IGFBP-2及IGFBP-3对卵巢上皮性癌患者体内IGF-Ⅱ的生物学功能调节及体外化疗耐药相关研究
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摘要
目的:卵巢上皮性癌(epithelial ovarian cancer, EOC,以下简称为卵巢癌)是导致妇女癌性死亡的主要原因。其特点为缺乏早期症状、就诊晚及生存率低。经肿瘤细胞减灭术及以铂类为主的化疗可产生初始疗效,但70%的晚期患者可因化疗耐药而导致治疗失败。寻求新的诊断和预后的生物学指标,并探讨化疗耐药相关机制将有助于指导卵巢癌临床治疗。胰岛素样生长因子(Insulin-like growth factor ,包括IGF-I和IGF-II )可促进细胞增殖并抑制凋亡,在恶性肿瘤的发生和发展中起着重要的作用。研究发现IGF-II蛋白酶解转化过程与IGF-II的生物学功能密切相关,IGF-II前体多肽(proIGF-II,156个氨基酸)可经修饰转化为两个大的IGF-II肽(big IGF-II,分别含有87、104个氨基酸)和成熟IGF-II(mIGF-II,67个氨基酸)。在某些病理状况下,IGF-II各肽比例发生改变,提示不同IGF-II肽可能具有不同的生物学活性。IGFs作用受其6个结合蛋白(Insulin-like growth factor-binding protein , IGFBP1～6)调节。IGF-I以及不同IGF-II肽与IGFBPs的结合活性以及相关生物功能各不相同。在所有IGF结合蛋白中,IGFBP-3在血浆中的浓度最高,其次为IGFBP-2。IGFBP-3是一种潜在的抗细胞增生蛋白,可以抑制肿瘤细胞增生,并诱导癌细胞凋亡。血浆IGFBP-3水平的降低与多种恶性肿瘤有关。IGFBP-3抗细胞增生的作用主要是可以和IGF-I以及IGF-II形成复合物,进而阻止它们激活IGF-I受体以及由此诱导的细胞增生,另外,IGFBP-3也可以通过不依赖于IGF的机制而起作用。IGFBP-2在许多恶性肿瘤中的表达都明显升高,且与肿瘤的生长和侵袭性有关。IGFBP-2水平升高亦可增加卵巢癌细胞的生长和侵袭性。体外研究表明卵巢上皮性癌细胞株可表达所有与IGF家族有关的因子。尽管IGF家族各因子在卵巢癌的发生、发展以及预后中分别起到一定的作用,但血循环中主要IGFBPs对IGF-II生物学功能的调节作用以及对卵巢癌细胞生物学行为的影响和化疗耐药性的相关性尚不清楚。本研究应用新近改良的Western Ligand Blot (WLB)以及Wertern Blot (WB)方法检测卵巢良性、交界性和恶性肿瘤患者微量血清(0.5μl)中IGF-II各肽和其主要结合蛋白IGFBP-2, IGFBP-3的含量以及相关性,探讨血循环中IGFBP-2, IGFBP-3改变对IGF-II生物学功能的调节作用,以及血循环与癌组织中IGFBP-2和IGFBP-3表达的相关性和相应的临床病理学特征,同时,应用敏感和耐药的卵巢上皮性癌细胞株研究IGFBP-3在卵巢癌化疗耐药中的作用以及相应机制,从而为探讨IGF-II以及IGFBP-2,IGFBP-3作为卵巢上皮性癌诊断、病情监测和疗效评价的生物学标记物提供依据。
本研究分为四部分
第一部分
卵巢肿瘤患者IGF-II以及IGFBP-2和IGFBP -3的检测及临床意义
目的:检测IGF-II (pro-, big-,mIGF-II), IGFBP-2及IGFBP-3在卵巢良性、恶性肿瘤患者血清中的含量,探讨其与卵巢肿瘤的鉴别诊断和临床病理学特征的相关性,研究是否联合检测IGF-II及其相应结合蛋白IGFBP-2,IGFBP-3可作为卵巢癌诊断和治疗监测的指标之一。
方法:抽提滴加于滤纸上的微量血清,并采用Western Ligand Blot (WLB)和Wertern Blot (WB)方法分别检测23例卵巢上皮性癌(浆液性囊腺癌15例,粘液性囊腺癌8例),7例交界性肿瘤,20例卵巢良性上皮性肿瘤(其中浆液性囊腺瘤11例,粘液性囊腺瘤9例),22例妇科其他良性疾病患者以及21例妇科其他恶性肿瘤患者微量血清(0.5μl)中IGF-II各不同肽, IGFBP-2及IGFBP-3含量,同时以20例相应年龄健康妇女血清做正常对照。另外,分别对其中8例卵巢癌患者进行术前、术后以及化疗1个疗程后血清进行IGF-II,IGFBP-2及IGFBP-3含量检测。
结果:
1.卵巢肿瘤患者血清IGF-II(pro-, big-, mIGF-II)测定
卵巢癌患者血清proIGF-II, big IGF-II和mIGF-II含量均明显下降,与正常对照组,良性肿瘤及交界性肿瘤患者血清相比均有显著性差异(P<0.01或P<0.001)
2.卵巢肿瘤患者血清IGFBP-2和IGFBP-3测定
卵巢癌患者血清IGFBP-2水平明显升高,与正常对照组、良性以及交界性卵巢肿瘤患者血清相比有显著性差异(P<0.001);而血清IGFBP-3含量明显降低甚至消失,与正常对照组、良性肿瘤以及交界性肿瘤组相比均有显著性差异(P<0.001);正常对照组与交界性肿瘤组亦有显著性差异(P<0.05)。
3. IGF-II以及IGFBP-2/IGFBP-3与卵巢肿瘤分期、病理分类的相关性
IGF-II各肽以及IGFBP-2,IGFBP-3含量在良性浆液性和粘液性肿瘤组中无统计学差异(P>0.05);在恶性浆液性和粘液性组中亦无统计学差异(P>0.05);在早期和晚期患者间也无统计学差异(P>0.05)。
4.卵巢癌患者血清IGF-II以及IGFBP-2,IGFBP-3水平与CA125相关性
卵巢癌患者术前血清CA125值与血清IGFBP-2值存在正相关性(P<0.0001);与IGFBP-3值存在负相关性(P<0.0001);卵巢癌患者术前血清CA125值分别与IGF-II各不同肽存在负相关性: proIGF-II(P<0.0001); big IGF-II (P<0.0001); mIGF-II (P<0.0001)。
5.卵巢癌患者术前、术后及化疗后血清IGF-II及IGFBP-2,IGFBP-3含量的变化
卵巢癌患者血清IGF-II(pro-, big-和mIGF-II)多数于术后1周左右恢复至正常水平;术后1周IGFBP-2水平较术前稍升高或不变,IGFBP-3继续降低或无明显改变,但经过化疗1疗程后,IGFBP-2及IGFBP-3含量均回复至正常水平。
6.卵巢癌患者及妇科其他良性疾病患者血清IGF-II以及IGFBP-2,IGFBP-3水平的相关性
卵巢癌患者血清IGF-II(pro-, big-和mIGF-II)水平均明显低于其他妇科良性疾病患者,经统计学处理有显著性差异(P<0.001),而妇科良性疾病组与正常对照组相比均无统计学差异(P>0.05);卵巢癌组血清IGFBP-2水平明显高于妇科良性疾病组(P<0.001),妇科良性疾病组与正常对照组相比无统计学差异(P>0.05);卵巢癌组血清IGFBP-3水平明显低于妇科良性疾病组(P<0.001),而妇科良性疾病组与正常对照组间无显著性差异(P>0.05)。
7.卵巢癌及妇科其他恶性肿瘤患者血清IGF-II以及IGFBP-2,IGFBP-3水平的相关性
妇科其他恶性肿瘤组,约80%的患者血清IGF-II(pro-, big-和mIGF-II)水平高于卵巢癌组, IGFBP-2,IGFBP-3水平变化与卵巢癌相似,但IGFBP-2明显升高且同时伴有IGFBP-3明显降低的发生率低于卵巢癌组。
结论:
1.卵巢癌患者血清中IGFBP-2水平明显升高,可能与瘤负荷有关,而IGFBP-3水平的明显降低则可能有助于肿瘤生长;
2.卵巢癌患者血清中IGF-I(I包括pro-, big-和mIGF-II)水平均显著降低,可能标志着IGF-II生物学活性的改变;
3.卵巢癌患者术后及化疗后血清pro-, big-和mIGF-II以及IGFBP-2,IGFBP-3相继恢复至正常水平,提示联合检测血清IGF-II各肽以及IGFBP-2和IGFBP-3含量有助于判断卵巢癌患者的治疗效果。
4. IGF-II各肽及IGFBP-2,IGFBP-3水平与CA125均有明显的相关性,联合检测上述指标有助于筛查导致CA125升高的一些良性妇科疾病及部分妇科恶性肿瘤,提高卵巢上皮性癌诊断的准确性和特异性。
5.本研究应用改良的WLB和WB方法检测从滤纸抽提的卵巢肿瘤患者微量血清中不同IGF-II肽以及IGFBP-2,IGFBP-3蛋白含量,与传统的蛋白检测方法相比具有高度灵敏度,为卵巢癌以及其他疾病大样本筛查以及地区性研究提供了可行性的血清学检测途径。
第二部分IGFBP-2及IGFBP-3对卵巢上皮性癌患者IGF-II生物学功能的调节
目的:检测卵巢癌患者血清IGFBP-2及IGFBP-3与IGF-II各肽的相关性,研究IGFBP-2和IGFBP -3对IGF-II生物学功能的调节作用。
方法:采用免疫沉淀(Immunoprecipitation, IP)方法确定血循环中IGF-II与IGFBP-2及IGFBP-3的结合状况。血清标本分别应用IGFBP-2,IGFBP-3特异性抗体吸附沉淀(IP)后,再分别经WB测定血清中IGF-II各肽的含量,并通过WLB确定血清中特异吸附沉淀的IGFBP-2及IGFBP-3含量,另外,通过应用IGF-II/IGFBP交叉连接复合物测定(Cross-link)方法分别检测卵巢癌患者血清和正常对照者血清中呈结合状态的IGF-IGFBPs。
结果:1. IGFBP-2与big IGF-II的结合能力明显低于IGFBP-3。无论在正常对照组和卵巢癌组,血清中big IGF-II均不与IGFBP-2结合。经过IGFBP-2 IP后,可检测到卵巢癌患者血清中升高的IGFBP-2以及与正常血清相比升高的mIGF-II水平;经过IGFBP-3 IP后,与正常对照组相比,卵巢癌患者血清IGFBP-3明显减少或消失,但可见升高的IGFBP-3降解片段,而big/m IGF-II均减少。
2.当bigIGF-II含量明显高于mIGF-II时,可与IGFBP-2结合。与正常血清相比,在卵巢癌患者血清中,bigIGF-II和mIGF-II水平均低于正常对照者,在低剂量外源性bigIGF-II存在的情况下,随着外源性bigIGF-II含量的增加,可检测到其水平增加,而给予相同浓度外源性bigIGF-II的正常血清虽可检测到较高水平bigIGF-II,但并不随剂量增加而明显改变。与其发生相应改变的是在正常血清中随着外源性bigIGF-II的增加,150kDa复合物含量明显增加;而卵巢癌患者可检测到明显增加的IGFBP-2与bigIGF-II连接物,以及因与IGFBP-2结合消耗而较正常血清体系明显降低的bigIGF-II含量。
结论:
1.卵巢癌患者血清IGFBP-2,IGFBP-3的改变与IGF-II各肽含量的下降明显相关,IGFBP-3明显降低而IGFBP-2明显升高的状况可改变其与big/m IGF-II的结合状况,进而导致游离IGF-II增加而到达相应靶组织发挥作用并降解。
2.本研究结果提示血循环中IGFBP-2和IGFBP-3的改变可导致IGF-II生物学活性增强,揭示IGFBP-2和IGFBP对卵巢癌患者IGF-II生物学功能的调节机制。
第三部分IGFBP-2及IGFBP-3在卵巢肿瘤患者组织中的表达及临床意义
目的:检测卵巢良性、恶性肿瘤患者组织中IGFBP-2以及IGFBP -3的表达,探讨其与卵巢癌临床病理学特征的相关性,并进一步探讨IGFBP-2及IGFBP-3在卵巢癌发生和发展中可能的作用以及作为卵巢上皮性癌生物学标记的可能性。
方法:采用免疫组化染色方法检测卵巢肿瘤组织(卵巢良性肿瘤8例,卵巢交界性肿瘤8例,卵巢癌上皮性癌23例,包括高、中度分化者15例,低度分化者8例)中IGFBP-2和IGFBP-3的表达。同时以4例正常卵巢组织(来自于因其他妇科疾病须行全部或部分卵巢切除的患者,并经病理证实无卵巢组织病变)作为对照。
结果:
1. IGFBP-2的表达位于细胞质或细胞核,卵巢恶性肿瘤IGFBP-2的表达率明显升高,与正常卵巢组织以及卵巢良性肿瘤相比均有显著性差异(P<0.0001);而正常对照组与卵巢良性肿瘤和交界性肿瘤、卵巢良性肿瘤与交界性肿瘤以及交界性与恶性肿瘤相比均无统计学差异(P>0.05)。
2. IGFBP-3表达位于细胞质,虽然卵巢恶性肿瘤IGFBP-3的表达率与正常对照组、卵巢良性肿瘤相比呈降低趋势,但经统计学处理无显著性差异(P>0.05)。
3.随着卵巢癌分化程度的降低,IGFBP-2的表达呈增强趋势,但低分化组与高中分化组相比无统计学意义(P>0.05)。
4.在卵巢上皮性癌组织中,随着卵巢癌细胞分化程度的降低,IGFBP-3的表达明显降低或呈阴性表达,低分化卵巢癌细胞与高、中分化卵巢癌细胞相比有显著性差异(P<0.05)。
结论:本研究中发现卵巢肿瘤组织细胞中IGFBP-2和IGFBP-3的表达不仅与肿瘤的良、恶性有关,而且与卵巢癌细胞的分化程度有一定相关性,说明在组织细胞中IGFBP-2及IGFBP-3的表达与卵巢癌患者的临床病理学特征有关。
第四部分IGFBP-3在卵巢癌化疗耐药中的调节作用
目的:应用铂类敏感和耐药的卵巢上皮性癌细胞株检测IGFBP-3在卵巢化疗耐药中的作用和调节机制。
方法:分别建立IGFBP-3完整段以及1-97N末端质粒cDNA,应用两对铂类敏感和耐药的卵巢上皮性癌细胞株( OV2008/ C13* ,A2780s/A2780cp)经过细胞培养、IGFBP-3转染伴有或不伴有DN-Akt病毒感染,再经过CDDP (0, 2.5, 5, 10μM; 24 h)处理后,检测卵巢癌细胞的凋亡以及相应Caspase-3改变情况。
结果:
1. CDDP可诱导敏感卵巢癌细胞凋亡,但对耐药卵巢癌细胞的凋亡无影响(p<0.01,P>0.05)。CDDP诱导的卵巢癌细胞的凋亡伴随着IGFBP-3的改变。典型的凋亡细胞表现为细胞变园,萎缩,核浓缩并出现分离形成凋亡小体。
2.过度表达IGFBP-3可以增强敏感性卵巢癌细胞对顺铂(10μM)诱导的凋亡,与对照组相比有显著性差异(p<0.05);过度表达IGFBP-3虽然可诱导部分耐药卵巢癌细胞对顺铂介导的凋亡,但与对照组相比无显著性差异(P>0.05)。
3.过度表达IGFBP-3,并通过DN-Akt病毒感染耐药卵巢癌细胞阻止Akt功能后,可启动并增强CDDP诱导的细胞凋亡。
4.过度表达IGFBP-3以及DN-Akt病毒感染介导的卵巢癌细胞的凋亡与激活Caspase-3相关。
结论:
1. CDDP诱导的卵巢癌细胞的凋亡与IGFBP-3有一定相关性。
2.过度表达IGFBP-3可增加CDDP诱导的细胞凋亡,且与DN-Akt病毒感染有协同作用,提示IGFBP-3对卵巢癌细胞的凋亡诱导作用可能与下调Akt介导的细胞信号传导通路有关。
Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. EOC is characterized by few early symptoms, presentation at an advanced stage, and poor survival. Despite the initial response to combined treatment of cytoreduction (debulking) and chemotherapy, about 70% of patients with advanced ovarian cancer ultimately develop drug-resistant disease (relapse). Nevertheless, seeking for the effective biomarkers for the early detection and prognostic evaluation of EOC, and investigating the mechanism of chemoresistance will benefit the treatment. The insulin-like growth factors (IGFs) play a key role in the tumorigenesis and progression of human malignancies mainly through their antiapoptotic activities or by enhancing tumor cell proliferation. IGF-II processing has been recognized as an important mechanism involved in the regulation of IGF-II’s biological activity. ProIGF-II peptide originally synthesized with 156 amino acids undergoes regulated endoproteolytic cleavage by proprotein convertases to generate its two big variants, IGF-II (1-87) and IGF-II (1-104), and mature form. In certain pathological conditions, a much greater proportion of big IGF-II has been detected whereas total IGF-II concentrations are unchanged, indicating that the different isoforms of IGF-II may have different biological properties. The activities of IGFs are regulated by six IGF-binding proteins (IGFBPs), namely IGFBP-1 to IGFBP-6. The binding activities of IGF-I and different isoforms of IGF-II to IGFBPs are different, resulting in the different bioavailabilities. Of all IGFBPs, IGFBP-3 is the predominant form found in the human circulation, followed by IGFBP-2. IGFBP-3 is a potent anti-proliferative protein that can induce apoptosis and inhibit cancer cells proliferation. Decreased expression and plasma levels of IGFBP-3 are associated with a number of malignancies. The anti-proliferative actions of IGFBP-3 resulted from its ability to form complexes with IGF-I and IGF-II could prevent the growth factors from activating IGF-I receptors to induce cell proliferation and survival. In addition, IGFBP-3 can also act through IGF-independent mechanisms. IGFBP-2 is overexpressed in many malignancies,and associated with increased aggressiveness and invasiveness of the tumor. Increased expression and level of IGFBP-2 can also promote ovarian cancer cell growth and invasiveness. In vitro models have demonstrated that EOC cell lines express all major components of the IGF family, and several studies have implicated the IGF system in the etiology of human ovarian epithelial cancer. Although the individual member of IGF family plays roles in the development, progression and prognosis of ovarian cancer, the predominant form of IGFBPs in the circulation of EOC and its proper roles in the regulation of IGF-II bioaviability and chemosensitivity have not been demonstrated in EOC. In current study, the serum levels of GFBP-2, IGFBP-3 and different isoforms of IGF-II were measured by modified Western ligand blot (WLB) and Western blot (WB) in patients with benign, borderline ovarian tumor and malignant tumor (epithelial ovarian cancer) respectively, and their proper roles in the regulation of IGF-II bioavailability were investigated. Meanwhile, the correlation between the serum levels of IGFBP-2, IGFBP-3 and their expressions in ovarian tumor tissues detected by immunohistochemistry as well as related clinicopathological characteristics were evaluated. Furthermore, the proper role and mechanism of IGFBP-3 in regulating chemoresistance was studied by overexpression of IGFBP-3 in both chemosensitive and resistant ovarian cancer cells in vitro. Our study will further demonstrate the possible roles of IGF-II, IGFBP-2 and IGFBP-3 in regulating the bioactivities of ovarian cancer, and provide evidences for their potential roles as the biomarkers in diagnosing, monitoring the progresses and evaluating the efficacy of chemotherapy in patients of ovarian cancer.
Part 1. The clinical significance of detecting the serum levels of IGF-II and IGFBP-2, IGFBP-3 in patients with ovarian tumor
Objective: To detect the serum levels of IGF-II (pro-, big and mIGF-II) and IGFBP-2, IGFBP-3 in patients with both benign and malignant ovarian tumors, and explore their proper role in differentiated diagnosis, their correlations with clinicopathological characteristics and, further evaluate whether IGF-II, IGFBP-2 and IGFBP-3 can be the biomarkers in diagnosis and surveillance of ovarian cancer.
Methods: The serum (0.5μl/each case) levels of IGF-II, IGFBP-2 and IGFBP-3 were detected by Western Ligand Blot (WLB) and Western Blot (WB) in 23 cases of epithelial ovarian cancer (15 serous and 8 mucinous ), 7 cases of borderline ovarian tumor, 20 cases benign tumor (11 serous and 9 mucinous), 22 patients with benign gynecological diseases and 21 cases of other gynecological cancer , and 20 cases of healthy women as normal control.
Results: 1. The serum levels of IGF-II in patients with ovarian tumors The serum levels of all forms of IGF-II were significantly decreased in EOC group compared with those of normal control group, benign and borderline ovarian tumor groups (P<0.01 or P<0.001).
2. The serum levels of IGFBP-2 and IGFBP-3 in patients with ovarian tumors The serum level of IGFBP-2 increased, while the level of IGFBP-3 decreased in patients with EOC, and there were significant difference compare with those of normal control group, benign and borderline ovarian tumor groups respectively. (P<0.001); The difference between normal control and borderline was also significant (P<0.05)
3. The correlation between IGF-II, IGFBP-2, -3 and clinical stages as well as pathological types of epithelial ovarian tumor The serum levels of IGF-II, IGFBP-2 and IGFBP-3 were not significantly different between benign serous epithelial ovarian tumor group and its mucnious counterpart (P>0.05), and there were also no significant difference between serous ovarian cancer and its mucnious counterpart (P>0.05).
4. The correlation of the serum levels of IGF-II, IGFBP-2, IGFBP-3 with CA125 in patients with epithelial ovarian cancer The serum levels of CA125 was positively correlated to IGFBP-2 (P<0.0001) and negatively correlated to IGFBP-3 (P<0.0001), respectively, and also was positive correlated to proIGF-II (P<0.0001); big IGF-II (P<0.0001) and mIGF-II (P<0.0001), respectively.
5. The changes of serum IGF-II, IGFBP-2 and IGFBP-3 pre-, postoperation and after chemotherapy in patients with epithelial ovarian cancer The serum levels of IGF-II (pro-, big and mIGF-II) were returning toward normal around one week postoperatively, while the serum levels of IGFBP-2 and IGFBP-3 either increased or decreased a little or without significant changes, but they were back to normal when measured after one cycle of chemotherapy.
6. Comparision of serum levels of IGF-II, IGFBP-2 and IGFBP-3 between EOC group and benign gynecological diseases (BGD) group The serum levels of IGF-II (pro-, big and mIGF-II) in patients with EOC were significantly lower than those of patients with BGD(P<0.001), while there were no difference between normal control group and BGD group (P>0.05). The serum level of IGFBP-2 in EOC group was significantly higher than those of BGD group(P<0.001), but there was no significant difference between normal control group and BGD group(P>0.05); The serum level of IGFBP-3 in EOC group was significantly lower than that of BGD group (P<0.001), while there was no significant difference between normal control and BGD group (P>0.05).
7. Comparesion of serum levels of IGF-II, IGFBP-2 and IGFBP-3 between EOC group and other gynecological cancer group The serum levels of IGF-II(pro-, big and mIGF-II) in 80% of patients with other gynecological cancer were higher than those with EOC; and some of them with either increased IGFBP-2 or decreased IGFBP-3 level, while increased IGFBP-2 together with decreaed IGFBP-3 was less happened in other gynecological cancers compared with that in ovarian cancer group.
Conclusion:
1. The serum level of IGFBP-2 increased markedly in patients with epithelial ovarian cancer, probably due to the correlation with the tumor burden, while decreased level of IGFBP-3 might enhance the availability of tumor growth.
2. The changes of different isoforms of serum IGF-II might affect the availability of IGF-II in patients with epithelial ovarian cancer.
3. There were obvious correlations between CA125 and serum IGF-II, IGFBP-2, IGFBP-3 in patients with EOC, indicating that detecting serum IGF-II, IGFBP-2 and IGFBP-3 could help screening some benign gynecological dieases with increased CA125 level and other gynecological cancers, and enhance the diagnositic accuracy of ovarian cancer.
4. The serum levels of IGF-II, IGFBP-2 and IGFBP-3 were returning toward normal following the surgery or chemotherapy, suggesting that detecting IGF-II, IGFBP-2 and IGFBP-3 together could be useful for predicting therapeutic efficacy and prognosis of epithelial ovarian cancer.
5. The modified WLB and WB have much higer sensitivity compared with their traditional ways in detecting IGFBP-2, IGFBP-3 and different isoforms of IGF-II by extracting serum protein from filter paper in patients with EOC, it also makes the large scale of serum research available including different regions with multiple samples.
Part 2. The role and regulation of IGFBP-2 and IGFBP-3 on the IGF-II bioaviability in patients with ovarian cancer
Objective: To detect the correlation between serum levels of IGFBP-2, IGFBP-3 and IGF-II in patients with EOC, and to investigate the possible mechanism of IGF-II bioaviability regulated by IGFBP-2 and IGFBP-3 in the development and progression of ovarian cancer.
Methods: 1. Immunoprecipitation (IP) to determine IGF-II binding to IGFBP-2 and IGFBP-3 in the circulation. Diluted serum samples were incubated with rabbit-anti-IGFBP-2 or anti-IGFBP-3,absorbed by A/G beads then dissociated by boiling. The serum level of IGF-II was determined by WB and specificity of immunoprecipitation of IGFBP-2 and IGFBP-3 was confirmed by WLB. 2. Determination of IGF-II/IGFBPs complex formation and biological availability of IGF-II isoforms. Two sets of aliquots of diluted samples (containing 1 ? ml serum) with different doses of big IGF-II were incubated for 1 hour at room temperature. One aliquot to be mixed with 2 l of 40 mM Disuccinimidyl suberate (DSS) cross-link overnight at 4oC. The comlex contents were determined by Western blotting in non-reducing condition.
Results: 1. The serum levels of IGFBP-2 and IGFBP-3 were positively correlated with all the forms of IGF-II respectively. 2. Formation of big IGF-II/IGFBP-2 complex does not occur in either normal control group or epithelial ovarian cancer sera. Increased IGFBP-2 and mIGF-II content were detected after IGFBP-2 IP. Decreased IGFBP-3 accpanied by increased IGFBP-3 fragment were detected, and both big and mIGF-II have decreased. 3. A higher proportion of big IGF-II relative to mature IGF-II is required for the formation of big IGF-II/IGFBP-2 complex.
Conclusion: Increased IGFBP-2 and decreased IGFBP-3 serum levels are associated with the decreased isoforms of IGF-II in ovarian cancer patients, which could alter the formation of big/mature IGF-II with their dominant IGFBPs in the circulation, leading to more free IGF-II available to the target tissues or cells. Our current results suggest that the bioaviability of IGF-II might be increased induced by the alteration of IGFBP-2 and IGFBP-3 in patients with ovarian cancer.
Part 3. The expressions and clinical significance of IGFBP-2 and IGFBP-3 in epithelial ovarian cancer tissues
Objective: To investigate the expressions and the clinical significance of IGFBP-2 and IGFBP-3 in patients with epithelial ovarian cancer, and to explore the roles of IGFBP-2 and IGFBP-3 in the progression of tumor as well as the possibility to serve as the potential biomarkers in epithelial cancer.
Methods: The expressions of both IGFBP-2 and IGFBP-3 were examined by immunochemistry in ovarian tumor tissues (8 cases of benign tumor, 8 cases of borderline tumor and 23 cases of epithelial ovarian cancer) and 4 cases of normal ovarian tissues.
Results: 1. The expression of IGFBP-2 was located in cytoplasm or nucleus. The expression rate significantly increased in ovarian cancer group compared with those of normal control,benign tumor and borderline tumor groups, while there were no significant difference between normal control vs benign, benign vs borderline, borderline vs malignant groups.
2. The expression of IGFBP-3 was located in cytoplasm. Although there was a decreased trend of the expression of IGFBP-3 with the increased malignancy, but there were no significant difference among different groups.
3. The expression of IGFBP-2 was increased with the decreased differentiation of tumors, but was not significant.
4. The expression of IGFBP-3 was decreased with the decreased differentiation of tumor tissues(P<0.05).
Conclusion: The expressions of IGFBP-2 and IGFBP-3 might be associated with the malignancy of the tumor as well as the degree of differentiation of cancer tissues, indicating that the expressions of IGFBP-2 and IGFBP-3 can be correlated with the clinicopathological characteristics.
Part 4. The role and regulation of IGFBP-3 in chemoresistance in ovarian cancer
Objective: To detect the proper role of IGFBP-3 in the regulating chemoresitivity of ovarian cancer by using both chemosensitive and chemoresistant ovaian cancer cells in vitro.
Methods: Both intact and 1-97 N-terminal IGFBP-3 plasmid cDNA were constructed, and two pairs of chemosensitive and resistant ovarian cancer cell line(sOV2008/ C13*,A2780s/A2780cp)were used in the current experiments. OV2008/ C13* were transfected with IGFBP-3 cDNA with or without DN-Akt virus infection (20 or 40 MOI) for 24 hours, followed by CDDP (0, 2.5, 5, 10μM; 24 h) treatment, the apoptotic cells and caspase-3 cleavage were examined by Hoechst staing and Western Blot respectively.
Results: 1. Apoptosis induced by CDDP increased in chemosensitive ovarian cancer cells in a dose dependent manner, but not in their resistant counterparts. IGFBP-3 protein content also increased in chemosensitive OV2008 cells when cells were treated with CDDP, but no significant changes happened in chemoresistant cells. Typical morphology changes of apoptotic cells are condensed nucleus and nuclear fragmentation etc.
2. Overexpression of IGFBP-3 could intensify chemosensitive ovarian cancer cells to CDDP-induced apoptosis, but can not significantly facilitate chemoresistant cells to CDDP-induced apoptosis.
3. Overexpression of IGFBP-3 and blocking the function of Akt by DN-Akt virus infection had synergetic role in enhancing CDDP-indued apoptosis.
4. Overexpression of IGFBP-3 and DN-Akt mediated,CDDP induced apoptosis was associated with caspase-3 activation.
Conclusion: CDDP-induced apoptosis can be associated with IGFBP-3, and overexperssion of IGFBP-3 and blocking the function of Akt can have synergetic role in facilitating ovarian cancer cells to CDDP-induced apoptosis, suggesting that Akt pathway might be involved in IGFBP-3-mediated apoptosis.
本研究分为四部分
第一部分
卵巢肿瘤患者IGF-II以及IGFBP-2和IGFBP -3的检测及临床意义
目的:检测IGF-II (pro-, big-,mIGF-II), IGFBP-2及IGFBP-3在卵巢良性、恶性肿瘤患者血清中的含量,探讨其与卵巢肿瘤的鉴别诊断和临床病理学特征的相关性,研究是否联合检测IGF-II及其相应结合蛋白IGFBP-2,IGFBP-3可作为卵巢癌诊断和治疗监测的指标之一。
方法:抽提滴加于滤纸上的微量血清,并采用Western Ligand Blot (WLB)和Wertern Blot (WB)方法分别检测23例卵巢上皮性癌(浆液性囊腺癌15例,粘液性囊腺癌8例),7例交界性肿瘤,20例卵巢良性上皮性肿瘤(其中浆液性囊腺瘤11例,粘液性囊腺瘤9例),22例妇科其他良性疾病患者以及21例妇科其他恶性肿瘤患者微量血清(0.5μl)中IGF-II各不同肽, IGFBP-2及IGFBP-3含量,同时以20例相应年龄健康妇女血清做正常对照。另外,分别对其中8例卵巢癌患者进行术前、术后以及化疗1个疗程后血清进行IGF-II,IGFBP-2及IGFBP-3含量检测。
结果:
1.卵巢肿瘤患者血清IGF-II(pro-, big-, mIGF-II)测定
卵巢癌患者血清proIGF-II, big IGF-II和mIGF-II含量均明显下降,与正常对照组,良性肿瘤及交界性肿瘤患者血清相比均有显著性差异(P<0.01或P<0.001)
2.卵巢肿瘤患者血清IGFBP-2和IGFBP-3测定
卵巢癌患者血清IGFBP-2水平明显升高,与正常对照组、良性以及交界性卵巢肿瘤患者血清相比有显著性差异(P<0.001);而血清IGFBP-3含量明显降低甚至消失,与正常对照组、良性肿瘤以及交界性肿瘤组相比均有显著性差异(P<0.001);正常对照组与交界性肿瘤组亦有显著性差异(P<0.05)。
3. IGF-II以及IGFBP-2/IGFBP-3与卵巢肿瘤分期、病理分类的相关性
IGF-II各肽以及IGFBP-2,IGFBP-3含量在良性浆液性和粘液性肿瘤组中无统计学差异(P>0.05);在恶性浆液性和粘液性组中亦无统计学差异(P>0.05);在早期和晚期患者间也无统计学差异(P>0.05)。
4.卵巢癌患者血清IGF-II以及IGFBP-2,IGFBP-3水平与CA125相关性
卵巢癌患者术前血清CA125值与血清IGFBP-2值存在正相关性(P<0.0001);与IGFBP-3值存在负相关性(P<0.0001);卵巢癌患者术前血清CA125值分别与IGF-II各不同肽存在负相关性: proIGF-II(P<0.0001); big IGF-II (P<0.0001); mIGF-II (P<0.0001)。
5.卵巢癌患者术前、术后及化疗后血清IGF-II及IGFBP-2,IGFBP-3含量的变化
卵巢癌患者血清IGF-II(pro-, big-和mIGF-II)多数于术后1周左右恢复至正常水平;术后1周IGFBP-2水平较术前稍升高或不变,IGFBP-3继续降低或无明显改变,但经过化疗1疗程后,IGFBP-2及IGFBP-3含量均回复至正常水平。
6.卵巢癌患者及妇科其他良性疾病患者血清IGF-II以及IGFBP-2,IGFBP-3水平的相关性
卵巢癌患者血清IGF-II(pro-, big-和mIGF-II)水平均明显低于其他妇科良性疾病患者,经统计学处理有显著性差异(P<0.001),而妇科良性疾病组与正常对照组相比均无统计学差异(P>0.05);卵巢癌组血清IGFBP-2水平明显高于妇科良性疾病组(P<0.001),妇科良性疾病组与正常对照组相比无统计学差异(P>0.05);卵巢癌组血清IGFBP-3水平明显低于妇科良性疾病组(P<0.001),而妇科良性疾病组与正常对照组间无显著性差异(P>0.05)。
7.卵巢癌及妇科其他恶性肿瘤患者血清IGF-II以及IGFBP-2,IGFBP-3水平的相关性
妇科其他恶性肿瘤组,约80%的患者血清IGF-II(pro-, big-和mIGF-II)水平高于卵巢癌组, IGFBP-2,IGFBP-3水平变化与卵巢癌相似,但IGFBP-2明显升高且同时伴有IGFBP-3明显降低的发生率低于卵巢癌组。
结论:
1.卵巢癌患者血清中IGFBP-2水平明显升高,可能与瘤负荷有关,而IGFBP-3水平的明显降低则可能有助于肿瘤生长;
2.卵巢癌患者血清中IGF-I(I包括pro-, big-和mIGF-II)水平均显著降低,可能标志着IGF-II生物学活性的改变;
3.卵巢癌患者术后及化疗后血清pro-, big-和mIGF-II以及IGFBP-2,IGFBP-3相继恢复至正常水平,提示联合检测血清IGF-II各肽以及IGFBP-2和IGFBP-3含量有助于判断卵巢癌患者的治疗效果。
4. IGF-II各肽及IGFBP-2,IGFBP-3水平与CA125均有明显的相关性,联合检测上述指标有助于筛查导致CA125升高的一些良性妇科疾病及部分妇科恶性肿瘤,提高卵巢上皮性癌诊断的准确性和特异性。
5.本研究应用改良的WLB和WB方法检测从滤纸抽提的卵巢肿瘤患者微量血清中不同IGF-II肽以及IGFBP-2,IGFBP-3蛋白含量,与传统的蛋白检测方法相比具有高度灵敏度,为卵巢癌以及其他疾病大样本筛查以及地区性研究提供了可行性的血清学检测途径。
第二部分IGFBP-2及IGFBP-3对卵巢上皮性癌患者IGF-II生物学功能的调节
目的:检测卵巢癌患者血清IGFBP-2及IGFBP-3与IGF-II各肽的相关性,研究IGFBP-2和IGFBP -3对IGF-II生物学功能的调节作用。
方法:采用免疫沉淀(Immunoprecipitation, IP)方法确定血循环中IGF-II与IGFBP-2及IGFBP-3的结合状况。血清标本分别应用IGFBP-2,IGFBP-3特异性抗体吸附沉淀(IP)后,再分别经WB测定血清中IGF-II各肽的含量,并通过WLB确定血清中特异吸附沉淀的IGFBP-2及IGFBP-3含量,另外,通过应用IGF-II/IGFBP交叉连接复合物测定(Cross-link)方法分别检测卵巢癌患者血清和正常对照者血清中呈结合状态的IGF-IGFBPs。
结果:1. IGFBP-2与big IGF-II的结合能力明显低于IGFBP-3。无论在正常对照组和卵巢癌组,血清中big IGF-II均不与IGFBP-2结合。经过IGFBP-2 IP后,可检测到卵巢癌患者血清中升高的IGFBP-2以及与正常血清相比升高的mIGF-II水平;经过IGFBP-3 IP后,与正常对照组相比,卵巢癌患者血清IGFBP-3明显减少或消失,但可见升高的IGFBP-3降解片段,而big/m IGF-II均减少。
2.当bigIGF-II含量明显高于mIGF-II时,可与IGFBP-2结合。与正常血清相比,在卵巢癌患者血清中,bigIGF-II和mIGF-II水平均低于正常对照者,在低剂量外源性bigIGF-II存在的情况下,随着外源性bigIGF-II含量的增加,可检测到其水平增加,而给予相同浓度外源性bigIGF-II的正常血清虽可检测到较高水平bigIGF-II,但并不随剂量增加而明显改变。与其发生相应改变的是在正常血清中随着外源性bigIGF-II的增加,150kDa复合物含量明显增加;而卵巢癌患者可检测到明显增加的IGFBP-2与bigIGF-II连接物,以及因与IGFBP-2结合消耗而较正常血清体系明显降低的bigIGF-II含量。
结论:
1.卵巢癌患者血清IGFBP-2,IGFBP-3的改变与IGF-II各肽含量的下降明显相关,IGFBP-3明显降低而IGFBP-2明显升高的状况可改变其与big/m IGF-II的结合状况,进而导致游离IGF-II增加而到达相应靶组织发挥作用并降解。
2.本研究结果提示血循环中IGFBP-2和IGFBP-3的改变可导致IGF-II生物学活性增强,揭示IGFBP-2和IGFBP对卵巢癌患者IGF-II生物学功能的调节机制。
第三部分IGFBP-2及IGFBP-3在卵巢肿瘤患者组织中的表达及临床意义
目的:检测卵巢良性、恶性肿瘤患者组织中IGFBP-2以及IGFBP -3的表达,探讨其与卵巢癌临床病理学特征的相关性,并进一步探讨IGFBP-2及IGFBP-3在卵巢癌发生和发展中可能的作用以及作为卵巢上皮性癌生物学标记的可能性。
方法:采用免疫组化染色方法检测卵巢肿瘤组织(卵巢良性肿瘤8例,卵巢交界性肿瘤8例,卵巢癌上皮性癌23例,包括高、中度分化者15例,低度分化者8例)中IGFBP-2和IGFBP-3的表达。同时以4例正常卵巢组织(来自于因其他妇科疾病须行全部或部分卵巢切除的患者,并经病理证实无卵巢组织病变)作为对照。
结果:
1. IGFBP-2的表达位于细胞质或细胞核,卵巢恶性肿瘤IGFBP-2的表达率明显升高,与正常卵巢组织以及卵巢良性肿瘤相比均有显著性差异(P<0.0001);而正常对照组与卵巢良性肿瘤和交界性肿瘤、卵巢良性肿瘤与交界性肿瘤以及交界性与恶性肿瘤相比均无统计学差异(P>0.05)。
2. IGFBP-3表达位于细胞质,虽然卵巢恶性肿瘤IGFBP-3的表达率与正常对照组、卵巢良性肿瘤相比呈降低趋势,但经统计学处理无显著性差异(P>0.05)。
3.随着卵巢癌分化程度的降低,IGFBP-2的表达呈增强趋势,但低分化组与高中分化组相比无统计学意义(P>0.05)。
4.在卵巢上皮性癌组织中,随着卵巢癌细胞分化程度的降低,IGFBP-3的表达明显降低或呈阴性表达,低分化卵巢癌细胞与高、中分化卵巢癌细胞相比有显著性差异(P<0.05)。
结论:本研究中发现卵巢肿瘤组织细胞中IGFBP-2和IGFBP-3的表达不仅与肿瘤的良、恶性有关,而且与卵巢癌细胞的分化程度有一定相关性,说明在组织细胞中IGFBP-2及IGFBP-3的表达与卵巢癌患者的临床病理学特征有关。
第四部分IGFBP-3在卵巢癌化疗耐药中的调节作用
目的:应用铂类敏感和耐药的卵巢上皮性癌细胞株检测IGFBP-3在卵巢化疗耐药中的作用和调节机制。
方法:分别建立IGFBP-3完整段以及1-97N末端质粒cDNA,应用两对铂类敏感和耐药的卵巢上皮性癌细胞株( OV2008/ C13* ,A2780s/A2780cp)经过细胞培养、IGFBP-3转染伴有或不伴有DN-Akt病毒感染,再经过CDDP (0, 2.5, 5, 10μM; 24 h)处理后,检测卵巢癌细胞的凋亡以及相应Caspase-3改变情况。
结果:
1. CDDP可诱导敏感卵巢癌细胞凋亡,但对耐药卵巢癌细胞的凋亡无影响(p<0.01,P>0.05)。CDDP诱导的卵巢癌细胞的凋亡伴随着IGFBP-3的改变。典型的凋亡细胞表现为细胞变园,萎缩,核浓缩并出现分离形成凋亡小体。
2.过度表达IGFBP-3可以增强敏感性卵巢癌细胞对顺铂(10μM)诱导的凋亡,与对照组相比有显著性差异(p<0.05);过度表达IGFBP-3虽然可诱导部分耐药卵巢癌细胞对顺铂介导的凋亡,但与对照组相比无显著性差异(P>0.05)。
3.过度表达IGFBP-3,并通过DN-Akt病毒感染耐药卵巢癌细胞阻止Akt功能后,可启动并增强CDDP诱导的细胞凋亡。
4.过度表达IGFBP-3以及DN-Akt病毒感染介导的卵巢癌细胞的凋亡与激活Caspase-3相关。
结论:
1. CDDP诱导的卵巢癌细胞的凋亡与IGFBP-3有一定相关性。
2.过度表达IGFBP-3可增加CDDP诱导的细胞凋亡,且与DN-Akt病毒感染有协同作用,提示IGFBP-3对卵巢癌细胞的凋亡诱导作用可能与下调Akt介导的细胞信号传导通路有关。
Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. EOC is characterized by few early symptoms, presentation at an advanced stage, and poor survival. Despite the initial response to combined treatment of cytoreduction (debulking) and chemotherapy, about 70% of patients with advanced ovarian cancer ultimately develop drug-resistant disease (relapse). Nevertheless, seeking for the effective biomarkers for the early detection and prognostic evaluation of EOC, and investigating the mechanism of chemoresistance will benefit the treatment. The insulin-like growth factors (IGFs) play a key role in the tumorigenesis and progression of human malignancies mainly through their antiapoptotic activities or by enhancing tumor cell proliferation. IGF-II processing has been recognized as an important mechanism involved in the regulation of IGF-II’s biological activity. ProIGF-II peptide originally synthesized with 156 amino acids undergoes regulated endoproteolytic cleavage by proprotein convertases to generate its two big variants, IGF-II (1-87) and IGF-II (1-104), and mature form. In certain pathological conditions, a much greater proportion of big IGF-II has been detected whereas total IGF-II concentrations are unchanged, indicating that the different isoforms of IGF-II may have different biological properties. The activities of IGFs are regulated by six IGF-binding proteins (IGFBPs), namely IGFBP-1 to IGFBP-6. The binding activities of IGF-I and different isoforms of IGF-II to IGFBPs are different, resulting in the different bioavailabilities. Of all IGFBPs, IGFBP-3 is the predominant form found in the human circulation, followed by IGFBP-2. IGFBP-3 is a potent anti-proliferative protein that can induce apoptosis and inhibit cancer cells proliferation. Decreased expression and plasma levels of IGFBP-3 are associated with a number of malignancies. The anti-proliferative actions of IGFBP-3 resulted from its ability to form complexes with IGF-I and IGF-II could prevent the growth factors from activating IGF-I receptors to induce cell proliferation and survival. In addition, IGFBP-3 can also act through IGF-independent mechanisms. IGFBP-2 is overexpressed in many malignancies,and associated with increased aggressiveness and invasiveness of the tumor. Increased expression and level of IGFBP-2 can also promote ovarian cancer cell growth and invasiveness. In vitro models have demonstrated that EOC cell lines express all major components of the IGF family, and several studies have implicated the IGF system in the etiology of human ovarian epithelial cancer. Although the individual member of IGF family plays roles in the development, progression and prognosis of ovarian cancer, the predominant form of IGFBPs in the circulation of EOC and its proper roles in the regulation of IGF-II bioaviability and chemosensitivity have not been demonstrated in EOC. In current study, the serum levels of GFBP-2, IGFBP-3 and different isoforms of IGF-II were measured by modified Western ligand blot (WLB) and Western blot (WB) in patients with benign, borderline ovarian tumor and malignant tumor (epithelial ovarian cancer) respectively, and their proper roles in the regulation of IGF-II bioavailability were investigated. Meanwhile, the correlation between the serum levels of IGFBP-2, IGFBP-3 and their expressions in ovarian tumor tissues detected by immunohistochemistry as well as related clinicopathological characteristics were evaluated. Furthermore, the proper role and mechanism of IGFBP-3 in regulating chemoresistance was studied by overexpression of IGFBP-3 in both chemosensitive and resistant ovarian cancer cells in vitro. Our study will further demonstrate the possible roles of IGF-II, IGFBP-2 and IGFBP-3 in regulating the bioactivities of ovarian cancer, and provide evidences for their potential roles as the biomarkers in diagnosing, monitoring the progresses and evaluating the efficacy of chemotherapy in patients of ovarian cancer.
Part 1. The clinical significance of detecting the serum levels of IGF-II and IGFBP-2, IGFBP-3 in patients with ovarian tumor
Objective: To detect the serum levels of IGF-II (pro-, big and mIGF-II) and IGFBP-2, IGFBP-3 in patients with both benign and malignant ovarian tumors, and explore their proper role in differentiated diagnosis, their correlations with clinicopathological characteristics and, further evaluate whether IGF-II, IGFBP-2 and IGFBP-3 can be the biomarkers in diagnosis and surveillance of ovarian cancer.
Methods: The serum (0.5μl/each case) levels of IGF-II, IGFBP-2 and IGFBP-3 were detected by Western Ligand Blot (WLB) and Western Blot (WB) in 23 cases of epithelial ovarian cancer (15 serous and 8 mucinous ), 7 cases of borderline ovarian tumor, 20 cases benign tumor (11 serous and 9 mucinous), 22 patients with benign gynecological diseases and 21 cases of other gynecological cancer , and 20 cases of healthy women as normal control.
Results: 1. The serum levels of IGF-II in patients with ovarian tumors The serum levels of all forms of IGF-II were significantly decreased in EOC group compared with those of normal control group, benign and borderline ovarian tumor groups (P<0.01 or P<0.001).
2. The serum levels of IGFBP-2 and IGFBP-3 in patients with ovarian tumors The serum level of IGFBP-2 increased, while the level of IGFBP-3 decreased in patients with EOC, and there were significant difference compare with those of normal control group, benign and borderline ovarian tumor groups respectively. (P<0.001); The difference between normal control and borderline was also significant (P<0.05)
3. The correlation between IGF-II, IGFBP-2, -3 and clinical stages as well as pathological types of epithelial ovarian tumor The serum levels of IGF-II, IGFBP-2 and IGFBP-3 were not significantly different between benign serous epithelial ovarian tumor group and its mucnious counterpart (P>0.05), and there were also no significant difference between serous ovarian cancer and its mucnious counterpart (P>0.05).
4. The correlation of the serum levels of IGF-II, IGFBP-2, IGFBP-3 with CA125 in patients with epithelial ovarian cancer The serum levels of CA125 was positively correlated to IGFBP-2 (P<0.0001) and negatively correlated to IGFBP-3 (P<0.0001), respectively, and also was positive correlated to proIGF-II (P<0.0001); big IGF-II (P<0.0001) and mIGF-II (P<0.0001), respectively.
5. The changes of serum IGF-II, IGFBP-2 and IGFBP-3 pre-, postoperation and after chemotherapy in patients with epithelial ovarian cancer The serum levels of IGF-II (pro-, big and mIGF-II) were returning toward normal around one week postoperatively, while the serum levels of IGFBP-2 and IGFBP-3 either increased or decreased a little or without significant changes, but they were back to normal when measured after one cycle of chemotherapy.
6. Comparision of serum levels of IGF-II, IGFBP-2 and IGFBP-3 between EOC group and benign gynecological diseases (BGD) group The serum levels of IGF-II (pro-, big and mIGF-II) in patients with EOC were significantly lower than those of patients with BGD(P<0.001), while there were no difference between normal control group and BGD group (P>0.05). The serum level of IGFBP-2 in EOC group was significantly higher than those of BGD group(P<0.001), but there was no significant difference between normal control group and BGD group(P>0.05); The serum level of IGFBP-3 in EOC group was significantly lower than that of BGD group (P<0.001), while there was no significant difference between normal control and BGD group (P>0.05).
7. Comparesion of serum levels of IGF-II, IGFBP-2 and IGFBP-3 between EOC group and other gynecological cancer group The serum levels of IGF-II(pro-, big and mIGF-II) in 80% of patients with other gynecological cancer were higher than those with EOC; and some of them with either increased IGFBP-2 or decreased IGFBP-3 level, while increased IGFBP-2 together with decreaed IGFBP-3 was less happened in other gynecological cancers compared with that in ovarian cancer group.
Conclusion:
1. The serum level of IGFBP-2 increased markedly in patients with epithelial ovarian cancer, probably due to the correlation with the tumor burden, while decreased level of IGFBP-3 might enhance the availability of tumor growth.
2. The changes of different isoforms of serum IGF-II might affect the availability of IGF-II in patients with epithelial ovarian cancer.
3. There were obvious correlations between CA125 and serum IGF-II, IGFBP-2, IGFBP-3 in patients with EOC, indicating that detecting serum IGF-II, IGFBP-2 and IGFBP-3 could help screening some benign gynecological dieases with increased CA125 level and other gynecological cancers, and enhance the diagnositic accuracy of ovarian cancer.
4. The serum levels of IGF-II, IGFBP-2 and IGFBP-3 were returning toward normal following the surgery or chemotherapy, suggesting that detecting IGF-II, IGFBP-2 and IGFBP-3 together could be useful for predicting therapeutic efficacy and prognosis of epithelial ovarian cancer.
5. The modified WLB and WB have much higer sensitivity compared with their traditional ways in detecting IGFBP-2, IGFBP-3 and different isoforms of IGF-II by extracting serum protein from filter paper in patients with EOC, it also makes the large scale of serum research available including different regions with multiple samples.
Part 2. The role and regulation of IGFBP-2 and IGFBP-3 on the IGF-II bioaviability in patients with ovarian cancer
Objective: To detect the correlation between serum levels of IGFBP-2, IGFBP-3 and IGF-II in patients with EOC, and to investigate the possible mechanism of IGF-II bioaviability regulated by IGFBP-2 and IGFBP-3 in the development and progression of ovarian cancer.
Methods: 1. Immunoprecipitation (IP) to determine IGF-II binding to IGFBP-2 and IGFBP-3 in the circulation. Diluted serum samples were incubated with rabbit-anti-IGFBP-2 or anti-IGFBP-3,absorbed by A/G beads then dissociated by boiling. The serum level of IGF-II was determined by WB and specificity of immunoprecipitation of IGFBP-2 and IGFBP-3 was confirmed by WLB. 2. Determination of IGF-II/IGFBPs complex formation and biological availability of IGF-II isoforms. Two sets of aliquots of diluted samples (containing 1 ? ml serum) with different doses of big IGF-II were incubated for 1 hour at room temperature. One aliquot to be mixed with 2 l of 40 mM Disuccinimidyl suberate (DSS) cross-link overnight at 4oC. The comlex contents were determined by Western blotting in non-reducing condition.
Results: 1. The serum levels of IGFBP-2 and IGFBP-3 were positively correlated with all the forms of IGF-II respectively. 2. Formation of big IGF-II/IGFBP-2 complex does not occur in either normal control group or epithelial ovarian cancer sera. Increased IGFBP-2 and mIGF-II content were detected after IGFBP-2 IP. Decreased IGFBP-3 accpanied by increased IGFBP-3 fragment were detected, and both big and mIGF-II have decreased. 3. A higher proportion of big IGF-II relative to mature IGF-II is required for the formation of big IGF-II/IGFBP-2 complex.
Conclusion: Increased IGFBP-2 and decreased IGFBP-3 serum levels are associated with the decreased isoforms of IGF-II in ovarian cancer patients, which could alter the formation of big/mature IGF-II with their dominant IGFBPs in the circulation, leading to more free IGF-II available to the target tissues or cells. Our current results suggest that the bioaviability of IGF-II might be increased induced by the alteration of IGFBP-2 and IGFBP-3 in patients with ovarian cancer.
Part 3. The expressions and clinical significance of IGFBP-2 and IGFBP-3 in epithelial ovarian cancer tissues
Objective: To investigate the expressions and the clinical significance of IGFBP-2 and IGFBP-3 in patients with epithelial ovarian cancer, and to explore the roles of IGFBP-2 and IGFBP-3 in the progression of tumor as well as the possibility to serve as the potential biomarkers in epithelial cancer.
Methods: The expressions of both IGFBP-2 and IGFBP-3 were examined by immunochemistry in ovarian tumor tissues (8 cases of benign tumor, 8 cases of borderline tumor and 23 cases of epithelial ovarian cancer) and 4 cases of normal ovarian tissues.
Results: 1. The expression of IGFBP-2 was located in cytoplasm or nucleus. The expression rate significantly increased in ovarian cancer group compared with those of normal control,benign tumor and borderline tumor groups, while there were no significant difference between normal control vs benign, benign vs borderline, borderline vs malignant groups.
2. The expression of IGFBP-3 was located in cytoplasm. Although there was a decreased trend of the expression of IGFBP-3 with the increased malignancy, but there were no significant difference among different groups.
3. The expression of IGFBP-2 was increased with the decreased differentiation of tumors, but was not significant.
4. The expression of IGFBP-3 was decreased with the decreased differentiation of tumor tissues(P<0.05).
Conclusion: The expressions of IGFBP-2 and IGFBP-3 might be associated with the malignancy of the tumor as well as the degree of differentiation of cancer tissues, indicating that the expressions of IGFBP-2 and IGFBP-3 can be correlated with the clinicopathological characteristics.
Part 4. The role and regulation of IGFBP-3 in chemoresistance in ovarian cancer
Objective: To detect the proper role of IGFBP-3 in the regulating chemoresitivity of ovarian cancer by using both chemosensitive and chemoresistant ovaian cancer cells in vitro.
Methods: Both intact and 1-97 N-terminal IGFBP-3 plasmid cDNA were constructed, and two pairs of chemosensitive and resistant ovarian cancer cell line(sOV2008/ C13*,A2780s/A2780cp)were used in the current experiments. OV2008/ C13* were transfected with IGFBP-3 cDNA with or without DN-Akt virus infection (20 or 40 MOI) for 24 hours, followed by CDDP (0, 2.5, 5, 10μM; 24 h) treatment, the apoptotic cells and caspase-3 cleavage were examined by Hoechst staing and Western Blot respectively.
Results: 1. Apoptosis induced by CDDP increased in chemosensitive ovarian cancer cells in a dose dependent manner, but not in their resistant counterparts. IGFBP-3 protein content also increased in chemosensitive OV2008 cells when cells were treated with CDDP, but no significant changes happened in chemoresistant cells. Typical morphology changes of apoptotic cells are condensed nucleus and nuclear fragmentation etc.
2. Overexpression of IGFBP-3 could intensify chemosensitive ovarian cancer cells to CDDP-induced apoptosis, but can not significantly facilitate chemoresistant cells to CDDP-induced apoptosis.
3. Overexpression of IGFBP-3 and blocking the function of Akt by DN-Akt virus infection had synergetic role in enhancing CDDP-indued apoptosis.
4. Overexpression of IGFBP-3 and DN-Akt mediated,CDDP induced apoptosis was associated with caspase-3 activation.
Conclusion: CDDP-induced apoptosis can be associated with IGFBP-3, and overexperssion of IGFBP-3 and blocking the function of Akt can have synergetic role in facilitating ovarian cancer cells to CDDP-induced apoptosis, suggesting that Akt pathway might be involved in IGFBP-3-mediated apoptosis.
引文
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10 Hudgins WR, Hampton B, Burgess WH, Perdue JF. The identification of O-glycosylated precursors of insulin-like growth factor II. J Biol Chem 1992; 267: 8153-8160.
11 Zapf J, Futo E, Peter M, Froesch ER. Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest 1992; 90: 2574-2584.
12 Daughaday WH, Trivedi B. Measurement of derivatives of proinsulin-like growth factor-II in serum by a radioimmunoassay directed against the E-domain in normal subjects and patients with nonislet cell tumor hypoglycemia. J Clin Endocrinol Metab 1992; 75: 110-115.
13 Hoekman K, van DJ, Gloudemans T, Maassen JA, Schuller AG, Pinedo HM. Hypoglycaemia associated with the production of insulin-like growth factor II and insulin-like growth factor binding protein 6 by a haemangiopericytoma. Clin Endocrinol (Oxf) 1999; 51: 247-253.
14 Elmlinger MW, Rauschnabel U, Koscielniak E, Weber K, Ranke MB. Secretion of noncomplexed 'Big' (10-18 kD) forms of insulin-like growth factor-II by 12 soft tissue sarcoma cell lines. Horm Res 1999; 52: 178-185.
15 Daughaday WH, Trivedi B, Baxter RC. Serum "big insulin-like growth factor II" from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing. Proc Natl Acad Sci U S A 1993; 90: 5823-5827.
16 Perdue JF, LeBon TR, Kato J, Hampton B, Fujita-Yamaguchi Y. Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors. Endocrinology 1991; 129: 3101-3108.
17 Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW, Wallace JC. Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR. Mol Endocrinol 2004; 18: 2502-2512.
18 Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005; 16: 421-439.
19 Scott CD, Firth SM. The role of the M6P/IGF-II receptor in cancer: tumor suppression or garbage disposal? Horm Metab Res 2004; 36: 261-271.
20 LeRoith D, Roberts CT, Jr. The insulin-like growth factor system and cancer. Cancer Lett 2003; 195: 127-137.
21 Zapf J, Schmid C, Froesch ER. Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 1984; 13: 3-30.
22 Wetterau LA, Moore MG, Lee KW, Shim ML, Cohen P. Novel aspects of the insulin-like growth factor binding proteins. Mol Genet Metab 1999; 68: 161-181.
23 Baxter RC, Binoux MA, Clemmons DR, Conover CA, Drop SL, Holly JM, Mohan S, Oh Y, Rosenfeld RG. Recommendations for nomenclature of the insulin-like growth factor binding protein superfamily. J Clin Endocrinol Metab 1998; 83: 3213.
24 Lassarre C, Binoux M. Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology 1994; 134: 1254-1262.
25 Blat C, Villaudy J, Binoux M. In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 1994; 93: 2286-2290.
26 Clemmons DR. Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev 1997; 8: 45-62.
27 Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75: 73-82.
28 Irving JA, Lala PK. Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-beta, IGF-II, and IGFBP-1. Exp Cell Res 1995; 217: 419-427.
29 Sibley CP, Coan PM, Ferguson-Smith AC, Dean W, Hughes J, Smith P, Reik W, Burton GJ, Fowden AL, Constancia M. Placental-specific insulin-like growth factor 2 (Igf2) regulates the diffusional exchange characteristics of the mouse placenta. Proc Natl Acad Sci U S A 2004; 101: 8204-8208.
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40 Baciuchka M, Remacle-Bonnet M, Garrouste F, Favre R, Sastre B, Pommier G. Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) proteolysis in patients with colorectal cancer: possible association with the metastatic potential of the tumor. Int J Cancer 1998; 79: 460-467.
41 Espelund U, Cold S, Frystyk J, Orskov H, Flyvbjerg A. Elevated free IGF2 levels in localized, early-stage breast cancer in women. Eur J Endocrinol 2008; 159: 595-601.
42 Mor G, Visintin I, Lai Y, Zhao H, Schwartz P, Rutherford T, Yue L, Bray-Ward P, Ward DC. Serum protein markers for early detection of ovarian cancer. Proc Natl Acad Sci U S A 2005; 102: 7677-7682.
43 Daughaday WH, Emanuele MA, Brooks MH, Barbato AL, Kapadia M, Rotwein P. Synthesis and secretion of insulin-like growth factor II by a leiomyosarcoma with associated hypoglycemia. N Engl J Med 1988; 319: 1434-1440.
44 Khosla S, Hassoun AA, Baker BK, Liu F, Zein NN, Whyte MP, Reasner CA, Nippoldt TB, Tiegs RD, Hintz RL, Conover CA. Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults. J Clin Invest 1998; 101: 2165-2173.
45 Singh SK, Moretta D, Almaguel F, Wall NR, De LM, De LD. Differential effect of proIGF-II and IGF-II on resveratrol induced cell death by regulating survivin cellular localization and mitochondrial depolarization in breast cancer cells. Growth Factors 2007; 25: 363-372.
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2 Yee D, Lee AV. Crosstalk between the insulin-like growth factors and estrogens in breast cancer. J Mammary Gland Biol Neoplasia 2000; 5: 107-115.
3 Qiu Q, Jiang JY, Bell M, et al. Activation of endoproteolytic processing of insulin-like growth factor-II in fetal, early postnatal, and pregnant rats and persistence of circulating levels in postnatal life. Endocrinology 2007; 148: 4803-4811.
4 Valenzano KJ, Heath-Monnig E, Tollefsen SE, et al. Biophysical and biological properties of naturally occurring high molecular weight insulin-like growth factor II variants. J Biol Chem 1997; 272: 4804-4813.
5 Khosla S, Ballard FJ, Conover CA. Use of site-specific antibodies to characterize the circulating form of big insulin-like growth factor II in patients with hepatitis C-associated osteosclerosis. J Clin Endocrinol Metab 2002; 87: 3867-3870.
6 Daughaday WH, Emanuele MA, Brooks MH, Barbato AL, Kapadia M, Rotwein P. Synthesis and secretion of insulin-like growth factor II by a leiomyosarcoma with associated hypoglycemia. N Engl J Med 1988; 319: 1434-1440.
7 Zapf J, Futo E, Peter M, et al. Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest 1992; 90: 2574-2584.
8 Khosla S, Hassoun AA, Baker BK, et al. Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults. J Clin Invest 1998; 101: 2165-2173.
9 Singh SK, Moretta D, Almaguel F, et al. Precursor IGF-II (proIGF-II) and mature IGF-II (mIGF-II) induce Bcl-2 And Bcl-X L expression through different signaling pathways in breast cancer cells. Growth Factors 2008; 26: 92-103.
10 Singh SK, Moretta D, Almaguel F, et al. Differential effect of proIGF-II and IGF-II on resveratrol induced cell death by regulating survivin cellular localization and mitochondrial depolarization in breast cancer cells. Growth Factors 2007; 25: 363-372.
11 Espelund U, Cold S, Frystyk J, et al. Elevated free IGF2 levels in localized, early-stage breast cancer in women. Eur J Endocrinol 2008; 159: 595-601.
12 Mor G, Visintin I, Lai Y, et al. Serum protein markers for early detection of ovarian cancer. Proc Natl Acad Sci U S A 2005; 102: 7677-7682.
13 Flyvbjerg A, Mogensen O, Mogensen B, et al. Elevated serum insulin-like growth factor-binding protein 2 (IGFBP-2) and decreased IGFBP-3 inepithelial ovarian cancer: correlation with cancer antigen 125 and tumor-associated trypsin inhibitor. J Clin Endocrinol Metab 1997; 82: 2308-2313.
14 Zapf J, Schmid C, Froesch ER. Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 1984; 13: 3-30.
15 Wetterau LA, Moore MG, Lee KW, et al. Novel aspects of the insulin-like growth factor binding proteins. Mol Genet Metab 1999; 68: 161-181.
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2 Flyvbjerg A, Mogensen O, Mogensen B, et al. Elevated serum insulin-like growth factor-binding protein 2 (IGFBP-2) and decreased IGFBP-3 in epithelial ovarian cancer: correlation with cancer antigen 125 and tumor-associated trypsin inhibitor. J Clin Endocrinol Metab 1997; 82: 2308-2313.
3 Fanayan S, Firth SM, Baxter RC. Signaling through the Smad pathway by insulin-like growth factor-binding protein-3 in breast cancer cells. Relationship to transforming growth factor-beta 1 signaling. J Biol Chem 2002; 277: 7255-7261.
4 Hong J, Zhang G, Dong F, et al. Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells. J Biol Chem 2002; 277: 10489-10497.
5 Lee HY, Chun KH, Liu B, et al. Insulin-like growth factor binding protein-3 inhibits the growth of non-small cell lung cancer. Cancer Res 2002; 62: 3530-3537.
6 Butt AJ, Fraley KA, Firth SM, et al. IGF-binding protein-3-induced growth inhibition and apoptosis do not require cell surface binding and nuclear translocation in human breast cancer cells. Endocrinology 2002; 143: 2693-2699.
7 Kim HS, Ingermann AR, Tsubaki J, et al. Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells. Cancer Res 2004; 64: 2229-2237.
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13 Lalou C, Lassarre C, Binoux M. A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin. Endocrinology 1996; 137: 3206-3212.
14 Giudice LC, Farrell EM, Pham H, et al. Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity. J Clin Endocrinol Metab 1990; 71: 806-816.
15 Hossenlopp P, Segovia B, Lassarre C, et al. Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy. J Clin Endocrinol Metab 1990; 71: 797-805.
16 Blat C, Villaudy J, Binoux M. In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 1994; 93: 2286-2290.
17 Salahifar H, Firth SM, Baxter RC, et al. Characterization of an amino-terminal fragment of insulin-like growth factor binding protein-3 and its effects in MCF-7 breast cancer cells. Growth Horm IGF Res 2000; 10: 367-377.
1 Fowden AL. The insulin-like growth factors and feto-placental growth. Placenta 2003; 24: 803-812.
2 Christofori G, Naik P, Hanahan D. A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis. Nature 1994; 369: 414-418.
3 DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 1991; 64: 849-859.
4 Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R, Stewart F, Kelsey G, Fowden A, Sibley C, Reik W. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 2002; 417: 945-948.
5 Duguay SJ, Jin Y, Stein J, Duguay AN, Gardner P, Steiner DF. Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis. J Biol Chem 1998; 273: 18443-18451.
6 Qiu Q, Basak A, Mbikay M, Tsang BK, Gruslin A. Role of pro-IGF-II processing by proprotein convertase 4 in human placental development. Proc Natl Acad Sci U S A 2005; 102: 11047-11052.
7 Valenzano KJ, Heath-Monnig E, Tollefsen SE, Lake M, Lobel P. Biophysical and biological properties of naturally occurring high molecular weight insulin-like growth factor II variants. J Biol Chem 1997; 272: 4804-4813.
8 Khosla S, Ballard FJ, Conover CA. Use of site-specific antibodies to characterize the circulating form of big insulin-like growth factor II in patients with hepatitis C-associated osteosclerosis. J Clin Endocrinol Metab 2002; 87: 3867-3870.
9 Zumstein PP, Luthi C, Humbel RE. Amino acid sequence of a variant pro-form of insulin-like growth factor II. Proc Natl Acad Sci U S A 1985; 82: 3169-3172.
10 Hudgins WR, Hampton B, Burgess WH, Perdue JF. The identification of O-glycosylated precursors of insulin-like growth factor II. J Biol Chem 1992; 267: 8153-8160.
11 Zapf J, Futo E, Peter M, Froesch ER. Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest 1992; 90: 2574-2584.
12 Daughaday WH, Trivedi B. Measurement of derivatives of proinsulin-like growth factor-II in serum by a radioimmunoassay directed against the E-domain in normal subjects and patients with nonislet cell tumor hypoglycemia. J Clin Endocrinol Metab 1992; 75: 110-115.
13 Hoekman K, van DJ, Gloudemans T, Maassen JA, Schuller AG, Pinedo HM. Hypoglycaemia associated with the production of insulin-like growth factor II and insulin-like growth factor binding protein 6 by a haemangiopericytoma. Clin Endocrinol (Oxf) 1999; 51: 247-253.
14 Elmlinger MW, Rauschnabel U, Koscielniak E, Weber K, Ranke MB. Secretion of noncomplexed 'Big' (10-18 kD) forms of insulin-like growth factor-II by 12 soft tissue sarcoma cell lines. Horm Res 1999; 52: 178-185.
15 Daughaday WH, Trivedi B, Baxter RC. Serum "big insulin-like growth factor II" from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing. Proc Natl Acad Sci U S A 1993; 90: 5823-5827.
16 Perdue JF, LeBon TR, Kato J, Hampton B, Fujita-Yamaguchi Y. Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors. Endocrinology 1991; 129: 3101-3108.
17 Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW, Wallace JC. Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR. Mol Endocrinol 2004; 18: 2502-2512.
18 Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005; 16: 421-439.
19 Scott CD, Firth SM. The role of the M6P/IGF-II receptor in cancer: tumor suppression or garbage disposal? Horm Metab Res 2004; 36: 261-271.
20 LeRoith D, Roberts CT, Jr. The insulin-like growth factor system and cancer. Cancer Lett 2003; 195: 127-137.
21 Zapf J, Schmid C, Froesch ER. Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin Endocrinol Metab 1984; 13: 3-30.
22 Wetterau LA, Moore MG, Lee KW, Shim ML, Cohen P. Novel aspects of the insulin-like growth factor binding proteins. Mol Genet Metab 1999; 68: 161-181.
23 Baxter RC, Binoux MA, Clemmons DR, Conover CA, Drop SL, Holly JM, Mohan S, Oh Y, Rosenfeld RG. Recommendations for nomenclature of the insulin-like growth factor binding protein superfamily. J Clin Endocrinol Metab 1998; 83: 3213.
24 Lassarre C, Binoux M. Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology 1994; 134: 1254-1262.
25 Blat C, Villaudy J, Binoux M. In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 1994; 93: 2286-2290.
26 Clemmons DR. Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev 1997; 8: 45-62.
27 Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75: 73-82.
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