Influence of ADAM10 Polymorphisms on Plasma Level of Soluble Receptor for Advanced Glycation End Products and The Association With Alzheimer's Disease Risk.

To determine the role of A disintegrin and metalloproteinase 10 (ADAM10) in genetic susceptibility to Alzheimer's disease (AD) in a representative Chinese sample, we genotyped 362 AD patients and 370 healthy controls for the rs514049A/C and rs653765C/T polymorphisms in the ADAM10 promoter using the SNaPshot technique. We also examined the potential impact of these polymorphisms on the plasma level of soluble receptor for advanced glycation end products (sRAGE), a decoy receptor whose reduction has been associated with a higher risk of AD. Additionally, a meta-analysis was performed using the present study and the largest GWAS from the International Genomics of Alzheimer's Project (IGAP). No significant differences were found in the distributions of genotypes or alleles between AD patients and control subjects. However, age-at-onset stratification analysis revealed that there were significant differences in the genotypes (P = 0.015) and alleles (P = 0.006) of the rs653765 SNP. Furthermore, patients with the rs653765 CC genotype showed a lower ADAM10 level and a faster cognitive deterioration than those in patients with the CT/TT genotype in late-onset AD (LOAD), and the rs653765 CC polymorphism was able to regulate the production of the ADAM10 substrate sRAGE. In contrast, the rs514049 polymorphism was not statistically associated with AD. In the meta-analysis, we observed that both rs514049 (A allele vs. C allele, P = 0.002) and rs653765 (C allele vs. T allele, P = 0.004) were associated with AD risk. The present study indicated that the rs653765 polymorphism might be associated with the risk and development of LOAD; in particular, the risk genotype, CC, may decrease the expression of ADAM10, influencing the plasma levels of sRAGE, and thus may be correlated with the clinical progression of AD.

[Huangjing Zanyu Capsule enhances sperm mitochondrial membrane potential in asthenozoospermia patients].

OBJECTIVE: To observe the effect of Huangjing Zanyu Capsule (HZC) on sperm mitochondrial membrane potential (MMP) in asthenozoospermia patients. METHODS: We assigned 70 asthenozoospermia patients to a treatment group (n = 39) and a control group (n = 31), the former treated with oral HZC at the dose of 4 capsules tid for 3 months while the latter left untreated. We obtained semen parameters from the patients and detected their sperm mitochondrial membrane potentials (MMP) by JC-1 staining and flow cytometry before and after medication, followed by comparison between the two groups. RESULTS: The total effectiveness rate was 71.05% in the treatment group and natural pregnancy was achieved in 3 cases during the medication. A total of 35 patients in the treatment group and 30 controls completed all the laboratory examinations after a 3-month observation. Compared with the controls, the patients treated with HZC exhibited significant improvement after medication in MMP (variation value: ï¼»1.19 ± 10.36ï¼½% vs ï¼»20.28 ± 14.21ï¼½%, P <0.01), total sperm motility (variation value: ï¼»3.46 ± 8.67ï¼½% vs ï¼»20.68 ± 14.12ï¼½%, P <0.01), the percentage of progressively motile sperm (variation value: ï¼»2.26 ± 8.29ï¼½% vs ï¼»17.58 ± 12.73ï¼½%, P <0.01), and the percentage of morphologically normal sperm (variation value: ï¼»0.23 ± 3.48ï¼½% vs ï¼»3.37 ± 3.99ï¼½%, P <0.01). MMP was significantly correlated with total sperm motility (r = 0.69, P <0.01), progressive sperm motility (r = 0.75, P <0.01) and normal sperm morphology (r = 0.26, P <0.01). CONCLUSIONS: Huangjing Zanyu Capsule can enhance sperm mitochondrial membrane potential and sperm mitochondrial function, thus improving total sperm motility, progressive sperm motility and normal sperm morphology. It is safe and effective for the treatment of asthenospermia.

Epigenetic inactivation of BRCA1 through promoter hypermethylation in ovarian cancer progression.

AIM: The BRCA1 promoter is hypermethylated in ovarian cancer patients. We postulated that this hypermethylation might be involved in ovarian cancer progression. METHODS: To confirm our hypothesis, tissue and serum samples were collected from ovarian carcinoma patients and categorized according to tumor stage. Healthy or benign ovarian disease tissue samples and corresponding serum samples were used as controls. Breast and ovarian cancer susceptibility gene 1 (BRCA1) promoter methylation levels were detected by real-time polymerase chain reaction (PCR). Real-time PCR was also used to evaluate BRCA1 gene expression, and Western blot was performed to assay the expression of BRCA1 protein. RESULTS: BRCA1 showed hypomethylation in 30 normal ovarian and 30 benign ovarian tumors, but showed hypermethylation or methylation in ovarian cancer patients. There was also a significant difference in the BRCA1 promoter methylation levels between different ovarian cancer stages. Compared to stage I and the control groups, there were higher BRCA1 promoter methylation frequencies in stage II and III ovarian cancers. BRCA1 methylation correlated with the loss of BRCA1 expression. BRCA1 promoter in stage I tumors showed hypomethylated. CONCLUSION: Promoter hypermethylation may act as a biomarker for sporadic ovarian cancer progression, but is unlikely to be helpful in the early diagnosis of ovarian cancer.

Estrogenic transmembrane receptor of GPR30 mediates invasion and carcinogenesis by endometrial cancer cell line RL95-2.

PURPOSE: The mechanisms underlying the effects of estrogen on endometrial cancer remain undefined. Although the classical mechanism of the action of estrogen involves binding to the estrogen receptors α and ß, and transduction of the signal into the cell, G protein-coupled receptor (GPR) 30 has been shown to mediate nongenomic estrogen signaling. The goal of this study was to determine the role of GPR30 signal in the basic process such as invasion and carcinogenesis of endometrial cancer. METHODS: We downregulated the expression of GPR30 in endometrial cancer cell line RL95-2 by transfection with shGPR30-pGFP-V-RS, a GPR30 antisense expression vector. The cells were then subjected to an MTT assay and a Transwell(®) migration assay. And an animal model was also used to investigate the influence of downregulation of GPR30 on oncogenesis. RESULTS: Downregulation of GPR30 led to reduced growth and invasion by cells treated with 17ß-estradiol. And the capacity of transfected RL95-2 cells to promote tumorigenesis was weakened in vivo. CONCLUSIONS: Our data suggest that, for the endometrial cancer cell line RL95-2, GPR30 plays important roles in mediating the proliferative and invasive effects of estrogen and in tumorigenesis.

Crosstalk between estrogen receptor and mitogen-activated protein kinase signaling in the development and progression of endometrial cancer.

OBJECTIVE: The objectives of the study were to evaluate the role of mitogen-activated protein kinase (MAPK) signaling in normal, hyperplastic, and neoplastic endometrium in relation to estrogen receptor (ER) status and to investigate whether 17ß-estradiol (E2) and tamoxifen (TAM) mediate the proliferation and apoptosis of endometrial cancer cells through the MAPK pathway. METHODS: The expressions of phosphorylated and total extracellular signal-regulated kinases 1/2 (phosphorylated extracellular signal-regulated kinase 1/2 [p-ERK1/2] and total ERK1/2 [t-ERK1/2]) were analyzed with immunohistochemistry in normal, hyperplastic, and neoplastic endometrium. The expression levels of p-ERK1/2 and t-ERK1/2 in RL95-2 and KLE after stimulation by E2, progesterone (P), and TAM were detected by Western blotting. The effects of E2 and TAM in combination with MAPK pathway inhibitors on the growth and apoptosis of endometrial cancer cells were examined by the MTS assay and flow cytometry analysis. RESULTS: The expression level of p-ERK1/2 was significantly associated with the International Federation of Gynecology and Obstetrics stage (P = 0.0072). The ratio of phosphorylated/total ERK1/2 was higher in ER-positive endometrial cancer tissues and cells (P < 0.05). 17ß-Estradiol increased ERK1/2 phosphorylation, and TAM decreased ERK1/2 phosphorylation in endometrial cancer cell lines within 30 minutes (P < 0.05). The MEK1/2 inhibitor, U0126, and the stress-activated protein kinase/c-Jun NH2-terminal kinase inhibitor, SP600125, significantly suppressed the proliferation of human endometrial cancer cell lines RL95-2 and KLE induced by E2 (P < 0.05). The level of TAM-induced apoptosis was greater in KLE than in RL95-2 cells, and the p38 cascade was involved in the TAM-induced apoptosis of both cell lines (P < 0.05). CONCLUSIONS: The cross-talk between MAPK signaling and ER status might exert a key role in progression of endometrial cancer. Furthermore, the effects of E2 or TAM on the proliferation or apoptosis of ER-positive and ER-negative endometrial cancer cells were mediated through distinct MAPK pathways. These mechanisms might contribute to ER-specific differences in MAPK activation for molecular-target therapies in endometrial carcinoma.

Aberrant methylation of breast and ovarian cancer susceptibility gene 1 in chemosensitive human ovarian cancer cells does not involve the phosphatidylinositol 3'-kinase-Akt pathway.

Methylation is an important silencing mechanism of breast and ovarian cancer susceptibility gene 1 (BRCA1) expression in sporadic ovarian cancer. However, the role of BRCA1 methylation in chemotherapy in sporadic ovarian cancer and the related pathways have not been understood completely. This study has determined the roles of BRCA1 hypermethylation in chemotherapy of sporadic ovarian cancer and its related signaling pathways. We used bisulfite sequencing, real-time polymerase chain reaction, and western blotting to check the methylation state and expression levels of BRCA1 of the following cell lines: platinum-sensitive human ovarian cancer cell line COC1, platinum-resistant cell line COC1/DDP, SKOV-3, and 5-Aza-dC treated COC1. The cisplatin sensitivity of ovarian cancer cells was examined by MTS (methyl-thiazol tetrazolium) assay. Tumorigenicity in vivo and DDP-based chemosensitivity were compared among the above cells. Phosphatidylinositol 3'-kinase (PI3K)-Akt pathway activation in ovarian cancer cells was studied by western blotting. The frequency of BRCA1 methylation in the COC1 cell line was higher than in COC1/DDP and SKOV-3 cell lines, whereas the mRNA and protein expression of BRCA1 were lower than in the COC1/DDP and SKOV-3 cell lines. DNA demethylation decreased the chemosensitivity of COC1 cells and partially increased the expression levels of BRCA1. The activation of the PI3K-Akt pathway was low in ovarian cancer cells. Our results indicate that hypermethylation of BRCA1 might play an important role in the chemosensitivity of ovarian cancer, and that the PI3K-Akt pathway is not involved in this response.

Kallikrein 4 overexpression in endometrial carcinoma and upregulation by estrogen via mitogen-activated protein kinase signal pathway.

OBJECTIVE: The aim of this study was to investigate the expression of kallikrein 4 (KLK4) and the potential signal pathway through which estrogen up-regulates KLK4 in endometrial cancer. METHODS: The expression of KLK4 was analyzed in 15 human normal endometrium, 13 hyperplasia endometrium, and 68 endometrioid adenocarcinoma by immunohistochemistry. After exposure to 17beta-estradiol and/or to the mitogen-activated protein kinase (MAPK) inhibitor U0126 and to the PI3K inhibitor LY294002, the expression of KLK4 in the endometrial cancer cell lines KLE and RL95-2 was detected with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: The expression of KLK4 protein was higher in endometroid endometrial cancer than in hyperplasia or normal endometrium (P < 0.001). Immunohistochemical staining revealed that 92.6% (63/68) of endometrial adenocarcinoma, 61.5% (8/13) of hyperplasia endometrium, and 26.7% (4/15) of normal endometrium were positive for KLK4 protein. The expression of KLK4 was significantly associated with tumor grade (P = 0.004), but not with ER status (P = 0.532). Quantitative reverse transcriptase PCR and Western blot analysis showed that estrogen can up-regulate the expression of KLK4 in endometrial cancer cell lines KLE and RL95-2, and the up-regulation effect of 17beta-estradiol on KLK4 can be inhibited by U0126 in the 2 endometrial cancer cell lines but not by LY294002. CONCLUSIONS: Kallikrein 4 is a new nuclear protein, and estrogen up-regulates the expression of KLK4 by activating the MAPK pathway in endometrial cancer cell lines, which may play an important role in the development of endometrial cancer.

Is caspase inhibition a valid therapeutic strategy in cryopreservation of ovarian tissue?

PURPOSE: The aim of this study is to determine whether inclusion of caspase inhibitor can improve the efficacy of cryopreservation of ovarian tissue. METHODS: Mice were randomly assigned to the Group A (fresh control group) Group B (inclusion of caspase inhibitor) and Group C (non-inclusion of caspase inhibitor). Ovarian tissue in Group B and Group C was vitrified-thawed. TUNEL assay and Bax protein detection were measured after cryopreservation. The mice in all groups received autotransplantation. The number of days before the resumption of estrous cycles was measured daily from the 5th day after surgery, and the percentage of cells expressing PCNA in grafts was measured one month following transplantation. RESULTS: The incidence of TUNEL positive follicles in Group B was significantly higher than that in Group C. Similarly, the percentage of follicles expressing Bax protein in Group B was significantly higher than that in Group C. The number of days before the resumption of estrous cycles in Group B was significantly less than that in Group C. In addition, the percentage of follicular and stromal cells expressing PCNA of grafts in Group B was significantly higher than that in Group C. CONCLUSIONS: The global caspase inhibitor Z-VAD-FMK decreases the incidence of apoptosis of ovarian tissue induced by cryopreservation, and inclusion of caspase inhibitor improves the efficacy of cryopreservation of ovarian tissue.

Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway.

The regulatory mechanism of endometrial carcinoma and the signal transduction pathways involved in hormone action are poorly defined. It has become apparent that the G protein-coupled receptor (GPR) 30 mediates the non-genomic signaling of 17beta-estradiol (E2). Here we show that GPR30 is highly expressed in endometrial cancer tissues and cancer cell lines and positively regulates cell proliferation and invasion. GPR30 expression was detected in 50 human endometrial carcinomas. The transcription level of GPR30 was significantly higher in the tissue of endometrial carcinoma than in normal endometrium (P < 0.05). Immunohistochemical assays revealed that the positive expression rate of GPR30 protein in endometrial carcinoma tissue (35/50, 70%) was statistically higher than in normal endometrium tissue (8/30, 26.67%) (chi2 = 14.16, P = 0.0002). GPR30 overexpression was correlated with high-grade endometrial carcinoma. GPR30 expression was also found in two human endometrial cancer cell lines: RL95-2 (estrogen receptor positive) and KLE (estrogen receptor negative). The roles of GPR30 in proliferative and invasive responses to E2 and G1, a non-steroidal GPR30-specific agonist, in RL95-2 and KLE cell lines were then explored. We showed that E2 and G1 could initiate the MAPK/ERK mitogen-activated protein kinase pathway in both cell lines. What's more, E2 and G1 promoted KLE and RL95-2 proliferation and stimulated matrix metalloproteinase production and activity via the GPR30-mediated MEK/ERK mitogen-activated protein kinase pathway, as well as increased interleukin-6 secretion. These findings suggest that GPR30-mediated non-genomic signaling could play an important role in endometrial cancer.

Sucrose affecting successful transplantation of vitrified-thawed mouse ovarian tissues.

PURPOSE: The aim of this experiment is to detect effects of varying levels of sucrose on vitrified ovarian tissues. METHODS: Ovarian tissues of mice were vitrified-thawed. Mice were randomly assigned to the fresh control group and experimental groups. According to different concentration of sucrose in vitrification solution, the experimental groups were randomly divided into Group I (0.2 M sucrose), Group II (0.4 M sucrose), Group III (0.8 M sucrose) and Group IV (1.6 M sucrose). Cytology was followed throughout the oophorectomy and transplantation period. Hormone levels and density of follicle were measured 1 month after transplantation. RESULTS: The number of days before the resumption of estrous cycles in control group was significantly smaller than those in all of experimental groups. The serum estradiol levels of mice and the follicular density of ovarian grafts in control group were significantly higher than those in all of experimental groups. In addition, the number of days before the resumption of estrous cycles in Group II and Group III were smaller than those in Group I and Group IV. The serum estradiol levels of mice and the follicular density of ovarian grafts in Group II and Group III were significantly higher than those in Group I and Group IV. However, no difference was observed in the number of days before the resumption of estrous cycles and the serum estradiol levels between Group II and Group III. A similar follicular density was also observed in Group II and Group III. CONCLUSION: Sucrose concentration of 0.4 M or 0.8 M in cryoprotective media is suitable for vitrifying mouse ovarian tissues.

[Anoikis-suppression and invasion induced by tyrosine kinase receptor B in OVCAR3 ovarian cancer cells].

OBJECTIVE: To study the relationship between tyrosine kinase receptor B (TrkB) expression and anoikis-suppression and invasion in OVCAR3 ovarian cancer cells. METHODS: The expression of TrkB mRNA in OVCAR3 ovarian cancer cells under two culture conditions: adhesive cells and cell-spheroids were evaluated by RT-PCR and real-time PCR. The relationship between TrkB expression and anoikis-suppression of OVCAR3 ovarian cancer cells was examined by RNA interference (RNAi) technic, anchorage independent culture and fluorescence-activated cell sorting analysis. The difference in invasion and metastatic ability of OVCAR3 cells under two culture conditions and with or without TrkB silenced by small interfering RNA (siRNA) was investigated by matrigel invasion assay and in vivo studies. RESULTS: The expression of TrkB mRNA was highest in OVCAR3 ovarian cancer cells, 0.0240 +/- 0.0017, compared with the other three cell lines, 0.0030 +/- 0.0006, 0.0027 +/- 0.0009 and 0.0087 +/- 0.0003 respectively, and the expression in OVCAR3 multicellular spheroids was significantly higher than that in cells under monolayer adhesive culture, 0.0437 +/- 0.0021 versus 0.0240 +/- 0.0017 (P < 0.01). TrkB mediated anoikis-suppression in OVCAR3 ovarian cancer cells. OVCAR3 multicellular spheroids had a higher invasion ability than OVCAR3 cells under monolayer adhesive culture, and the penetrating cells of the two groups were 71.8 +/- 0.8 and 47.7 +/- 0.8 respectively (P < 0.01). The metastatic ability of OVCAR3 cells was attenuated when TrkB was silenced, and the volume of the tumors developed by OVCAR3 adhesive cells and OVCAR3 adhesive cells with TrkB silenced were (16.3 +/- 4.7) mm(3) and (6.0 +/- 1.4) mm(3) respectively (P < 0.01). CONCLUSION: As an anoikis-suppressor, TrkB may increase the invasion and metastasis of OVCAR3 ovarian cancer cells.

[Study of differential gene expression in different stage human endometrial cancer].

OBJECTIVE: To study the gene expression profile of different stage endometrial cancer and identify genes related to the progression and metastasis of endometrial cancer. METHODS: cDNA microarray was used to analyze the differentially expressed genes between different stage endometrial cancers. The hierarchical cluster analysis was applied to the gene expression profile of 32 endometrial cancers. RESULTS: Twelve differentially expressed genes were identified between different stage endometrial cancers. The conformity rate was 66% between the result of hierarchical cluster analysis and the operative-histological stages of International Federation of Gynecology and Obstetrics (FIGO). CONCLUSION: Genes related to the endometrial cancer progression and metastasis can be identified by differential gene expression profile with cDNA microarray and high-risk endometrial cancer may be distinguished before surgery by hierarchical cluster analysis.

[Establishment of the model of heterotopic uterine transplantation in syngeneic rats].

OBJECTIVE: To develop a surgical method for establishment of a heterotopic uterine transplantation model in syngeneic rats and evaluate its feasibility. METHODS: Thirty pairs of Wistar rats aged 8 - 10 weeks were used as donor and recipient. Heterotopic uterine transplantation was conducted, and the duration of the operation was recorded. The recipient rats were killed 7 days after surgery. The morphology of the transplanted uteri was evaluated. RESULTS: Thirty heterotopic uterine transplantations were conducted. The survival rate increased from 40% (6/15) in the first 15 pairs of recipient rats to 75% (12/15) in the last 15 pairs of recipient rats. Among the 18 living recipient rats, 15 transplanted uteri were viable. The viable rate of uterine transplantation was 83%. Compared with the first 15 pairs of rats, the duration of donor procedures, recipient procedures vascular anastomosis and the total time of the surgery decreased to (65 +/- 10) min, (89 +/- 22) min, (36 +/- 8) min and (154 +/- 23) min respectively in the last 15 pairs of rats. CONCLUSION: It is feasible to establish the model of heterotopic uterine transplantation in Wistar rats.