| Introduction:Epithelial carcinoma of the ovary, also called ovarian cancer is the most lethal form of gynecologic cancer among women in the United States, accounting for an estimated 21,880 new cases and 13,850 deaths in 2010. Its case fatality rate is the highest among all of the malignant gynecologic tumors. Survival rates can approach 90% when ovarian cancer is diagnosed at an early stage. However, early detection is challenging, as the relatively nonspecific symptoms of ovarian lesions may be overlooked until advanced disease is manifested by abdominal distension of ascitic fluid, or large tumor mass. Even with extensive surgical debulking and aggressive chemotherapy, the prognosis for women with ovarian cancer currently is grim, and the 5-year survival rate for women with ovarian carcinoma has not apparently increased.Several studies have noted that different histologic subtypes of ovarian carcinoma are associated with different causes and underlying mechanisms, including gene amplification, genetic predisposition, and various carcinogens. In addition, accumulating evidence has strongly implicated that epithelial cells derived from the fimbriated ends of the fallopian tube, as the likely origin for high grade serous carcinoma. However, the exact origin and causes of various types of epithelial carcinoma of the ovary remain to be determined.Human chorionic gonadotropin (hCG) holds a physiologically significant role during pregnancy, and is produced as a heterodimeric glycoprotein complex by the placenta. The heterocomplex consists of an a subunit and a hormone-specificβsubunit, which collectively act as a ligand in activating the luteinizing hormone/hCG receptor in gonadal cells to regulate sex hormone synthesis and reproductive processes. The beta subunit of the hCG complex (β-hCG) is an accurate marker for diagnosis and monitoring of trophoblastic tumors and ovarian germ cell tumors. Recently, the expression and function ofβ-hCG in non- trophoblastic tumors from diverse primary tissue origin has attracted more attentions and studies demonstrated that increasedβ-hCG expression simultaneously stimulates proliferation and inhibits apoptosis of cancer cells derived from the urinary bladder and the cervix in vitro. Similarly, silencing expression ofβ-hCG promotes apoptosis in human cervical carcinoma cells. Finally,β-hCG promotes mitotis of human mammary epithelial cells indicating thatβ-hCG plays a role in the malignant transformation of mammary epithelial cells. Furthermore, ovarian carcinoma tissue displays an over-expression not only of the hormone-specificβ-hCG subunit, but also of the cognate receptor hCGR, predicting thatβ-hCG subunit plays an essential role in the origin and developing of epithelial carcinoma of the ovary. Besides, relative researches demonstrate that, elevated levels ofβ-hCG in serum, urine, or tumor tissues correlates with patient's outcome in a variety of non-trophoblastic tumors of diverse primary tissue origin. In addition, elevatedβ-hCG in urine predicted severe aggressive grade, resistant to therapy and poor prognosis in patients with non-trophoblastic cancers including urinary bladder cancer patients. However, the functional role and molecular mechanism ofβ-hCG tumorigenesis in non-trophoblastic tumors including ovarian cancer has not been characterized.In a separate project, in order to identify the most common transforming oncogenes in primary ovarian cancer, we constructed a retrovirus cDNA expression library from a pool of 10 high-grade serous ovarian cancers. The library was infected into NIH3T3 mouse embryonic fibroblast cells, seeded on soft agar, the anchorage independent clones were picked for sequence analysis, and from 193 of clones analyzed,11 containedβ-hCG sequences. More recently, Nowak-Markwi et,al reported that the level of transcription and expression ofβ-hCG increased in several cases of epithelial carcinoma of the ovary. That promoted us to investigate the role ofβ-hCG in ovarian tumorigenesis.Objective:We transfectedβ-hCG into stable surface epithelium ovarian cells and established two cell lines:T29β-hCG and T80β-hCG cells, which are stable at expressing human chorionic gonadotropinβsubunit cDNA, We used these cells to determined the effect ofβ-hCG expression on cell proliferation and cell cycle progression; as well its potential role in exploring the underling molecular mechanism ofβ-hCG regulating apoptosis and cell cycle progression. By using agar growth plates and by injecting T29β-hCG and T80β-hCG cells into mice, we tested the role ofβ-hCG; testing whether this molecule plays a direct role in facilitating ovarian epithelial cell activation and tumorigenic potential, by using both in vitro and vivo methods, evaluating the different expression status ofβ-hCG in normal surface ovarian epithelium, fallopian tube epithelium, benign and malignant ovarian carcinoma epithelial cells. To forther explore theβsubunit of human chorionic gonadotropin (β-hCG) in promotion of immortalized human ovarian surface epithelial cells malignant transformation, and the molecular pathogenesis of its oncogenic activity.Method:Brief introduction about the cell lines used in this research: T29 and T80 cells were immortalized non-tumorigenic cell lines derived from normal ovarian surface epithelial cell lines IOSE-29 and IOSE-80 infected by retrovirus carrying SV40 T/t antigens and human telomerase catalytic subunit (hTERT) respectively, and confirmed that they were non-tumorigenic. These cell lines were designed as control groups in this study. T29β-hCG and T80β-hCG cells were derived from immortalized non-tumorigenic normal ovarian surface epithelial cell lines T29 and T80 cell lines which were double infected by retrovirus carryingβ-hCG cDNA, and were stable-selection with puromycin. These cell lines were designed as the experimental group in this study. The brief, our methodology was as follows:1. Apoptosis. Evaluate and compare the In vitro comparatively investigate the apoptotic index of immortalized human ovarian surface epithelial cells T29, T80, T29β-hCG and T80β-hCG cells, as well as the differences of expression of pro-survival proteins and pro-death proteins, which may be related with regulation of the cell cycle progression. We used flow cytometric detereminations to analyze the apoptotic index in T29, T80, T29β-hCG and T80β-hCG cell lines; We used western blotting to determine the expression levels of pro-survival protein Bcl- XL and pro-death protein Bad respectively in T29, T80, T29β-hCG and T80β-hCG cells.2. Cell proliferation. To explore rates of cell proliferation, we evaluated the expression of cell cycle cyclins and cyclin-dependent kinases in T29, T80, T29β-hCG, and T80β-hCG cells.Cell proliferation was assessed at 24,48,60, and 72 hrs in triplicate in T29, T80, T29β-hCG and T80β-hCG cells, based on using the colorimetric MTT cell proliferation assay. The cell cycle progression status of T29, T80, T29β-hCG and T80β-hCG cell lines was evaluated by analyzing the percentage of cells at the Gland G2 stages by using flow cytometer; We used western blotting to determine the expression levels of cyclin E, cyclin D1, CDK2, CDK4, and CDK6 in T29,T80,T29β-hCG and T80β-hCG cells.3. In vitro and in-vivo tumorigenicity. To determine potential of tumorigenicity in vitro, we determined the ratio of colony formation using soft agarose. To determine the in-vivo tumorigenesis, we used subcutaneous xenografts of T29, T80, T29β-hCG and T80β-hCG cells, and determined the incidence of tumor formation in athymic nude mice. We performed immunohistochemical analysis of tumors arising in xenografts to determine the presence ofβ-hCG and the immune expression pattern in xenograft tumor of nude mice. 4.β-hCG expression in clinical specimens. We performed immunohistochemical analysis ofβ-hCG in a tissue microarray containing normal human gynecologic tissues and ovarian tumors. We perform statistical analysis to determine the presence of significant differences of expression ofβ-hCG in normal surface epithelium, normal fallopian tube epithelium, non-neoplastic ovarian cystic lesions, cystadenomas of ovary, and low and high grade of ovarian serous carcinomas. We also determined and compared expression ofβ-hCG in several major histotypes of ovarian epithelial carcinoma. Expression ofβ-hCG was correlated with overall survival rate and disease free survival rate for epithelial ovarian cancer patientsResults:1. Apoptosis. Flow cytometric analysis of the apoptotic index revealed a significant decrease rate in T29β-hCG cells as compared with T29 cells (2.9% vs 12.3% p<0.05), and also in T80β-hCG cells as compared with T80 cells (3.1%vs 9.2%, p<0.05); Western blot analysis revealed that over-expression ofβ-hCG associated with upregulation of the anti-apoptotic, pro-survival protein Bcl-XL, as well as a significant decrease of the active form of the pro-apoptotic protein Bad, without altering total Bad protein relative to parental control cell lines of T29 and T80 respectively.2. Cell Proliferation rate. The cell proliferation rate evaluated by the colorimetric MTT cell proliferation assay demonstrated that T29β-hCG cells had increased cell proliferation at 24,48, and 60 hrs, and T80β-hCG cells had increased proliferation at 24,48, and 72 hrs, relative to control parental cell lines, p< 0.05 at 24 hrs, p< 0.001 at 48 and 60 hrs) respectively; Flow cytometric analysis of cell cycle progression revealed decreased G1 cell populations in T29β-hCG cells(39.5% vs 48.8%) and T80β-hCG cells (43.1% vs 52.5%) as well as increased G2 cell populations in T29β-hCG cells (33.3% vs 22.6%) and T80β-hCG cells (29.8% vs 19.7%), relative to control parental cell lines T29 and T80. Western blot analysis revealed that over-expression ofβ-hCG associated with upregulation of cyclins E and D1 and their associated partner cyclin-dependent kinases Cdk2 and Cdk4, as well as Cdk6, relative to control parental cell lines T29 and T80.3. Analysis of tumorigenesis. The potential for tumorigenesis using the cell colony-formation in soft agar evaluated the in vitro associated with anchorage-independent assay. T29β-hCG and T80β-hCG cells displayed increased anchorage-independent colony-formation in soft agar when compared with parental T29 and T80 cells (p< 0.01 and p<0.05, respectively); The effect ofβ-hCG on xenograft tumor formation and growth in vivo were observed by injecting T29, T80, T29β-hCG and T80β-hCG cells as subcutaneous xenografts into athymic nude mice. Tumor formation was detected in 13 of 24 T29β-hCG cell injections in 10 mice, and 6 of 14 T80β-hCG cell injections in 7 mice. All tumors occurred within 4-6 weeks of follow up. However, no tumor growth was observed in the 4 mice injected with T29 or T80 parental control cells after 13 weeks of observation. A histopathologic analysis of xenograft tumors produced by the injection of T29β-hCG or T80β-hCG cells revealed poorly differentiated carcinoma. Immunohistochemical analysis ofβ-hCG in representative T80β-hCG-derived tumors demonstrated intense epithelial membrane and cytoplasmic reactivity pattern. We also demonstrated immuoreactivity for SV40 T/t antigens indicating tumor formation due to immortalized human cells, as well as a high density of CD34-positive microvessels, strong cytoplasmic cytokeratin reactivity indicative of epithelial lineage, positive nuclear progesterone receptor immunostaining, and negative immunoreactivity for nuclear estrogen receptor.4.β-hCG expression in clinical specimens. A survey ofβ-hCG expression was conducted using 518 tissue samples from the normal ovary, fallopian tube, as well as all ovarian tumor histological types.β-hCG immunoreactivity, detected as diffuse cytoplasmic staining with moderate-to-high intensity in varying proportions of epithelial cells, was significantly higher in high-grade, low-grade, and borderline tumors of low malignant potential (all p<0.01), as compared to normal ovarian surface epithelium, fallopian tube epithelium, as well as benign ovarian cystic lesions. However,β-hCG expression was not distinct among the eight major ovarian carcinoma histotypes in 539 patient samples. P>0.05. In addition,β-hCG expression was not a significant independent predictor of overall survival (P>0.05) or disease-free survival (P>0.05) among the major ovarian cancer histotypes.Conclusions:1.β-hCG over-expression decreases apoptosis rate in association with elevated pro-survival and decreased pro-death proteins in ovarian epithelial cell lines transfected withβ-hCG.2.β-hCG over-expression induces cell cycle progression in human immortalized ovarian surface epithelial cell lines, through elevated expression of CyclinE,Cyclin D 1, and cyclin-dependent kinases cdk2,cdk4, and cdk6.3. Over-expression ofβ-hCG in human immortalized ovarian surface epithelial cells promotes anchorage-independent colony growth in soft agar and enhanced xenograft tumorigenesis in nude mice, demonstrating thatβ-hCG over-expression transforms immortalized ovarian surface epithelial cells resulting in anchorage-independent colony formation in vitro and xenograft tumorigenesis in vivo, and yields an oncogenic functions on immortalized ovarian surface epithelial cells.4.β-hCG is overexpressed significantly in ovarian epithelial carcinomas and borderline tumors of ovary, as compared with normal ovarian surface epithelium, fallopian tube epithelium and benign ovarian cystic lesions.β-hCG expression did not significantly vary among the common major histotypes of ovarian carcinoma. Variation inβ-hCG expression was not a significant independent predictor of overall survival rate and disease-free survival rate among the major histotypes of ovarian carcinoma, and didn't show the predictive value on cancer patient's outcome.
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