Antiangiogenesis therapy of endometriosis using PAMAM as a gene vector in a noninvasive animal model.

OBJECTIVE: To evaluate the characteristics and antiangiogenic effects of endostatin-loaded PAMAM on endometriosis in a noninvasive animal model. MATERIALS AND METHODS: A noninvasive animal model was established by injecting adenovirus-GFP transfected endometrial stromal and glandular epithelial cells subcutaneously into nude mice. Endostatin-loaded PAMAM was prepared and identified by transmission electron microscopy. For in vitro studies, the DNA protection and cytotoxicity of PAMAM were investigated and compared with Lipofectamine 2000. For in vivo study, endostatin-loaded PAMAM was injected into the noninvasive model and evaluated by continuously observing the fluorescent lesion, lesion weight, microvessel density and VEGF immunostaining. RESULTS: Compared with Lipofectamine 2000, PAMAM and HC PAMAM-ES group, MC PAMAM-ES group and LC PAMAM-ES group demonstrated a better stromal cells protective such that MC PAMAM-ES group of CCK8 was 0.617 ± 0.122 at 24 hr and 0.668 ± 0.143 at 48 hr and LC PAMAM-ES group of CCK8 was 0.499 ± 0.103 at 24 hr and 0.610 ± 0.080 at 48 hr in stromal cells (P < 0.05) but similar cytotoxicity in glandular epithelial cells in vitro. After 16 hrs of digestion, DNA decreased slightly under the protection of PAMAM. Endostatin-loaded PAMAM of HD PAMAM-ES group and LD PAMAM-ES group inhibited the growth of the endometriotic lesion in vivo at days 15, 20, 25 and 30 detected by noninvasive observation after injecting one dose endostatin of various medicines into the endometrial lesion in each mouse on day 10 (P < 0.05) and confirmed by lesion weight at day 30 with HD PAMAM-ES group being 0.0104 ± 0.0077 g and LD PAMAM-ES group being 0.0140 ± 0.0097 g (P < 0.05). Immunohistochemistry results showed that endostatin-loaded PAMAM reduced the microvessel density 3.8 ± 2.4 especially in HD PAMAM-ES group in the lesion (P < 0.05). CONCLUSION: Endostatin-loaded PAMAM inhibits the development of endometriosis through an antiangiogenic mechanism and can be observed through the noninvasive endometriosis model.

Lentivirus-mediated RNA interference targeting the long noncoding RNA HOTAIR inhibits proliferation and invasion of endometrial carcinoma cells in vitro and in vivo.

OBJECTIVE: The overexpression of long noncoding RNA HOTAIR is associated with various aggressive solid carcinomas. However, its relationship with endometrial carcinoma has not been reported. The present study aimed to investigate the expression of the long noncoding RNA HOTAIR in endometrial carcinoma, its relationship with the carcinoma's clinicopathologic features, and the biological function of HOTAIR in regulating endometrial cancer cell proliferation and invasion in vitro and in vivo. METHODS: The expression of HOTAIR was detected in different tissues and cell lines by real-time PCR. Lentivirus-mediated HOTAIR-specific shRNAvectors were transfected into endometrial cancer HEC-1A cells. Cell proliferation and colony formation were examined by CCK-8 assays and colony formation assays, respectively. Invasion and migration were examined by Transwell assays. Flow cytometry assay was used to examine the cell cycle. In addition, xenograft model assays were performed to analyze the growth of endometrial cancer cells in vivo. RESULTS: Our data showed that HOTAIR expression was higher in endometrial cancer cells and tissues than in normal endometrial tissues. HOTAIR expression was closely related to the tumor stage (P = 0.045), myometrial invasion (P = 0.014), and lymph node metastasis (P = 0.033). The down-regulation of HOTAIR resulted in a significant inhibition of cell proliferation, migration, and invasion and in cell cycle arrest at the G0/G1 phase. Furthermore, HOTAIR depletion significantly suppressed the endometrial cancer tumorigenesis in vivo. CONCLUSIONS: This study is the first to suggest that HOTAIR plays an important role in the carcinogenesis of endometrial cancer. Targeting HOTAIR may be a novel therapeutic strategy for endometrial cancer.

A red fluorescent nude mouse model of human endometriosis: advantages of a non-invasive imaging method.

OBJECTIVES: To establish red fluorescent human endometriosis lesions in a nude mouse model and dynamically and non-invasively to compare intraperitoneal and subcutaneous injection models. STUDY DESIGN: Primary cultures of endometrial stromal cells (ESCs) and epithelial cells (EECs) isolated from 24 patients with a normal uterine cavity were transfected with 2.5×10(8) (Group 1) and 1.25×10(8) (Group 2) plaque-forming units (PFU) of adenovirus encoding red fluorescent protein (Ad-RFP). Transfection efficiencies, fluorescence intensity and apoptosis rate of the two types of cells were compared in vitro. A mixture of 2.5×10(8) PFU Ad-RFP-infected approximately 400 EECs cell mass and 2×10(6) ESCs for 36h was injected individually into 24 female nude mice subcutaneously (Group A) or intraperitoneally (Group B). From Day 5 after injection, an in vivo imaging system (IVIS) was used to non-invasively observe and compare the lesions of the two groups every week until Day 33. Specifically, the fluorescent intensity, positive rates, persistence time and lesion weight in the implanted human endometriosis lesions were compared. A parametric Student's t-test and two-way analysis of variance were used for statistical analysis. RESULTS: Compared with 1.25×10(8) PFU RFP, a titre of 2.5×10(8) PFU RFP ESCs and EECs incubated for 36h exhibited higher transfection efficiencies and higher fluorescence intensities in vitro. In vivo imaging of the fluorescent human endometriosis lesions originating from an RFP titre of 2.5×10(8) PFU showed that the intensity and lesion weight in Group A were significantly higher than in Group B. However, the two groups had the same RFP-positive rates and fluorescence persistence. The structure of each lesion was evaluated by immunohistochemistry to confirm its human endometrial origin. CONCLUSIONS: The red fluorescent human endometriosis model established by subcutaneously injecting 2.5×10(8) PFU RFP-transfected stromal cells and epithelial cells into nude mice had a higher fluorescent positive rate from Day 5, higher intensity and weight but the same persistence as the intraperitoneal injection model.

Predictive value of excision repair cross-complementing rodent repair deficiency complementation group 1 and ovarian cancer risk.

OBJECTIVE: We aimed to analyze the association between excision repair cross-complementing rodent repair deficiency complementation group 1 (XRCC1) and ovarian cancer risk. METHODS: We performed a hospital-based case-control study with 155 cases and 313 controls in China. All Chinese cases with newly diagnosed primary ovarian cancer between May 2005 to May 2010 in our hospital were invited to participate within 2 months of diagnosis. Controls were randomly selected from people who requested general health examinations in the same hospital during the same period. SNPs in EXCC1, ERCC1 C8092A and ERCC1 T19007C, were analyzed by PCR-RFLP method. RESULTS: We observed a non-significantly increased risk of ovarian cancer among individuals with ERCC1 8092TT compared with those with the 8092CC genotype (adjusted OR=1.55, 95% CI%=0.74-2.97). Moreover, 19007TT genotype carriers also showed a non-significant increased risk of ovarian cancer over those with the 19007CC genotype (adjusted OR=1.78, 95% CI%=0.91-3.64). CONCLUSION: Our firstly investigation of links between polymorphisms in the ERCC1 gene and the risk of ovarian cancer in Chinese population demonstrated no significant association. Further large sample studies in Chinese populations are needed.

[Effect of levonorgestrel-releasing intrauterine system combined with GnRH analogue for treatment of large adenomyosis].

OBJECTIVE: To evaluate the effects of levonorgestrel-releasing intrauterine system (LNG-IUS) combined with GnRH analogue (GnRH-a) in the treatment of adenomyosis with uterine body enlargement. METHODS: Twelve women (mane age 40.3 years) with adenomyosis and uterine cavity depth over 11 cm received injections of GnRH-a every 4 weeks, and after the uterine cavity depth was reduced to below 10 cm, LNG-IUS was deployed. VAS pain score, PBAC bleeding score, uterine volume, and hemoglobin levels of the women were measured before the treatment and at 6 and 12 months after LNG-IUS placement. RESULTS: The VAS pain score was significantly lowered at 6 and 12 month after LNG-IUS placement (P<0.05), and the PBAC bleeding score also showed significant reductions (P<0.05). The uterine volume decreased significantly at 6 and 12 months after LNG-IUS placement as compared with that before the treatment, but was significantly greater at 6 month in comparison with that at the time of LNG-IUS placement (P<0.05). Serum hemoglobin levels underwent significant increments after LNG-IUS placement (P<0.05). CONCLUSION: LNG-IUS combined with GnRH analogue injection can be effective in the treatment of adenomyosis with dysmenorrhea and hypermenorrhea.

[Expression and methylation of adenomatous polyposis coli gene in endometrioid adenocarcinoma].

BACKGROUND & OBJECTIVE: Endometrial carcinoma is a common malignant tumor of female reproductive system, with an increasing incidence in China. Adenomatous polyposis coli (APC) gene, a tumor suppressor gene, is expressed in many tissues, and has a certain relationship with ovarian cancer. This study was to observe the expression and DNA methylation of APC gene in endometrioid adenocarcinoma, and explore its correlations to the occurrence and development of this disease. METHODS: The methylation, mRNA and protein expression of APC gene were detected by methylation-specific polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in 30 specimens of normal proliferative endometrium, 30 specimens of atypical hyperplastic endometrium and 60 specimens of endometrioid adenocarcinoma. RESULTS: The methylation rate of APC gene was significantly higher, the positive rates of APC mRNA and protein were significantly lower in endometrioid adenocarcinoma than in atypical hyperplastic endometrium and normal proliferative endometrium (65.0% vs. 33.3% and 23.3%, 33.3% vs. 63.3% and 73.3%, 30.0% vs. 50.0% and 66.7%,P<0.05). There was no significant difference between atypical hyperplastic endometrium and normal endometrium (P>0.05). APC methylation was positively correlated to APC mRNA expression. CONCLUSION: The expression and DNA methylation of APC gene are certainly related with the occurrence and development of endometrioid adenocarcinoma.

Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.

BACKGROUND: In an attempt to allow for acquisition of oocyte cytoplasmic maturation, PDE3 specific inhibitor, cilostamide and adenylate cyclase activator, forskolin were used to extend pre-maturation culture of immature human oocytes. METHODS: Cumulus-oocyte complexes retrieved from unstimulated ovaries were continuously cultured under 20 microM cilostamide or 50 microM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication (GJC) and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) were subject to intracytoplasmic sperm injection and embryos were cultured for five more days. RESULTS: Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes. Combined treatment of cilostamide and forskolin significantly lowered the rates of germinal vesicle breakdown (GVBD) at 6, 12, 24 or 48 h after meiotic inhibitory culture, when compared with the control (all P < 0.05). A delay of 6 h for the loss of GJC was also observed under the combined treatment of cilostamide and forskolin. The fertilization rate was significantly higher under the combined treatment of cilostamide and forskolin than that of the control. Although the rates of oocyte maturation and embryo cleavage were similar among groups, there was a slight but non-significant increase in blastocyst formation rate with the treatment of cilostamide and forskolin. CONCLUSIONS: Combined treatment of cilostamide and forskolin positively influences oocyte developmental competence by exhibiting a synergistic effect on the prevention of GJC loss and resumption of meiosis.