The Role Of IGFBP-2 And IGFBP-3 In The Regulation Of IGF-â…¡ Bioavailability In Patients With Epithelial Ovarian Cancer And Related Study On Chemosensitivity In Vitro

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 tumorObjective: 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 cancerObjective: 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 tissuesObjective: 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 cancerObjective: 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.