Expression of octamer-4 in serous and mucinous ovarian carcinoma.

BACKGROUND: Octamer-4 (Oct4) is a well known regulator of self-renewal in embryonic stem cells; it has been detected in several human cancers and may play a critical role in carcinogenesis. AIMS: To assess the expression of Oct4 in epithelial ovarian tumours. METHODS: Expression of Oct4 was evaluated by immunohistochemistry in 460 cases of various epithelial ovarian lesions as well as 35 cases of normal fallopian tube epithelium. The association between Oct4 expression and various clinical pathological parameters was analysed. RESULTS: Oct4 expression was significantly increased from normal epithelium (both ovarian epithelium and fallopian tube epithelium) to benign and borderline cystadenoma to carcinoma in the serous lesion subgroup. Oct4 overexpression was associated with more advanced FIGO stage and higher histological grade in serous adenocarcinoma. Conversely, Oct4 expression did not differ among mucinous lesions or correlate with clinicopathological parameters in patients with mucinous adenocarcinoma. CONCLUSION: Results suggest that Oct4 expression may contribute to the initiation, promotion and progression of serous ovarian carcinoma; it might be a useful biomarker for the diagnosis and outcome prediction of serous ovarian carcinoma.

Hes1/Hes5 gene inhibits differentiation via down-regulating Hash1 and promotes proliferation in cervical carcinoma cells.

INTRODUCTION: Hairy and enhancer of split 1 (Hes1) and Hes5 are target genes for the mammalian Notch pathway, which are highly expressed in epithelia in the process of embryogenesis or in neural stem cells, inhibit cell differentiation via the Notch-Hes-Hash signaling, and promote the survival of stem cells. Either Hes1 or Hes5 overactivation is likely to affect cell differentiation, thereby resulting in carcinogenesis. METHODS: We transfected the diced small interference RNA into SiHa cells and detected cell differentiation and proliferation by immunocytochemistry, Western blot, and methyl thiazolyl tetrazolium assay. RESULTS: Knockdown of Hes1 and Hes5 would up-regulate the downstream gene Hash1, but not the upstream gene Notch1 in the Notch-Hes-Hash pathway. After Hes1/Hes5 RNA interference, expression of differentiation-associated proteins (including Nanog, stage-specific embryonic antigen 4, and tumor rejection antigen-1-60) was reduced, and the cell differentiation was promoted; meanwhile, the cell proliferation was inhibited, which was verified by detecting proliferation-associated proteins (eg, Ki-67, proliferating cell nuclear antigen) and methyl thiazolyl tetrazolium assay. CONCLUSIONS: Our findings suggest that Hes1/Hes5 gene would inhibit the cell differentiation via down-regulating Hash1 and promote the cell proliferation in cervical carcinoma cells; the cell differentiation and proliferation can be reversed simultaneously by Hes1/Hes5 knockdown using RNA interference.

Methotrexate induces apoptosis of human choriocarcinoma cell line JAR via a mitochondrial pathway.

OBJECTIVE: To investigate methotrexate (MTX)-induced apoptosis and the involved pathways in human choriocarcinoma cells. STUDY DESIGN: MTX-induced apoptosis of human choriocarcinoma cell line JAR was examined using a PI/Annexin V stain with flow cytometer (FCM). Mitochondrial apoptosis was detected by fluorescence microscopy, and analyzed by FCM using a MitoCapture mitochondrial apoptosis detection kit. The activities of caspase-8 and caspase-9 were quantified by microtiter plate reader at 405 nm using FLICE/Caspase-8 colorimetric assay kit and Caspase-9/Mch6 colorimetric assay kit. The changes in Bax and Bcl-2 expression were detected during apoptosis using immunocytochemistry and Western blot analysis. RESULTS: JAR cells underwent apoptosis after exposure to 0.1-2.5 microg/ml MTX for 48 h. Decreased mitochondrial membrane potential was observed both by fluorescence microscopy and FCM. The activation of caspase-9 was increased 4.35+/-0.76-fold in MTX-incubated JAR, while there was no obvious change in the activation of caspase-8. When JAR cells underwent apoptosis, the expression of Bcl-2 was decreased and the expression of Bax was increased; both were detected by immunocytochemistry assay. CONCLUSION: Methotrexate in lower concentrations induces apoptosis of human choriocarcinoma cells via mitochondrial-initiated pathways, including reduction of mitochondrial membrane potential, activation of caspase-9, and up-regulation of Bax/Bcl-2 expression.

[Study of apoptosis and Fas expression of peritoneal fluid and peripheral blood T lymphocytes in patients with epithelial ovarian cancer and their relationship with CA125].

OBJECTIVE: To investigate the apoptosis and Fas (CD95) expression of T lymphocytes from the peripheral blood and peritoneal fluid of the patients with ovarian cancer and their relationship with CA125. METHODS: Apoptosis and Fas expression of peritoneal fluid and peripheral blood T lymphocytes were assessed by flow cytometry. Peripheral blood samples were obtained from the following objects respectively: patients with stage III - IV ovarian cancer (n = 18) before and after treatment, patients with stage I - II ovarian cancer (n = 15), patients with benign ovarian tumor (n = 18), patients with Krukenberg tumor (n = 6) and normal control (n = 20). Peritoneal fluids were obtained from all the patients with ovarian cancer, Krukenberg tumor and ten patients with benign ovarian tumor. Level of serum CA125 of the patients with ovarian cancer was assessed. RESULTS: In the patients with stage III - IV ovarian cancer, the apoptosis level of the peripheral blood T lymphocytes was 5.55 (3.57 - 9.62)%, significantly higher than those from the patients with stage I - II ovarian cancer, patients with benign ovarian tumor, controls (P < 0.008 in all instances) and the patients with stage III - IV ovarian cancer after treatment (P < 0.05). The intensity of Fas expression of the peripheral blood T lymphocytes from the patients with stage III - IV ovarian cancer was 51 +/- 10, significantly higher than that from controls (P < 0.05). In peritoneal fluid, the apoptosis rates of T lymphocytes, positive rate and intensity of Fas expression on T lymphocytes from patients with stage I - II and stage III - IV ovarian cancer were 17.41 (7.06 - 24.56)%, (57 +/- 16)%, (55 +/- 11)% and 34.06 (17.03 - 44.65)%, (66 +/- 12)%, (70 +/- 24)%, respectively, increased significantly compared with those from patients with benign ovarian tumor, which were 0.78 (0.67 - 1.44)%, (37 +/- 6)%, 43 +/- 6, respectively (P < 0.01 in all instances). The apoptosis level and positive rate of Fas expression on peritoneal fluid T lymphocytes from patients with stage III - IV ovarian cancer were significantly higher than those from patients with Krukenberg tumor (P < 0.01). There was a positive correlation between the serum CA125 level and the apoptosis level of peritoneal fluid T cell in the patients with stage I - II ovarian cancer (r = 0.77, P = 0.009). For ovarian cancer, the apoptosis level of peritoneal fluid T lymphocytes from patients with the serum CA125 > 500 KU/L was higher than that from the patients with the serum CA125 < or = 500 KU/L (P = 0.009). CONCLUSIONS: (1) Extraordinarily increased apoptosis of T cells may play an important role in the development of systemic and celiac immunodeficiency in the patients with ovarian cancer. In contrast with the patients with Krukenberg tumor, the patients with advanced ovarian cancer hare higher percentage of apoptotic peritoneal fluid T lymphocytes, which shows the particularity of local immunity defect. (2) For the patients with ovarian cancer, efficient treatment can decrease the percentage of apoptotic peripheral blood T lymphocytes. (3) The increased positive rate and intensity of Fas expression on peritoneal fluid T lymphocytes stressed the significance of Fas interference in the treatment of ovarian cancer. (4) Level of serum CA125 can reflect the celiac immunity defection in patients with ovarian cancer.

Fas-mediated pathway and apoptosis in normal cervix, cervical intraepithelial neoplasia and cervical squamous cancer.

The aim of this study was to examine the expression patterns of apoptosis-related antigens, such as Fas, FasL, and cFLIP, in cervical squamous cells, and investigate the role of Fas-mediated apoptosis in the pathogenesis of cervical neoplasia. Using specific antibodies for Fas, FasL, and cFLIP, we examined protein expression in 19 specimens of normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma (SCC) by immunohistochemistry. We detected the apoptotic indices by TUNEL in the same specimens. Fas expression levels were quite similar in CIN I, CIN II and the normal cervix. Though Fas expression tended to increase in grade 2 cancer compared to grade 1 cancer and CIN III, and a slight decline was present in grade 3 compared with grade 2 cancer, these differences did not reach statistical significance. Almost all CINs did not express FasL, while FasL expression increased with the grade of the tumor. Statistically significant differences could be observed between grade 1 and grade 2 (p<0.01) and between grade 2 plus grade 3 and grade 1 (p<0.001). All cases of normal cervix and CIN, except two cases of CIN III, did not express cFLIP. cFLIP expression tended to increase with the grade of the tumor. Apoptosis was determined in all samples by TUNEL. There was a decreasing tendency of apoptotic cells from normal cervix to cancers. A negative correlation between cFLIP and apoptosis (r=-0.499 and p=0.000) was observed. Deregulated Fas/FasL system and constitutive expression of cFLIP in SCC may be an important mechanism by which SCCs escape apoptosis during the malignant transformation and progression of these tumors.

[Deletion of OPCML gene and promoter methylation in ovarian epithelial carcinoma].

OBJECTIVE: To investigate the expression of OPCML gene in ovarian epithelial carcinoma and determine the relationship between mRNA expression and methylation of their promoters. METHOD: Twenty normal ovarian tissues and 89 ovarian epithelial tumor specimens (72 malignant, 17 benign), as well as 3 ovarian carcinoma cell lines (SKOV-3, CAOV3, and 3AO), were collected for detection of OPCML gene expression by reverse transcription-polymerase chain reaction and for detection of promoter methylation by restriction enzyme cut analysis from 7. 1999 to 7. 2003. RESULTS: Among ovarian epithelial carcinoma 19.4% expressed OPCML mRNA, while 85% of normal ovarian tissue and 76.5% of benign ovarian tumor. The ratio of expression of OPCML mRNA in ovarian epithelial carcinoma was significantly lower than those of normal (chi2 = 30.108, P = 0.0000) and benign tumors (chi2 = 21.162, P = 0.000). No OPCML mRNA expression was found in SKOV-3 and CAOV3, but was found in 3AO. Methylations were detected in 44.4% of cancer cells promoter, while 0% in normal ovarian tissue and benign ovarian tumors. The ratio of methylation of ovarian epithelial carcinoma was significantly higher than those of normal (chi2 = 13.630, P = 0.0000) and benign tumors (chi2 = 11.797, P = 0.000). Methylation was found in SKOV-3 and CAOV3, but not in 3AO. The relationship between gene expression and promoter methylation was correlated (r = 11.589, P = 0.002), especially at Hap I1 site (r = 11.640, P = 0.004). Methylation was also found in SKOV-3 and CAOV3 cell lines, but not in 3AO cell line. CONCLUSION: Deletion of OPCML gene exists in ovarian epithelial carcinoma cell. The gene promoter methylations, especially Hap II motif, may be one of pathways that contribute the inhibition of OPCML expression.

[The accuracy of diagnosing cervical intraepithelial neoplasia with colposcopically directed biopsy].

OBJECTIVE: To evaluate the accuracy of colposcopically directed biopsy in the diagnosis of cervical intraepithelial neoplasia (CIN). METHODS: The clinical data of 153 patients with CIN diagnosed by colposcopically directed biopsy and treated with cervical loop electrosurgical procedure (LEEP) shortly after were retrospectively studied. The consistency of pathological examination between colposcopically directed biopsy and LEEP was evaluated. The factors of missed diagnosis of cervical invasive carcinoma were analyzed. RESULTS: The diagnosis of 106 out of the 153 patients by colposcopically directed biopsy was consistent with that by LEEP as confirmed by pathologic diagnosis with a consistence rate of 69.3%, however, the diagnoses of 47 of the 153 patients by colposcopically directed biopsy were not consistent with those by LEEP, with an inconsistent rate of 30.7%, including 22 cases of overdiagnosis (14.4%, 22/153) and 25 cases of underdiagnosis (16.3%, 25/153). Eighteen patients were confirmed as with cervical invasive carcinoma by LEEP/hysterectomy at last with a missed diagnosis rate of 11.8%. The missed diagnosis rate of invasive carcinoma by colposcopically directed biopsy was significantly higher in the patients with cytologic diagnosis of with > or = HSIL than in the patients cytologically diagnosed as with < HSIL (chi(2) = 8.208, P < 0.05). CONCLUSION: The accuracy of diagnosing CIN with colposcopically directed biopsy is still unsatisfying. Attention should be paid on the patients with cytological diagnosis of > or = HSIL so as to avoid missed diagnosis of cervical carcinoma.

[Evaluation of neoadjuvant chemotherapy for locally advanced cervical cancinoma].

OBJECTIVE: To investigate the effect of neoadjuvant chemotherapy on locally advanced cervical cancer, the factors affecting outcome of chemotherapy and the long-term survival rate in the patients. METHODS: A total of 64 patients with stageIb2-IIb of locally advanced cervical cancers treated with neoadjuvant chemotherapy between June 1999 and October 2004 in Women's Hospital, School of Medicine, Zhejiang University were retrospectively analysed. The effect of chemotherapy, as well as survival rate was evaluated. The related factors of effect, including age, histology, clinical stage, grade of differentiation, initial tumor size, chemotherapy regimens, number of courses were analysed. RESULTS: Overall effective rate of neoadjuvant chemotherapy was 80%. The effective rate was associated with histology. The patients with squamous cell cancer had significantly higher 5-year survival rate than those with adenocarcinoma (82% vs 6/9, P < 0.05). The rates of positive pelvic lymph node metastasis and parametrial invasion were significantly higher in complete or partial response patients than those in stable patients (P < 0.05). The overall 5-year survival rate was 89%. The 5-year survival rate in complete or partial response group was 100%, in stable disease group was 46% (P < 0.05). The 3, 5-year disease-free survival rate in complete or partial response group was 95% and 83%, respectively, while in stable disease group was 33% and 0, respectively. The disease-free survival rates in complete or partial response group were higher than those of stable disease group (P < 0.05). CONCLUSIONS: The clinical response to neoadjuvant chemotherapy is related to histology. The patients who have good response to chemotherapy should choose surgery. Chemotherapy can decrease the high risk factors of pathology in complete or partial response patients. Neoadjuvant chemotherapy for locally advanced cervical cancer patients seems to improve the long-term survival rate.

Altered expression of cellular membrane molecules of HLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias and invasive squamous cell carcinoma.

OBJECTIVE: The aim of this study was to investigate the role of HLA-DR, HLA-G and CD99 during cervical carcinogenesis and to examine the prognostic significance of these protein expressions in invasive squamous cell carcinoma (SCC). METHODS: Using specific antibodies for HLA-DR, HLA-G and CD99, we examined protein expressions in 19 normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma by immunohistochemistry. And we detected the expression of Ki67 in the same specimens. RESULTS: None of normal cervix and CINs except three cases of CIN III expressed HLA-DR. HLA-DR expression increased progressively with the grade of the tumor, and significant differences could be observed between grade 1 and grade 2 (P<0.01) and between grade 1 and grade 3 (P<0.05). In all normal epithelial control samples, HLA-G expression was seen in ectocervical squamous and endocervical columnar epithelium and the staining was strong and uniform. Only a small proportion of CINs and SCCs showed reduced expression of HLA-G. Compared with the results in the control samples, CINs and SCCs showed significantly reduced expression of HLA-G (P<0.001). SCCs showed significantly increased expression of CD99 when compared with normal cervix and CINs (P<0.05). Ki67 was expressed in all specimens. Significant differences were observed between CINs and normal cervix (P<0.001) and SCCs and controls (P<0.001), but no significant differences could be observed between SCCs and CINs. None of the expressions of these proteins was associated with any of clinicopathological parameters. CONCLUSIONS: These results indicate that increased expression of HLA-DR and CD99 may be related to the evolution of cervical cancer. All protein expressions were not associated with clinicopathological parameters.

Ovarian carcinoma cells effectively inhibit differentiation and maturation of dendritic cells derived from hematopoietic progenitor cells in vitro.

Loco-regional dissemination of ovarian carcinoma is associated with immunosuppression of the peritoneal cavity. One marked characteristic of the peritoneal immunity in this disease is the defective function of dendritic cells (DCs). In this study, the affect of ovarian carcinoma cells on DCs derived from hematopoetic progenitor cells was observed. The study demonstrated that the expansion, phenotype, and function of DCs generated from CD34+ precursors were significantly altered by the supernatant secreted by ovarian carcinoma cells, and this effect could be partly explained by tumoral overproduction of Vascular Endothelial Growth Factor (VEGF). The results indicated that a role of ovarian carcinoma cells in the differentiation and function of DCs could be associated with the immunosuppression and development of ovarian carcinoma.

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma.

OBJECTIVE: To study the frequency of the CD4+CD25+ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. METHODS: The percentages of CD4+CD25+ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4+PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4+CD25+ T cells was detected. RESULTS: CD4+CD25+ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4+CD25+ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4+PBLs from the patients was negative. The percentages of CD4+CD25+ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P=0.001 and 0.001, respectively). CONCLUSION: There is an increasing of the proportion of CD4+CD25+ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.

[Tumor lymphangiogenesis in cervical squamous cell carcinoma and its clinical significance].

OBJECTIVE: To investigate the clinical significance of lymphangiogenesis in cervical cancer. METHODS: Monoclonal podoplanin was used to immunostain the lymphatic microvessels in the paraffin sections of cervical squamous cancer tissues at the I(b) and II(a) stages from 35 cases kept in the archives. Computer-assisted morphometric analysis was used to quantitatively analyze the lymphatic vessels in intratumor and peritumor areas. The association with clinicopathologic data was analyzed. RESULTS: (1) The lymphatic microvessels were larger and denser in the peritumor areas than the intratumor areas. Cancer cells were found in the intratumoral lymphatic vessels of the lymph metastatic patients. (2) The lymphatic vessel density (LVD) of the peritumor areas was (31 +/- 10) vessels/mm(2), significantly greater than that in the intratumor areas [(20 +/- 10) vessels/mm(2), P < 0.01]. The relative lymphatic vessel area (LVA) in the peritumor areas was (0.75 +/- 0.40)%, significantly larger than that of the intratumor areas [(0.19 +/- 0.11)%, P < 0.01]. (3) The intra- and peri-tumoural LVD and LVA in the patients with lymph node metastasis were (29 +/- 7) vessels/mm(2) and (40 +/- 5) vessels/mm(2), and 0.27% +/- 0.10% and 1.23% +/- 0.36% respectively, all greater than those of the patients without lymph node metastasis [(16 +/- 8) vessels/mm(2) and (28 +/- 9) vessels/mm(2), and (0.16 +/- 0.09)%, (0.56 +/- 0.20)% respectively, P < 0.01, < 0.01, < 0.05, and < 0.01]. (4) The peri-tumoral LVA and intra-tumoral LVD and LVA in the patients with histological grade I, II, and III were (0.42 +/- 0.10)%, (9 +/- 3) vessels/mm(2), and (0.06 +/- 0.04)%; (0.77 +/- 0.37)%, (21 +/- 8) vessels/mm(2), and (0.21 +/- 0.09)%; and (0.83 +/- 0.46)%, (22 +/- 11) vessels/mm(2), and (0.21 +/- 0.12)% respectively. There were significant differences among the values of the grade II patients and grade I patients (all P < 0.05) and the grade III patients and grade I patients (all P < 0.05) and there were not significant differences between the values of the grade II patients and the grade III patients (all P > 0.05), III and grade I, no statistic significances between grade II and III. CONCLUSION: Higher lymphangiogenesis in early cervical squamous cell cancer may be associated with the risk of lymph node metastasis.

[Analysis of prognostic factors in patients with cervical squamous cell carcinoma of stage Ib and IIa].

OBJECTIVE: To analyze the prognostic factors in patients with cervical squamous cell carcinoma of stage Ib and IIa treated by surgery, and to investigate their guide roles in available post-operation adjuvant therapy. METHODS: The clinicopathologic records of 306 patients with cervical squamous cell carcinoma of stage Ib and IIa who underwent radical hysterectomy and pelvic lymphadenectomy were retrospectively analyzed, and the prognostic factors were explored by univariate and multivariate methods. Independent prognostic factors were identified by COX proportional hazards regression model. RESULTS: The overall 5-year survival rate of these 306 patients was 78.1%. In univariate survival analysis, the poor prognostic factors included poor differentiation, positive pelvic lymph nodes, deep stromal invasion, parametrial extension, tumor size > or = 4 cm, and lymph vascular space involvement (P < 0.05). While in multivariate survival analysis, the independent prognostic factors included positive pelvic lymph nodes, deep stromal invasion and parametrial extension (P < 0.05). According to the risk factors, all patients were divided into low, intermediate and high risk groups with 5-year survival rate of 90.3%, 83.9% and 43.1%, respectively. In low risk group, no risk factor or only parametrial extension was involved, pelvic recurrence rate was only 2.2%. In intermediate risk group, deep stromal invasion or together with parametrial extension was involved. Pelvic recurrence rate and extra pelvic metastasis rate were 13.5% and 1.3%, respectively. In high risk group, lymph node metastasis or together with other high risk factors was involved, pelvic recurrence rate and extra pelvic metastasis rate were 25.9% and 48.3%, respectively, and 10.3% had both pelvic recurrence and extra pelvic metastasis. CONCLUSIONS: Lymph node metastasis, deep stromal invasion and parametrial extension were independent prognostic factors of stage Ib and IIa cervical squamous cell carcinoma patients undergoing radical hysterectomy and lymphadenectomy. The establishment of prognosis-predicting system based on high risk factors may be helpful to predict the risk of recurrence and metastasis, and guide adjuvant therapy after surgery.

[Expressions of VEGF/VEGFRs and activation of STATs in ovarian carcinoma].

OBJECTIVE: To study the expressions of VEGF/VEGFRs and activation of STATs in ovarian epithelial carcinoma, and to elucidate direct effect of VEGF on ovarian carcinoma cells. METHODS: Tissue samples from 42 women with primary ovarian epithelial carcinoma (OVCA), 29 with begnin ovarian tumor (OVBT) and 11 with normal ovarian tissue (NOV) were collected. LSAB immunohistochemical staining was used to determine the expression of VEGF, VEGFR1, VEGFR2 and activated STATS (P-STAT1, P-STAT3, P-STAT5, P-STAT6) proteins. RESULTS: (1) Semi-quantitative scoring showed that VEGF expression in OVCA was significantly higher than that in OVBT and NOV (P < 0.01). Expressions of VEGFR1 and VEGFR2 were significantly elevated in OVCA, including tumor cells and stromal vascular endothelial cells (P < 0.01, compared with OVBT and NOV). There was no difference in VEGFRs expressions between OVBT and NOV. (2) In OVCA, tumor cells and endothelial cells expressed P-STAT3 and P-STAT5 at significantly higher levels than those in OVBT and NOV (P = 0.000). The staining of P-STAT1 and P-STAT6 was weak with no significant differences among OVCA, OVBT and NOV. (3) Expressions of VEGFR1 and VEGFR2 in endothelial cells were significantly correlated with P-STAT5 and P-STAT3, respectively (P = 0.006 and 0.001). In cancer cells, VEGF, VEGFR1 and VEGFR2 were all significantly correlated with P-STAT3 and P-STAT5 (P = 0.000), but not with P-STAT1 or P-STAT6. CONCLUSION: VEGF affects ovarian carcinoma cells via VEGFRs, and STATs probably participate in intracellular signaling of VEGF.

[Establishment of two-dimensional electrophoresis of uterine leiomyoma for the proteome analysis].

OBJECTIVE: To establish and optimize the two-dimensional electrophoresis (2-DE) of uterine leiomyoma for the proteome analysis. METHODS: Run immobilized pH gradient (IPG)-isoelectric focusing electrophoresis as the first dimension, then vertical SDS-PAGE electrophoresis as the second dimension. A series of important steps,such as sample solubility, volume of loading, electrophoresis parameters and protocol for staining were optimized. RESULTS: The 2-DE patterns of uterine leiomyoma and myometrium with good quality were obtained. CONCLUSION: With optimal condition the two-dimensional electrophoresis of uterine leiomyoma can be obtained.

[Effects of vascular endothelial growth factor on differentiation and function of dendritic cells generated from CD34+ hematopoietic progenitor cells in vitro].

OBJECTIVE: To investigate the effects of vascular endothelial growth factor (VEGF) on differentiation and function of dendritic cells derived from CD34+ hematopoietic progenitor cells. METHODS: After isolation from umbilical cord blood with a high-gradient magnetic cell sorting system (MACS), the CD34+ cells were cultured with a cocktail cytokines for differentiating into dendritic cells (DC). The cells were stimulated by VEGF (25 ng/ml) either at the beginning or at day 9 of culture. Kinetics analysis of cell proliferation was performed during the process of cell culture, and the expression of DC differentiation antigens including CD1alpha, CD83, CD80, CD54 and HLA-DR was examined by flow cytometry. DC function was evaluated by the ability to induce proliferation of allogeneic T cells in mixed lymphocyte reaction (MLR) assay, and the production of IL-12 by ELISA. RESULTS: VEGF added at day 1 of culture induced an increase of total cell numbers by (1.51 +/- 0.23)-folds (P = 0.001). VEGF added at the initial but not the late stage of culture could dramatically down-regulate the expression of CD1a [(33.00 +/- 2.12)% vs (81.20 +/- 6.93)%], CD83 [(42.23 +/- 1.15)% vs (87.98 +/- 7.97)%], CD80 (42.93 +/- 1.32)% vs (94.53 +/- 0.87)%], and HLA-DR [(37.93 +/- 5.30)% vs (74.15 +/- 3.74)%], while obviously up-regulate the expression of CD14. Moreover, the inhibitory effect of VEGF on DC function was confirmed by a reduced ability to induce proliferation of allogeneic T cells and production of IL-12 (P < 0.01). CONCLUSIONS: VEGF could induce the expansion of hematopoietic progenitor cells and inhibit at the early stage their differentiation into mature DC.

[Development of HPV16 positive cervical cancer model in the hu-PBL-SCID mouse and its immunological features].

OBJECTIVE: To develop a HPV16 positive cervical cancer model in the hu-PBL-SCID mouse and investigate its immunological features. METHODS: Thirty-two CB17SCID mice were randomly divided into 4 groups: group A (5 mice) subcutaneously injected with phosphate-buffered saline, group B (5 mice) intraperitoneally injected with human peripheral blood lymphocyte (PBL) for immune reconstruction, group C (11 mice) subcutaneously injected with human cervical carcinoma cell line SiHa, and group D (11 mice) intraperitoneally injected with PBL and subcutaneously injected with SiHa cells after 24 hours of PBL transplantation. The tumor growth, behaviors and status of xenogeneic graft versus host disease (XGVHD) were observed. Human immunoglobulins G (IgG) in mouse serum, the percentage of human CD3(+), CD4(+) and CD8(+) T cells in peripheral blood and spleen, spleen weight, tumor infiltrating lymphocytes and human CD4(+) T cells, and cytotoxicity test of spleen cells were detected. RESULTS: The rate of successful tumor transplantation was 100%. XGVHD was not found. On the 5th day, human IgG level in the group B (0.98 microg/ml +/- 0.20 microg/ml) and group D (1.39 microg/ml +/- 0.25 microg/ml) was significantly higher than that in the group A (t = 7.655, 9.937, both P = 0.000). Human IgG level in group D was significantly higher than that in the group B (t = 3.200, P = 0.006). Only very low levels of human serumal IgG were detected in the group C and group A with no significantly difference. The level of human serumal IgG was gradually elevated in all the humanized SCID mice as the the time after PBL transplantation went on, and was significantly higher than that in non-humanized mice (P < 0.05). The percentage of human CD3(+), CD4(+) and CD8(+) T cells was significantly increased in the peripheral blood and spleen of immunoreconstituted SCID mice. The weight of spleen was markedly increased in the group D. TIL infiltrating in the tumor were remarkable and human CD4(+) T cells was detected by immunohistochemistry in the group D but not in the group C. The spleen cells in the group D displayed stronger cytotoxicity to the target cells (P < 0.05). CONCLUSION: Human immune function can be successfully reconstructed in SCID mouse via intraperitoneally injecting with human PBL, and induce anti-tumor immune response to the transplantated tumor of HPV16 positive cervical cancer.

[Phosphorylation of signal transducer and activators of transcription 3 induced by vascular endothelial growth factor in ovarian carcinoma cell line in vitro].

OBJECTIVE: To observe phosphorylation of signal transducer and activators of transcription 3 (STAT3) in Caov-3 induced by vascular endothelial growth factor (VEGF), and to investigate molecular mechanisms of the effect of VEGF on ovarian carcinoma cells. METHODS: The expressions of phosphorylated STAT3 in Caov-3 induced by VEGF were detected by immunocytochemistry and Western blot methods. Furthermore, the relationship between STAT3 phosphorylation and VEGF stimulation in ovarian carcinoma cells was investigated using a peptide which could specifically bind VEGFR2 and thus block the binding of VEGF to its receptors. RESULTS: With VEGF stimulation, the expressions of phosphorylated STAT3 were significantly higher than those without VEGF stimulation in Caov-3. And 50 ng/ml was the effective dose resulting in a significant increase of phosphorylated STAT3 (2.20 +/- 0.28/1.37 +/- 0.17) compared to 0 ng/ml (0.56 +/- 0.15/0.47 +/- 0.19) (P < 0.001). Translocation into nuclei of activated STAT3 occurred after 30 min, while STAT3 phosphorylation decreased after 60 min of stimulation (0.95 +/- 0.18/0.66 +/- 0.20). A small peptide specific for VEGFR2, blocked the increase of STAT3 phosphorylation induced by VEGF in a dose dependent manner and 80 micro mol/L was the effective dose (P < 0.001). CONCLUSIONS: VEGF could induce STAT3 phosphorylation and translocation into nuclei of activated STAT3 in Caov-3, and the small peptide could effectively inhibit these effects of VEGF. It suggests that STAT3 participates in the VEGF signal transduction mediated by VEGFR2 in ovarian carcinoma cells.

Vascular endothelial growth factor (VEGF) and ovarian carcinoma cell supernatant activate signal transducers and activators of transcription (STATs) via VEGF receptor-2 (KDR) in human hemopoietic progenitor cells.

OBJECTIVE: To investigate the STATs signaling pathway activated by VEGF in human hemopoietic progenitor cells. METHODS: CD34(+) hemopoietic progenitor cells, which isolated from umbilical cord blood, were treated with VEGF or culture supernatant of ovarian carcinoma cell line which could secrete large amount of VEGF, phosphorylation and nuclear translocation of STAT3 and STAT5 were then detected by Western Blot and immunocytochemistry. Expression of VEGFR2/KDR on CD34(+) cells was studied by immunocytochemistry. The specific VEGFR2/KDR heptapeptide antagonist ATWLPPR was used to identify whether the activation of STATs signaling pathway was specifically mediated by VEGFR2/KDR. RESULTS: The concentration of VEGF in SKOV3-supernatant was 4024.84+/- 505.59 pg/ml. CD34(+) progenitor cells could express VEGFR2/KDR. When CD34(+) cells were stimulated by VEGF and SKOV3-supernatant, STAT3 appeared tyrosine-phosphorylation and nuclear translocation, but STAT5 was only phosphorylated, and not translocated. When ATWLPPR was used to block the binding of VEGF to KDR, VEGF and the SKOV3-supernatant failed to activate the phosphorylation of STAT3 and STAT5. CONCLUSIONS: STAT3 may participate in the signal transduction pathways activated by VEGF specifically mediated by VEGFR2/KDR in human hemopoietic progenitor cells, and the aforementioned signaling pathway participated in the interaction of ovarian carcinoma cells and progenitor cells.

[Relationship between interleukin-10 level in ascites of patients with primary ovarian epithelial carcinoma and immune defect in peritoneal cavity].

BACKGROUND & OBJECTIVE: As a multifunctional Th2-cytokine, interleukin-10 (IL-10)plays a major role in the immune response. It is well known that IL-10 is an immunosuppressive cytokine, and participates in the development and progression of various tumors. In this study, we investigated the relationship between the IL-10 level in the ascites of the patients with primary ovarian epithelial carcinoma (POEC) and immune defect in the peritoneal cavity. METHODS: The IL-10 levels in serum and ascites of 32 patients with POEC, in culture supernatants of 4 different ovarian carcinoma cell lines and in serum of 10 patients with ovarian epithelial benign tumor and 10 health women (control) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: (1) IL-10 level in ascites was significantly higher than that in serum of patients with POEC, (159.78+/-51.20 ng/L vs 12.01+/-4.38 ng/L, P=0.000). IL-10 level in serum of the patients with POEC (12.01+/-4.38 ng/L) was significantly higher than that of the patients with benign tumor (3.79+/-2.40 ng/L, P=0.000) and control (4.45+/-2.69 ng/L, P=0.003). There was no significant difference of IL-10 level in serum between ovarian benign tumor and control (P=0.529). (2) IL-10 level in ascites of the patients with POEC was correlated with FIGO stage but not correlated with histological grade. (3) IL-10 was detectable in culture supernatants of 4 different ovarian cancer cell lines (3ao, SKOV3, CAOV3 and OVCAR). CONCLUSION: High level of IL-10 in ascites of the patients with POEC is probably associated with immune defect in their peritoneal cavity, ovarian cancer cells may promote metastasis in peritoneal cavity by secreting IL-10.