皮下荷瘤,随即分为6组进行治疗：生理盐水对照组,空载体组,PDCD4组,顺铂组,空载体+顺铂组,PDCD4+顺铂组。结果显示：PDCD4+顺铂组肿瘤的大小和重量(0.0375士0.0069 mm3,0.0300+0.0044 g)都明显的低于顺铂组(0.1386±0.0185 mm3,0.0860±0.0204 g), PDCD4组(0.0779±0.0115 mm3,0.0533±0.0042 g),和空载体+顺铂组(0.1621±0.0090 mm3,0.07667±0.0126 g)。通过免疫组化检测以上各组肿瘤中PDCD4的表达显示,有PDCD4载体治疗组的PDCD4表达明显增高,说明体内表达成功。体内实验结果显示PDCD4和顺铂联合治疗能明显抑制卵巢癌的生长,提示PDCD4在体内仍具有增强卵巢癌对顺铂的化疗敏感性的作用。
5. PDCD4增强顺铂的化疗敏感性是通过诱导细胞凋亡,而不是改变细胞周期
为了进一步检测PDCD4增强卵巢癌对顺铂化疗敏感性的机制,我们首先采用流式细胞术检测PDCD4对顺铂作用后细胞周期的影响,结果显示顺铂能明显诱导SKOV3细胞S期阻滞,这和其他有关顺铂的作用机制的研究报道是相一致的,但是PDCD4过表达对顺铂诱导的细胞周期改变没有明显的影响。进一步,我们通过Hoechst染色和Tunnel染色检测了细胞凋亡,结果显示在相同浓度顺铂作用下,与SKOV3-MOCK对照组(凋亡率为1.5%)相比,PDCD4过表达组SKOV3-PDCD4细胞的核固缩、碎裂明显增多,Tunnel染色流式细胞术检测显示PDCD4过表达组细胞凋亡率(30.1%)明显增高。这些结果说明PDCD4增强卵巢癌对顺铂化疗敏感性的机制主要是通过增强顺铂诱导的细胞凋亡,而对细胞周期的变化没有什么影响。
6. PDCD4增强顺铂诱导的凋亡通路主要通过死亡受体通路
介导细胞凋亡主要的信号通路分为线粒体通路和死亡受体通路。为了确定PDCD4增强顺铂诱导的凋亡通路,我们通过western blot检测了凋亡相关蛋白caspase-3,8,9以及Bcl-2,Bax的变化。结果显示,在相同浓度顺铂作用下,与SKOV3-MOCK对照组相比,PDCD4过表达组SKOV3-PDCD4细胞的caspase-3和caspase-8活性蛋白表达明显升高,而caspase-9和Bax具有轻微的升高,Bcl-2的表达没有明显变化,免疫组化检测肿瘤切片中的表达也得到了相似结果,提示死亡受体通路可能是PDCD4的主要作用通路。采用caspase-8抑制剂能够逆转PDCD4对顺铂的化疗增敏作用,进一步验证了这个结论。另外,在顺铂作用下,PDCD4过表达能明显上调死亡受体DR5的表达,内源性caspase-8的抑制蛋白c-FLIP-L的表达明显下调,可能是casapse-8活性蛋白升高的原因之一。根据这些结果,我们认为PDCD4主要通过死亡受体介导的凋亡通路促进了顺铂诱导的凋亡,从而增强了卵巢癌细胞对顺铂的化疗敏感性。
三、PDCD4在细胞发生自噬过程中的作用及其机制研究
尽管我们和其它学者的研究显示PDCD4可能通过诱导细胞凋亡或干扰细胞周期发挥作用,但其作用并不明显,不能完全解释PDCD4对肿瘤的有效抑制。自噬是近几年发现的细胞死亡的另一种形式,故进一步探讨了PDCD4对自噬的影响。
1.外源性PDCD4表达抑制卵巢癌细胞发生自噬
为了研究PDCD4对细胞自噬的作用,我们在SKOV3中过表达PDCD4之后,用饥饿诱导因素EBSS诱导自噬的发生,检测自噬的标记分子LC3蛋白的表达。结果发现,PDCD4过表达组LC3-Ⅱ明显降低,说明其自噬水平降低。进一步检测体内实验显示空载体和PDCD4表达载体治疗的肿瘤组织中LC3蛋白的表达也得到了相似的结果,说明PDCD4
确实具有抑制卵巢癌细胞自噬发生的作用。
2. PDCD4敲除后巨噬细胞自噬明显增强
为了进一步确定PDCD4在自噬发生中的作用,我们获取了C57和PDCD4-/-小鼠的腹腔巨噬细胞和骨髓来源的巨噬细胞,分别用EBSS, LPS和雷帕霉素诱导自噬的发生,结果都显示PDCD4-/-小鼠的两种来源的巨噬细胞中LC3-Ⅱ表达明显升高。进一步在C57和PDCD4-/-两种小鼠的腹腔巨噬细胞中转染GFP-LC3质粒,LPS诱导自噬之后发现,PDCD4-/-小鼠的巨噬细胞中绿色荧光斑点明显多于C57小鼠,说明自噬小体形成增多,自噬水平升高。通过上述实验,我们得出结论：PDCD4敲除之后,自噬水平明显升高。
3. PDCD4抑制LPS诱导的自噬可能是通过p38MAPK通路
PDCD4本身可以抑制自噬的发生,PDCD4-/-小鼠的自噬也明显升高,其作用机制是什么呢?我们主要研究PDCD4对LPS诱导的自噬通路的影响,对LPS诱导自噬的几个通路进行了分析。我们通过流式细胞术检测ROS活性,结果发现PDCD4过表达对氧化应激通路没有明显的影响。进一步检测LPS诱导自噬作用下,自噬相关蛋白Beclin-1的表达,结果发现虽然LPS确实可以诱导Beclin-1的表达,但是PDCD4对这种诱导表达没有什么明显的影响,说明PDCD4不是通过ROS和Beclin-1介导的通路影响自噬。进一步探索发现LPS可以诱导磷酸化p38MAPK的表达,并且在PDCD4-/-小鼠的巨噬细胞中,这种诱导作用更加明显,磷酸化p38MAPK的表达明显高于C57小鼠,因此p38MAPK通路可能是PDCD4调控自噬发生的主要通路。
结论
1. PDCD4在人卵巢癌中表达明显降低甚至缺失,PDCD4的这种表达状态和卵巢癌高的病理级别密切相关。
2. PDCD4过表达在体内外均可以抑制卵巢癌的恶性增殖能力。
3. PDCD4在体内外都可以增强卵巢癌对顺铂的化疗敏感性。
4. PDCD4增强顺铂化疗敏感性的机制主要是通过促进顺铂诱导的细胞凋亡,其
通路主要是死亡受体介导的凋亡通路。
5. PDCD4具有抑制自噬发生的作用,其作用可能是通过调控与自噬发生相关的p38MAPK通路来实现的。
创新性和意义
1.发现PDCD4在卵巢癌中表达降低甚至缺失,并且与肿瘤的病理级别密切相关。进一步通过体内外实验证明PDCD4过表达确实具有抑制肿瘤生长和恶性行为的能力,为PDCD4的临床治疗提供了理论和实验依据。
2.国内外首次系统地研究了PDCD4与卵巢癌化疗敏感性的关系,并深入阐明了其机制。PDCD4在体内外均能够明显的增强卵巢癌对顺铂的化疗敏感性,其作用机制主要是通过促进顺铂诱导的细胞凋亡,其作用通路主要是死亡受体介导的凋亡通路。此研究不仅深入阐明了PDCD4的作用机制,而且为PDCD4应用与临床化疗药物联合应用提供了更有力的依据。
3.国内外首次证明了PDCD4对细胞自噬的影响。细胞自噬是存在于所有细胞的一个广泛现象,生理水平对维持细胞的稳定有重要的作用,自噬失调将会参与很多疾病的发生。我们证明了PDCD4具有抑制自噬发生的作用,这不仅是对PDCD4的功能进行了补充,而且对深入研究很多疾病的发生机制具有重要的意义。
研究的局限性
1. PDCD4诱导死亡受体表达及降低c-FLIP-L的表达机制还有待于进一步研究。
2. PDCD4抑制自噬发生的机制还有待于深入的研究。
第二部分PDCD5在卵巢癌中的表达及其临床意义目的
程序化死亡因子5基因(PDCD5)是北京大学人类疾病基因研究中心从白血病细胞株TF—1细胞中克隆的新基因。PDCD5蛋白在凋亡过程中不仅表达明显增加,而且在凋亡早期出现明显核转位现象,可能是一种细胞凋亡的早期信号。PDCD5单独不能对细胞产生明显的影响,只有在诱导凋亡的因素的存在下可明显促进凋亡,表明PDCD5是凋亡促进剂,而不是凋亡诱导剂。PDCD5作为促凋亡因子,已经证实在人类某些肿瘤如胃癌、胶质瘤等中表达降低,并且与病人的病理和预后相关,提示PDCD5可能和肿瘤的发生发展相关。但PDCD5在卵巢癌中表达和意义尚未见报道。
本研究的目的主要是通过检测PDCD5在卵巢癌细胞系,浆液性囊腺癌、浆液性囊腺瘤以及正常卵巢中的表达水平,分析PDCD5表达与临床病理特征及预后之间的关系,为下一步研究其机制及与PDCD5的关系提供实验基础。
方法
一. PDCD5在人卵巢癌细胞和组织中的表达
1.病例收集：收集山东大学齐鲁医院妇产科2001-2007年间41例浆液性囊腺癌,26例浆液性囊腺瘤,和20例正常对照(取自手术切除卵巢的非卵巢疾病患者)。所有患者手术前未接受任何放、化疗治疗。
2.分别用RT-PCR、western blot及免疫组化的方法检测卵巢癌细胞系(SKOV3、CAOV3和OVCAR3)以及上述组织中PDCD5mRNA及蛋白质的表达。
二、PDCD5在人卵巢癌组织中表达的临床意义
1.利用x2检验及Fisher's精确概率分析PDCD5表达和浆液性囊腺癌患者的年龄,发病部位,转移情况,病理分级和FIGO分期之间的关系。
2.用Kaplan-Meier生存曲线分析PDCD5的表达与卵巢癌患者生存率之间的关系。
结果
一、PDCD5mRNA和蛋白在人卵巢癌细胞系中的表达
为了探讨PDCD5在卵巢癌发生发展中的作用,我们首先用RT-PCR及western blot的方法检测了PDCD5在来源于浆液性囊腺癌的细胞系SKOV3、CAOV3和OVCAR3中的表达。结果显示,OVCAR3中表达较高的PDCD5mRNA和蛋白,SKOV3中表达较低,在CAOV3中虽然mRNA水平相对较高,但是蛋白水平仍然较低,表明在人浆液性囊腺癌细胞系中PDCD5的表达是降低的,提示PDCD5可能在卵巢癌中表达下调。
二、PDCD5mRNA和蛋白在人浆液性囊腺癌组织中的表达
进一步我们检测了正常卵巢,浆液性囊腺瘤(良性卵巢瘤)和浆液性囊腺癌组织中PDCD5的表达情况,结果显示在mRNA和蛋白水平,浆液性囊腺癌组织中的PDCD5表达下调甚至缺失,而在正常卵巢和浆液性囊腺瘤中均存在中等或高水平的PDCD5表达。进一步我们采用免疫组化的方法检测了20例正常卵巢,26例浆液性囊腺瘤和41例浆液性囊腺癌组织,结果显示所有的正常卵巢和浆液性囊腺瘤中PDCD5的表达都是阳性的,80%(16/20)的正常卵巢组织和76.9%(20/26)的浆液性囊腺瘤组织中PDCD5呈现中度或高度表达,然而,46.3%(19/41)的浆液性囊腺癌组织中呈现PDCD5低表达状态,甚至有22%(9/41)的浆液性囊腺癌组织中没有检测到PDCD5蛋白的表达。这些结果表明,PDCD5在卵巢癌组织中表达下调甚至缺失。
三、PDCD5在浆液性囊腺癌的低表达状态和卵巢癌的FIGO分期相关
为了进一步确定PDCD5在浆液性囊腺癌中低表达或缺失的临床意义,我们用χ2和Fisher's精确检验法分析了PDCD5表达水平和患者的年龄,发病部位,转移情况,病理分级和FIGO分期之间的相关性。结果显示,PDCD5的表达水平和其他临床指标都没有明显的相关性,但是和患者的FIGO分期有显著的相关性,PDCD5高表达的比率在FIGO分期Ⅲ期和Ⅳ期病人中为19.2%(5/26),明显低于其在FIGO分期Ⅰ期和Ⅱ期病人中的比率53.3%(8/15)。这些结果提示PDCD5在卵巢癌中的低表达状态和卵巢癌的进展相关。
四、PDCD5在浆液性囊腺癌的低表达或缺失和病人的预后密切相关
进一步,我们分析了PDCD5的表达和病人预后之间的关系。根据PDCD5的表达情况将病人分为两组：PDCD5高表达组(3-6分,n=16),PDCD5低表达组(0-3分,n=14)。将两组的生存时间应用Kaplan-Meier生存曲线及生存率log-rank检验进行分析之后发现,PDCD5高表达患者的生存时间明显长于低表达组(p<0.05),提示PDCD5的表达水平和患者的预后密切相关。
结论
1. PDCD5在人卵巢浆液性囊腺癌中表达显著降低甚至缺失。
2. PDCD5在卵巢癌中的降低表达和卵巢癌病人的FIGO分期密切相关。
3. PDCD5在卵巢癌中的表达水平和病人的预后有显著相关性。
创新性和意义
1.本研究发现PDCD5在卵巢浆液性囊腺癌中表达降低甚至缺失,并且和患者的FIGO分期密切相关,提出PDCD5可能在卵巢癌的发病中发挥重要作用。
2.发现了PDCD5和卵巢癌病人的预后密切相关,为判断病人预后的依据提供了理论基础。并且初步提出PDCD5在卵巢癌发生发展的作用,为进一步研究PDCD5的作用和将来在临床治疗中的应用提供了依据和线索。
局限性
1. PDCD5对卵巢癌的生长及其恶性行为的影响有待于进一步研究。
2. PDCD5在肿瘤中的作用机制至今还不清楚,有待于深入的研究。
Part 1 The effect and mechanism of PDCD4 in ovarian cancer
Objective
Ovarian cancer is the leading cause of death from gynecological malignancy worldwide and serous cystadenocarcinomas is the most common histologic type of epithelial ovarian carcinomas. Because early symptoms of patients with serous cystadenocarcinomas are frequently nonspecific and early detection is lack of reliable methods, the majority of ovarian cancer patients were diagnosed at late stage, and the prognosis is very poor. Platinum-based chemotherapy is the most commonly used method for treatment of advanced ovarian cancer. However, although the initial efficacy is 50-80%, its final efficacy is often limited to less than 30% because the existence or development of chemoresistance. Factors that enhance sensitivity of ovarian cancer cells to chemotherapeutic drugs could provide predictive biomarkers or targets for therapy. Therefore, it is necessary to develop drugs to target specific molecular pathways and improve the chemosensitivity of ovarian cancer.
PDCD4 (programmed cell death 4) is new tumor suppressor, which can inhibit tumor growth through suppressing protein translation and transcriptional activity of AP-1. It has been reported that PDCD4 expression was decreased in some cancers and PDCD4 played important roles in the development of cancer. Recently, some studies indicated that PDCD4 was also involved in inflammatory diseases. However, whether PDCD4 expression was decreased in ovarian cancer? What roles did PDCD4 play in the formation and development of ovarian cancer? Whether PDCD4 can change the chemosensitivity of ovarian cancer? What is the mechanism of PDCD4, and whether PDCD4 played roles in diseases by regulating autophagy? All these problems remain to be investigated.
In this study, we detected expression of PDCD4 in ovarian cancer, study the effect of PDCD4 on the development and chemosensitivity of ovarian cancer, and further explore the mechanisms of PDCD4, such as whether PDCD4 can promote apoptosis and regulate autophagy.
Methods
1. The expression status and role of PDCD4 in ovarian cancer
(1) We detected PDCD4 mRNA and protein expression in normal ovarian tissue, serous cystadenomas and serous cystadenocarcinomas by RT-PCR, western blot and immunohistochemistry. The Chi-square test was used to compare the expression of PDCD4 with clinico-pathological parameters.
(2) The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established by Lipofectamine 2000 transfection and G418 selection. Cell proliferation and colony formation assay were used to detected the effect of PDCD4 on ovarian cancer cells.
(3) SKOV3, SKOV3-MOCK, SKOV3-PDCD4 cells were injected subcutaneously in the left flank of nude mice. The effect of PDCD4 expression on tumorigenesis of ovarian cancer cells was detected.
2. The effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer
(1) The chemosensitivity of four kinds of ovarian cancer cells (SKOV3,CAOV3, OVCAR3 and 3AO) to cisplatin were detected by MTT assay. The relationship of PDCD4 expression and chemosensitivity was analyzed.
(2) PDCD4 was overexpressed in SKOV3 and CAOV3 cells, and whether PDCD4 overexpression can enhance chemosensitivity to cisplatin and other drugs in ovarian cancer cell lines was investigated by microscope and MTT assay.
(3) Two siRNAs targeted PDCD4 and non-silencing control siRNA were synthesized, then siRNAs were transfected into OVCAR-3 cells, after 48h, cells were subjected to RT-PCR and immunoblotting analysis. The effect of knockdown of PDCD4 with PDCD4-specific siRNAs on the chemosensitivity was detected by MTT assay.
(4) SKOV3 cells were inoculated subcutaneously into the left flank of nude mice. When tumors reached to about 100 mm3 one week later, these mice were randomly divided into six groups as follows:Normal saline; MOCK; PDCD4; cisplatin; MOCK+cisplatin; PDCD4+cisplatin. The tumor volume and weight was detected to investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo.
(5) The effect of PDCD4 expression on cisplatin-induced alterations in cell cycle was analyzed by PI staining. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays.
(6) To further examine the particular apoptotic pathways by which PDCD4 promotes cisplatin-induced apoptosis, we detected expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, c-FLIP by western blot. The expression of DR4 and DR5 was analyzed by flow cytometer. A specific caspase-8 inhibitor Z-ITED-FMK was used to inhibit the activity of csapase-8, then cisplatin-induced apoptosis in PDCD4-overexpressing ovarian cancer cells was determined.
3. The role and mechanism of PDCD4 in autophagy
(1) PDCD4 was overexpressed in ovarian cancer cell line SKOV3,48h later, autophagy was induced by starvation, and then marker protein of autophagy (LC3) was detected by western blot.
(2) The abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice were obtained, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/-mice, and expression of GFP-LC3 was observed by fluorescence microscope after autophagy induction.
(3) The activity of ROS was analyzed by flow cytometer, and expression of Beclin-1 and p38MAPK were detected by western blot after autophagy were induced in abdominal macrophages of C57 and PDCD4-/- mice.
Results
1. The expression status and role of PDCD4 in ovarian cancer
(1) Expression and significance of PDCD4 in human ovarian carcinomas
We first examined the expression of PDCD4 in ovarian cancer cell lines by semi-quantitative RT-PCR and western blot. The result showed that PDCD4 expression was low in SKOV3 and 3AO, moderate in CAOV3 and high in OVCAR3 cells.
To explore the expression level of PDCD4 in ovarian carcinoma, we detected PDCD4 expression in normal ovarian tissues, serous cystadenomas and serous cystadenocarcinomas by RT-PCR and immunohistochemistry (IHC). The results showed that 57.1%(8/14) of serous cystadenocarcinomas samples showed loss or reduction of PDCD4 mRNA expression. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD4 expression. However, 39.5%(17/43) of serous cystadenocarcinomas had no detectable PDCD4 protein expression. PDCD4 protein expression was found to be lost or significantly lower in serous cystadenocarcinomas compared with normal ovaries and serous cystadenomas (P<0.05). Lost or decreased PDCD4 expression in serous cystadenocarcinomas was associated significantly with higher pathological grade (P=0.0118).
(2) Overexpression of PDCD4 in ovarian cancer cells inhibited their proliferation and colony formation capacity.
The stably expressing PDCD4 cells (SKOV3-PDCD4) and control cells (SKOV3-MOCK) were established, and the effect of PDCD4 on ovarian cancer cells was further investigated. Our results showed that SKOV3-PDCD4 cells grew significantly slower than control cells (SKOV3 and SKOV3-MOCK) (P<0.001) detected by live cell counting. In addition, the colony number of SKOV3-PDCD4 cells was significantly decreased compared with that of SKOV3-MOCK cells. All these results show that PDCD4 overexpression significantly prevents the proliferation and colony formation capacity of ovarian cancer cells.
(3) PDCD4 overexpression in ovarian cancer cells decreased their tumorigenic capacity in nude mice.
To determine the effect of PDCD4 on proliferation of SKOV3 in vivo, we inoculated SKOV3, SKOV3-MOCK and SKOV3-PDCD4 cells into nude mice. One week after inoculation, all mice in SKOV3 and SKOV3-MOCK group developed palpable tumors and grew rapidly, however, none of the mice in SKOV3-PDCD4 group showed any obvious sign of tumor formation. Three weeks later, only two of eight mice developed small tumors. The overall mean tumor volume and weight in SKOV3-PDCD4 group were much smaller than those in SKOV3-MOCK or SKOV3 group. Moreover, there was obvious PDCD4 expression in tumors of SKOV3-PDCD4 group compared with those of SKOV3 or SKOV3-MOCK group detected by IHC. These results indicate that overexpression of PDCD4 decreases tumorigenic capacity and delays tumor growth in vivo.
2. PDCD4 Enhances Chemosensitivity of Ovarian Cancer Cells by Activating Death Receptor Pathway in Vitro and Vivo
(1) PDCD4 expression correlates with the cytotoxic activity of cisplatin in different ovarian cancer cell lines.
In the above part, we indicated that PDCD4 expression was significantly decreased in ovarian cancer, and PDCD4 expression could inhibit proliferation of ovarian cancer cells in vitro and in vivo. However, the effect of PDCD4 expression on the sensitivity of ovarian cancer cells to cisplatin needs to be further investigated. We detected the sensitivity of three kinds of ovarian cancer cell lines to cisplatin by MTT assay, the results showed that OVCAR3 and CAOV3 cells with relatively high or moderate levels of PDCD4 expression were more sensitive to cisplatin. However, SKOV3 and 3AO cells with relatively low levels of PDCD4 expression were resistant to cisplatin. These results suggest that PDCD4 expression may be associated with high sensitivity to cisplatin in ovarian cancer cell lines.
(2) PDCD4 overexpression enhances chemosensitivity of ovarian cancer cells.
To investigate the direct impact of PDCD4 on chemosensitivity, we overexpressed PDCD4 in SKOV3 and CAOV3 cells. The results showed that in the presence of cisplatin (25μg/ml), SKOV3-PDCD4 cells appeared marked cell death while there was no observable cell death in SKOV3-MOCK cells. This result was confirmed by MTT assay which showed that cell viability of SKOV3-PDCD4 cells was significantly decreased (p<0.05).Similarly, CAOV3 cells transfected with PDCD4 were more sensitive to cisplatin than control cells. Moreover, our results showed that the cell viability of SKOV3-PDCD4 cells was more decreased than that of SKOV3-MOCK cells at the same concentration of carboplatin (p<0.05). However, there was no significant difference between the SKOV3-PDCD4 cells and SKOV3-MOCK cells when the cells were treated with CTX, VP-16 or paclitaxcl. These results indicate that PDCD4 expression selectively enhances platinum-based chemotherapy in ovarian cancer cells.
(3) Knockdown of PDCD4 confers resistance to cisplatin in ovarian cancer cells.
To further confirm the effect of PDCD4 on the chemosensitivity of ovarian cancer cells, we knocked down PDCD4 expression by PDCD4-specific siRNAs in OVCAR3 cells. Two PDCD4-specific siRNAs markedly inhibit expression of PDCD4 mRNA by 77% and 76% and PDCD4 protein by 80% and 88%, whereas nonspecific siRNA had no significant effect on PDCD4 expression. Knockdown of PDCD4 with PDCD4-specific siRNAs significantly increased cell viability when compared with controls treated with nonspecific siRNA (p<0.05). This indicates that downregulation of PDCD4 enhances resistance to cisplatin.
(4) Combination of PDCD4 and cisplatin significantly inhibits xenograft growth in vivo.
To investigate the effect of PDCD4 expression on chemosensitivity of ovarian cancer in vivo, we established a xenograft animal model and mice were randomly divided into six groups and treated as follows:Normal saline (NS); MOCK; PDCD4; cisplatin; MOCK+cisplatin; and PDCD4+cisplatin. After treatment, mice were sacrificed, and tumor size and weight were measured. Tumor volume and weight in PDCD4+cisplatin group (0.0375±0.0069 mm3 and 0.0300±0.0044 g) were markedly lower when compared with that in cisplatin group (0.1386±0.0185 mm3 and 0.0860±0.0204 g) or PDCD4 (0.0779±0.0115 mm3 and 0.0533±0.0042 g) or MOCK+cisplatin (0.1621±0.0090 mm3 and 0.07667±0.0126 g). These data indicate that PDCD4 can also enhance the sensitivity of ovarian cancer to cisplatin in vivo.
(5) PDCD4 expression promotes cisplatin-induced apoptosis, but not alterations in cell cycle.
To determine the mechanism by which PDCD4 enhances sensitivity of ovarian cancer cells to cisplatin, we first analyzed the effect of PDCD4 expression on cisplatin-induced alterations in cell cycle. Cisplatin treatment markedly increased cell number of S phase, however, PDCD4 overexpression had no significant impact on platinum-induced alterations in cell cycle. To explore whether PDCD4 expression might alter platinum-induced apoptosis, we measured apoptosis by Hoechst staining and Tunnel assays. Staining with Hoechst showed that PDCD4 overexpression increased the number of apoptotic cells, characterized by chromatin condensation and nuclear fragmentation after treatment with cisplatin. Likewise, results with the Tunnel assay revealed that the combination of PDCD4 expression with cisplatin resulted in a significant increase in apoptosis (30.1%), whereas control cells treated with cisplatin resulted in minimal apoptosis(1.5%). Therefore, we can conclude that PDCD4 expression promotes cisplatin-induced apoptosis.
(6) PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathways.
To further explore the apoptotic pathway by which PDCD4 promotes cisplatin- induced apoptosis, we next detected the expression of several apoptosis-related proteins by western blot. Our results showed that the expression of cleaved caspase-3,-8 in SKOV3-PDCD4 cells treated with cisplatin obviously elevated compared with those in SKOV3 and SKOV3-MOCK cells. However, a slight increase of cleaved caspase-9 and Bax was observed in SKOV3-PDCD4 cells compared with the control cells while there was nearly no difference about expression of Bcl-2. When Z-ITED-FMK was used to inhibit the activity of caspase-8, cisplatin-induced apoptosis was attenuated in SKOV3-PDCD4 cells. Furthermore, we detected the expression of these apoptosis-related proteins in the tumor tissues from tumor-bearing nude mice, similar results were found. In addition, we detected altered expression of caspase-8 inhibitory protein c-FLIP-L and death receptors DR5 in PDCD4-overexpressing cells. PDCD4 decreased the expression of c-FLIP-L and increased the expression of DR5. Taken together, these results suggest that PDCD4 promotes cisplatin-induced apoptosis mainly by the activation of death receptor pathway.
3. The role and mechanism of PDCD4 in autophagy
(1) Overexpression of PDCD4 inhibit autophagy of ovarian cancer cells
To investigate the effect of PDCD4 on autophagy, we overexpressed PDCD4 in SKOV3 cells,48h later, autophagy was induced by starvation, and LC3 (autophagy marker) expression was detected by western blot. Our results showed that overexpression of PDCD4 could inhibit the expression of LC3-Ⅱ, which indicated that PDCD4 could inhibit autophagy of SKOV3 cells. We further determined the LC3 expression of tumor tissue from tumor-bearing nude mice treated with MOCK and PDCD4 vector, similar results were found.
(2) Autophagy was significantly increased in macrophage of PDCD4-/- mice
To further confirm the effect of PDCD4 on autophagy, we obtained the abdominal macrophages and bone marrow derived macrophages (BMDM) of C57 and PDCD4-/- mice, and autophagy were induced by starvation, LPS or rapamycin, and then LC3 expression was detected by western blot. Our results showed that LC3-Ⅱexpression of macrophagy from PDCD4-/- was significantly higher than that of C57 mice. In addition, GFP-LC3 was transfected to abdominal macrophages of C57 and PDCD4-/- mice, and the expression of GFP-LC3 was observed by fluorescence microscope after autophagy. The formation of GFP-LC3-labeled structures (GFP-LC3 "dots"), which represents autophagosomes, was extensively induced in PDCD4-/-macrophage exposed to LPS, which indicated that knockout of PDCD4 could enhance autophagy.
(3) PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.
PDCD4 could inhibit autophagy, and knockout of PDCD4 could enhance autophagy. However, the mechanism of PDCD4 regulating autophagy is unknown. We mainly study the mechanism of PDCD4 regulating LPS-induced autophagy. We found that there were no difference about the activity of ROS between C57 and PDCD4-/- mice induced by LPS. Next, we detected the expression of Beclin-1 and p38MAPK by western blot, the results showed that although LPS could induce the expression of Beclin-1, there was no difference of Beclin-1 expression between C57 and PDCD4-/- mice induced by LPS. Interestingly, we found that LPS induced the expression of pho-p38MAPK, and its expression was significantly increased in PDCD4-/- mice compared with that of C57 mice, which indicated that PDCD4 may inhibit LPS-induced autophagy through p38MAPK pathway.
Conclusions
1. PDCD4 expression was significantly decreased or lost in ovarian cancer, which correlated with the high pathological grade ovarian cancer.
2. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo.
3. PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo.
4. PDCD4 promotes cisplatin-induced apoptosis by mainly activating death receptor pathway.
5. PDCD4 may inhibit autophagy through p38MAPK pathway.
Originality
1. We demonstrate, for the first time, that loss of PDCD4 expression is a frequent event in human ovarian cancer and significantly correlated with the pathological grade of ovarian cancer. Overexpression of PDCD4 can inhibit malignant proliferation of ovarian cancer cells in vitro and in vivo, which provide first hand data for the gene therapy targeted PDCD4 for ovarian cancer.
2. We studied the effect and mechanism of PDCD4 on chemosensitivity of ovarian cancer for the first time. We found that PDCD4 expression can enhance the chemosensitivity of ovarian cancer cells to cisplatin in vitro and in vivo, the mechanism was that PDCD4 promoted cisplatin-induced apoptosis by mainly activating death receptor pathway.
3. We demonstrate, for the first time, PDCD4 may inhibit autophagy through p38MAPK pathway. It was not only provides the new research ideas for investigating the function of PDCD4, but also reveals the mechanism of many diseases, seeking new methods to prevent and treat the disease.
Limitations of this study
1. The mechanism of PDCD4 inducing DR5 expression and PDCD4 inhibiting c-FLIP-L need to be further investigated.
2. The mechanism of PDCD4 inhibiting autophagy needs to be further investigated.
Part 2 Clinical and prognostic significance of lost or decreased PDCD5 expression in human epithelial ovarian carcinomas
Objective
Programmed cell death 5 (PDCD5), also designated as TF-1 cell apoptosis-related gene-19 (TFAR19), is a novel gene cloned from TF-1 cells undergoing apoptosis, and it is up-regulated in the process of cell apoptosis and translocation from the cytoplasm to the nucleus. PDCD5 is a strong candidate of apoptosis-regulated protein and its overexpression promotes apoptosis triggered by certain stimuli, while blocking PDCD5 action suppresses apoptosis. Recent studies have demonstrated that PDCD5 is down-regulated in some human tumor tissues such as gastric cancer, hepatocellular carcinoma, breast cancer, acute and chronic myeloid leukemia and glioma. However, PDCD5 expression and its association with prognosis in gynecological cancer have not been evaluated.
In this study, we detected the status of PDCD5 expression in three kinds of human serous cystadenocarcinoma cell lines, normal ovarian tissues and serous ovarian tumors using RT-PCR, western blot and immuohistochemistry. Furthermore, we analyzed the association of PDCD5 expression with clinico-pathological features and survival of patients.
Methods
1. Expression of PDCD5 in human ovarian cancer cell lines and ovarian carcinomas.
(1) Case collection:Sixty-seven serous ovarian tumor samples (26 serous cystadenomas and 41 serous cystadenocarcinomas) were obtained from patients who underwent surgical operations at the Department of Gynecology, Qilu Hospital from 2001 to 2007. None of the patients studied had received adjuvant immunosuppressive treatments such as radiotherapy or chemotherapy prior to surgery. Twenty normal ovarian tissues were obtained from the normal ovaries of donors during surgery for other gynecological diseases in Qilu Hospital, Shandong University.
(2) Expression of PDCD5 mRNA and protein in ovarian cancer cell lines(SKOV3,CAOV3,OVCAR3) and above tissues were detected by RT-PCR, western blot and immunohistochemistry.
2. Clinical significance of PDCD5 expression in human ovarian carcinomas.
(1) The Chi-square and Fisher's exact test was used to compare the expression of PDCD5 with clinico-pathological parameters.
(2) Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.
Results
1. Expression of PDCD5 mRNA and protein in human ovarian cancer cell lines.
To explore the potential roles of PDCD5 in ovarian cancer, we firstly detected the status of PDCD5 expression in three ovarian cancer cell lines which derived from serous cystadenocarcinomas by RT-PCR and western blot. Our results showed that the OVCAR3 cell line expressed high levels of PDCD5 mRNA and protein, whereas the SKOV3 cell line showed low levels of PDCD5 expression. Although CAOV3 cells showed relative high PDCD5 mRNA expression, their protein expression was relative low. These results suggested that the expression of PDCD5 may be decreased in human serous cystadenocarcinomas.
2. Expression of PDCD5 mRNA and protein in human normal ovarian tissues and ovarian carcinomas.
To explore the expression of PDCD5 in ovarian carcinoma, we firstly detected PDCD5 expression in normal ovarian tissues and serous ovarian tumors by RT-PCR and western blot. We found that PDCD5 expression was obviously reduced or even lost in serous cystadenocarcinomas compared with normal ovarian tissues and serous cystadenomas. The results from IHC showed that all normal ovaries and serous cystadenomas were positive for PDCD5 expression. Among them,80%(16/20) of normal ovarian tissues and 76.9%(20/26) of serous cystadenomas showed moderate or strong PDCD5 protein expression. In contrast,46.3%(19/41) of serous cystadenocarcinomas exhibited weak PDCD5 expression and 22%(9/41) had no detectable PDCD5 protein expression. The overall expression of PDCD5 in serous cystadenocarcinoma was significantly lower compared with normal ovarian tissues or serous cystadenomas (p<0.01).
3. Correlation of the expression level of PDCD5 with the FIGO stage of serous cystadenocarcinoma.
To determine the clinical significance of lost or reduced PDCD5 expression in serous cystadenocarcinoma, we examined the correlation of PDCD5 expression with the clinico-pathological parameters of serous cystadenocarcinoma by Chi-square and Fisher's exact test. The results showed that there was no significant correlation among PDCD5 expression and age, site of origin, metastasis and pathological grade. However, the expression of PDCD5 correlated significantly with clinical FIGO stage. The percentage of cases with PDCD5 high expression is lower in FIGO stageⅢand IV patients (19.2%,5 of 26) than that in FIGO stageⅠandⅡpatients (53.3%,8 of 15) (p<0.05).
4. Loss or reduction of PDCD5 expression was significantly associated with survival of patients with serous cystadenocarcinoma.
To assess the association of PDCD5 expression with patient survival, the survival data from 30 patients with serous cystadenocarcinomas were generated by follow-up. According to the final PDCD5 staining score in the results of IHC, these patients were divided into a high expression group (score 3-6) and a low expression group (score 0-2). The difference in survival time between patients with high PDCD5 expression tumors (n=16) and those with low PDCD5 expression tumors (n=14) was evaluated by Kaplan-Meier method and log-rank test. The result demonstrated that patients with a low level of PDCD5 expression had a significantly poorer disease-specific survival than those with a high level of PDCD5 expression (p<0.05). This indicates that the level of PDCD5 expression significantly correlated with prognosis of patients with serous cystadenocarcinoma.
Conclusions:
1. PDCD5 expression is clearly reduced or lost in serous cystadenocarcinoma compared with normal ovaries and serous cystadenomas.
2. lost or decreased PDCD5 expression in serous cystadenocarcinomas was associated significantly with FIGO stage and poorer disease-specific survival of patients.
Originality
1. We found that PDCD5 expression was lost or decreased in serous cystadenocarcinomas, and was associated significantly with FIGO stage patients, which suggest that PDCD5 expression may contribute to the pathogenesis of human serous cystadenocarcinomas.
2. We demonstrate that PDCD5 expression in serous cystadenocarcinomas was associated significantly with disease-specific survival of patients, which provide new method for diagnosis and prognosis for evaluation of serous cystadenocarcinomas.
Limitations of this study
1. The effect of PDCD5 on malignant proliferation of ovarian cancer cells needs to be further investigated.
2. The effector mechanism of PDCD5 in tumor remains unknown.
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