Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer.

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells.

Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

3'-epi-12ß-hydroxyfroside-mediated autophagy degradation of RIPK1/RIPK3 necrosomes leads to anergy of immunogenic cell death in triple-negative breast cancer cells.

Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12ß-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.

Immunotherapy combining tumor and endothelium cell lysis with immune enforcement by recombinant MIP-3α Newcastle disease virus in a vessel-targeting liposome enhances antitumor immunity.

BACKGROUND: Several agents for oncolytic immunotherapy have been approved for clinical use, but monotherapy is modest for most oncolytic agents. The combination of several therapeutic strategies through recombinant and nanotechnology to engineer multifunctional oncolytic viruses for oncolytic immunotherapy is a promising strategy. METHODS: An endothelium-targeting iRGD-liposome encapsulating a recombinant Newcastle disease virus (NDV), which expresses the dendritic cell (DC) chemokine MIP-3α (iNDV3α-LP), and three control liposomes were constructed. MIP-3α, HMGB1, IgG, and ATP were detected by western blotting or ELISA. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells were analyzed by flow cytometry. The antitumor efficiency was investigated in B16 and 4T1 tumor-bearing mice. Immunofluorescence and immunohistochemistry were used to observe the localization of liposomes, molecular expression and angiogenesis. Synergistic index was calculated using the data of tumor volume, tumor angiogenesis and tumor-infiltrating lymphocytes. RESULTS: Compared with NDV-LP, treatment with iNDV3α-LP and NDV3α-LP induced stronger virus replication and cell lysis in B16 and 4T1 tumor cells and human umbilical vein endothelial cells (HUVECs) with the best response observed following iNDV3α-LP treatment. B16 and 4T1 cells treated with iNDV3α-LP produced more damage-associated molecular pattern molecules, including secreted HMGB1, ATP, and calreticulin. Moreover, iNDV3α-LP specifically bound to αvß3-expressing 4T1 cells and HUVECs and to tumor neovasculature. Tumor growth was significantly suppressed, and survival was longer in iNDV3α-LP-treated B16-bearing and 4T1-bearing mice. A mechanism study showed that iNDV3α-LP treatment initiated the strongest tumor-specific cellular and humoral immune response. Moreover, iNDV3α-LP treatment could significantly suppress tumor angiogenesis and reverse the tumor immune suppressive microenvironment in both B16-bearing and 4T1-bearing mice. CONCLUSIONS: In this study, iNDV3α-LP had several functions, such as tumor and vessel lysis, MIP-3α immunotherapy, and binding to αvß3-expressing tumor and its neovasculature. iNDV3α-LP treatment significantly suppressed tumor angiogenesis and reversed the tumor immunosuppressive microenvironment. These findings offer a strong rationale for further clinical investigation into a combination strategy for oncolytic immunotherapy, such as the formulation iNDV3α-LP in this study.

Engineered porous/hollow Burkholderia pseudomallei loading tumor lysate as a vaccine.

Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.

A recombinant oncolytic Newcastle virus expressing MIP-3α promotes systemic antitumor immunity.

BACKGROUND: The oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an excellent gene-engineering vector. The macrophage inflammatory protein-3α (MIP-3α) is a specific chemokine for dendritic cells (DCs). Thus, we constructed a recombinant NDV expressing MIP-3α (NDV-MIP3α) as an in vivo DC vaccine for amplifying antitumor immunities. METHODS: The recombinant NDV-MIP3α was constructed by the insertion of MIP-3α cDNA between the P and M genes. Western blotting assay and ELISA were used to detect MIP-3α, HMGB1, IgG, and ATP in the supernatant and sera. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells (eg, DCs) were analyzed by flow cytometry. The antitumor efficiency of NDV-MIP3α was observed in B16 and CT26 tumor-bearing mice. Immunofluorescence and immunohistochemistry were applied to observe the ecto-calreticulin (CRT) and intratumoral attraction of DCs. Adoptive transfer of splenocytes and antibodies and depletion of T-cell subsets were used to evaluate the relationship between antitumor immunities and the role of the T-cell subtype. RESULTS: The findings show that NDV-MIP3α has almost the same capabilities of tumor lysis and induction of ICD as the wild-type NDV (NDV-WT). MIP-3α secreted by NDV-MIP3α could successfully attract DCs in vitro and in vivo. Both B16 and CT26 cells infected with NDV-MIP3α could strongly promote DC maturation and activation. Compared with NDV-WT, intratumoral injection of NDV-MIP3α and the adoptive transfer of T lymphocytes from mice injected with NDV-MIP3α resulted in a significant suppression of B16 and CT26 tumor growth. The NDV-MIP3α-induced production of tumor-specific cellular and humoral immune responses was dependent on CD8+ T cells and partially on CD4+ T cells. A significant reversion of tumor microenvironments was found in the mice injected with NDV-MIP3α. CONCLUSIONS: Compared with NDV-WT, the recombinant NDV-MIP3α as an in vivo DC vaccine demonstrates enhanced antitumor activities through the induction of stronger system immunities and modulation of the tumor microenvironment. This strategy may be a potential approach for the generation of an in vivo DC vaccine.

Toxicarioside O Inhibits Cell Proliferation and Epithelial-Mesenchymal Transition by Downregulation of Trop2 in Lung Cancer Cells.

Toxicarioside O (TCO), a natural product derived from Antiaris toxicaria, has been identified to be a promising anticancer agent. In this study, we aimed to investigate the effect of TCO on the proliferation and epithelial-mesenchymal transition (EMT) of lung cancer cells and its molecular mechanisms. Here, we indicated that TCO inhibits the proliferation of lung cancer cells both in vitro and in vivo. Our results demonstrated that TCO induces apoptosis in lung cancer cells. Moreover, we found that TCO suppresses EMT program and inhibits cell migration in vitro. Mechanistically, TCO decreases the expression of trophoblast cell surface antigen 2 (Trop2), resulting in inhibition of the PI3K/Akt pathway and EMT program. Overexpression of Trop2 rescues TCO-induced inhibition of cell proliferation and EMT. Our findings demonstrate that TCO markedly inhibits cell proliferation and EMT in lung cancer cells and provides guidance for its drug development.

Binocular vision and abnormal head posture in children when watching television.

AIM: To determine the association between the binocular vision and an abnormal head posture (AHP) when watching television (TV) in children 7-14y of age. METHODS: Fifty normal children in the normal group and 52 children with an AHP when watching TV in the AHP group were tested for spherical equivalents, far and near fusional convergence (FC) and fusional divergence (FD) amplitudes, near point of convergence, far and near heterophoria, accommodative convergence/ accommodation ratio and stereoacuity. The values of these tests were compared between the two groups. The independent t test was applied at a confidence level of 95%. RESULTS: The far and near FC amplitudes and far FD amplitudes were lower in the AHP group (the far FC amplitudes: break point 13.6±5.4(Δ), recovery point 8.7±5.4(Δ). The near FC amplitudes: break point 14.5±7.3(Δ), recovery point 10.3±5.1(Δ). The far FD amplitudes: break point 3.9±2.7(Δ), recovery point 2.6±2.3(Δ)) compared with those in the normal group (the far FC amplitudes: break point 19.1±6.2(Δ), recovery point 12.4±4.5(Δ). The near FC amplitudes: break point 22.3±8.0(Δ), recovery point 16.1±5.7(Δ). The far FD amplitudes: break point 7.0±2.1(Δ), recovery point 4.6±1.9(Δ)). Other tests presented no statistically significant differences. CONCLUSION: An association between the reduced FC and FD amplitudes and the AHP in children when watching TV is proposed in the study. This kind of AHP is considered to be an anomalous manifestation which appears in a part of puerile patients of fusional vergence dysfunction.

miR-342-3p suppresses proliferation, migration and invasion by targeting FOXM1 in human cervical cancer.

FOXM1 is a well-established oncogenic factor that has been reported to be involved in multiple biological processes including cell proliferation, growth, angiogenesis, migration and invasion. It can also be regulated by miRNAs. In this study, we reported that FOXM1 is directly targeted by miR-342-3p, which is down-regulated along with its host gene, EVL, in human cervical cancer tissues compared to the adjacent normal tissues. Functional studies suggested that the overexpression of miR-342-3p inhibits cell proliferation, migration and invasion in cervical cell lines. FOXM1 is upregulated and negatively correlates with miR-342-3p in cervical cancer tissues, and the overexpression of FOXM1 rescues the phenotype changes induced by the overexpression of miR-342-3p.

Integration sites and genotype distributions of human papillomavirus in cervical intraepithelial neoplasia.

OBJECTIVES: To analyse HPV integration prevalence and genotype distributions in cervical intraepithelial neoplasia (CIN) in east part of China, furthermore to assess preferential sites for common HPV integrations and provide baseline information for cervical abnormality screening and prevention. METHODS: Integration of HPV in 113 paraffin-embedded cervical intraepithelial neoplasia samples was assessed using Gencap technology in Key Laboratory of Biotechnologies in BGI-Shenzhen. RESULTS: 64 samples were HPV-integrated and as the cervical lesions increased, the integration rate became higher significantly (P=0.002). Fifteen different HPV genotypes were detected, 14 high-risk (16, 18, 31, 33, 51, 52, 56, 58, 66, 68) and 1 low-risk (11). The most common genotypes were HPV-16, 58, 33, 52, 66, and 56. Thirteen patients had co-integration involving mainly HPV-16 and 58. The frequency of HPV gene disruption was higher in L1 and E1 genes than in other regions of the viral genomes. CONCLUSION: Some 56.6% of CIN lesions in Qingdao had HPV integrations, and 67.2% of HPV-integrated patients were HPV-16 and 58, more prone to be integrated in younger patients below 45 years old. There exist preferential sites for HPV-16 and HPV-58 integration, and they are more likely to be disrupted in the L1 and E1 loci.

Effect of Salvia miltiorrhiza on retinopathy.

OBJECTIVE: To explore the effect of Salvia miltiorrhiza on diabetic retinopathy (DR). METHODS: Diabetic mice of natural incidence type with monogenic inheritance were selected. Alloxan was injected into the caudal vein of mice once to induce DR. The structural changes of retina tissue in normal mice, DR mice and mice with high, medium and low dose of Salvia miltiorrhiza injection were observed under microscope. Then the blood glucose concentration and malonaldehyde (MDA) content were detected. RESULTS: There were some microaneurysms in retina of DR group, number of gangliocyte was decreased significantly, and cells were sparse and in disorder. After modeling, the blood glucose level of high-dose Salvia miltiorrhiza group (SM III group) was significantly different from DR group (P<0.01). Till the tenth week, the blood glucose level of all SM groups was decreased significantly compared with DR group (P<0.01). The effective rates of three SM groups were 93.8%, 76.4% and 50.3%, respectively. After ten weeks, MDA content of DR group was significantly higher than those of the normal control group and SM group (P<0.01), and medium and low dose SM groups had significantly higher MDA than that of normal control group (P<0.01). CONCLUSIONS: Salvia miltiorrhiza had certain protective effect on DR mice through the blood-ocular barrier.

[Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA' against CLDN-3,4-overexpressing ovarian cancer cells].

OBJECTIVE: The aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer. METHODS: CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. RESULTS: Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml. CONCLUSIONS: The C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

[Association of EGFR expression with angiogenesis and chemoresistance in ovarian carcinoma].

OBJECTIVE: To clarify the association of EGFR expression with angiogenesis and chemoresistance in ovarian cancer. METHODS: Immunohistochemical PV-6000 staining was used to detect the expression of EGFR, LRP protein and MVD in 102 ovarian tumor specimens. RESULTS: EGFR, LRP positive rates and MVD in borderline and malignant ovarian specimens were significantly higher than those in the normal and benign ones (P < 0.01). EGFR positive expression rate in stage III-IV carcinoma tissues, poor differentiation and with ascites was higher than that in stage I-II carcinomas of well differentiation and without ascites (P < 0.05). MVD was related to histological grade, residual tumor and ascites, LRP positive expression had no correlation with the clinicopathologic parameters (P > 0.05). The effective rate of chemotherapy in patients with EGFR and LRP-positive expression were 57.1% and 53.7%, respectively, significantly lower than that in cases with EGFR and LRP-negative expression (85.0% and 90.9%, P < 0.05). In the 64 cases with complete data, the three-year survival rate was 53.0%. The survival time was shorter in the cases with EGFR and LRP-positive expression, poor differentiation, ascites and chemoresistance (P < 0.01), and only LRP-positive expression and chemotherapeutic effect were independently related to survival time (P < 0.05). There was a correlation between EGFR and MVD (r = 0.548, P < 0.01), EGFR and LRP positive expression (P = 0.020). CONCLUSION: The expression of EGFR in ovarian cancer is related to angiogenesis and chemoresistance. EGFR and LRP-positive expression are related to chemoresistance, and detection of the two proteins may be helpful in guiding chemotherapy choice for ovarian cancer. LRP-positive expression and chemotherapeutic effect may be independent prognostic factors.

[Correlations of EGFR and LRP to chemotherapy resistance and prognosis of ovarian cancer].

BACKGROUND & OBJECTIVE: Abnormal expression and activation of epidermal growth factor receptor (EGFR), which is closely related to the recurrence and poor prognosis of ovarian cancer, can promote chemotherapy resistance of tumor cells. Lung resistance protein (LRP), a multidrug resistance protein causing platinum-resistance, is an independent factor in predicting chemotherapy sensitivity to ovarian cancer. This study was to explore the correlations of EGFR and LRP to chemotherapy resistance and prognosis of ovarian cancer. METHODS: Expressions of EGFR and LRP in 76 specimens of ovarian malignant tumor, nine borderline tumor, 17 benign tumor and 15 normal ovary were studied using immunohistochemistry. Patients with ovarian cancer were followed up. Correlations of EGFR and LRP to chemotherapy efficacy and survival time of patients with ovarian cancer after operation were analyzed. RESULTS: The positive rates of EGFR and LRP in malignant specimens (73.68% and 71.79%) were significantly higher than those in normal and benign ones (P <0.01). EGFR was highly expressed in ovarian cancer patients at late stage (III-IV), with poor differentiation and ascites (P <0.05). The short-term efficacy rates of ovarian cancer were lower in patients with positive expressions of EGFR and LRP (57.14% and 53.70%) than in those with negative expressions (P<0.05). The positive rates of EGFR and LRP were significant higher in patients with chemotherapy resistance (92.86% and 85.71%) than in those sensitive to chemotherapy (P<0.05). The three-year survival rate of ovarian cancer patients was 53.00%. Patients with positive EGFR and LRP and poor short-term efficacy after chemotherapy had short survival time (P<0.01). CONCLUSION: The expression of EGFR and LRP could be used to predict chemotherapy resistance and prognosis of ovarian cancer.

[Expression of mesothelin mRNA and protein in ovarian carcinomas].

OBJECTIVE: To investigate the expression of mesothelin (MESO) mRNA and protein and its significance in ovarian carcinomas. METHODS: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the expression level of MESO mRNA and protein, respectively, in 124 samples of ovarian tumor and normal tissues, including 84 epithelial ovarian carcinomas, 12 borderline ovarian tumors, 16 benign ovarian tumors and 12 normal ovarian tissues. RESULTS: The expression of MESO mRNA and protein in epithelial ovarian carcinomas (1.4005 +/- 0.4646, 2.7857 +/- 2.2712) and borderline ovarian tumors (1.0650 +/- 0.3100, 2.9167 +/- 2.391) were significantly higher than that in benign ovarian tumors (0.6463 +/- 0.2419, 1.2500 +/- 1.6125) and normal ovarian tissues (0.6439 +/- 0.2729, 0.9167 +/- 1.2401) (P < 0.05), and also significantly higher in serous cystadenocarcinoma (1.5255 +/- 0.4151, 3.3036 +/- 2.6141) and endometrioid carcinoma (1.5250 +/- 0.5419, 3.0000 +/- 2.3094) than that in mucinous cystadenocarcinoma (1.0675 +/- 0.3149, 1.0556 +/- 1.9242) (P < 0.05). The expression of MESO mRNA and protein in stages II and IV carcinomas (1.5100 +/- 0.4142, 3.6087 +/- 3.3959) was significantly higher than that in stages I and II carcinomas (1.1190 +/- 0.4909, 1.7895 +/- 2.6320; P < 0.05), and also significantly higher in grade 3 carcinomas than that in grade 1 and 2 ones (P < 0.05), but was not correlate with age or serum CA125 of the patients (P > 0.05). CONCLUSION: The results of this study demonstrated that the expression of MESO mRNA and protein is increased in ovarian carcinomas and borderline ovarian tumors, and MESO may play a role in the adhesion and dissemination of ovarian carcinomas.

Angiographic uterine artery embolization followed by immediate curettage: an efficient treatment for controlling heavy bleeding and avoiding recurrent bleeding in cervical pregnancy.

Two cases of cervical pregnancy with heavy bleeding successfully treated by uterine artery embolization (UAE) followed by immediate curettage are described in this report. Case 1 demonstrated intermittent bleeding after serious bleeding was successfully controlled by UAE. Serum beta human chorionic gonadotropin (beta-hCG) level rose remarkably after a short time decline. Transvaginal sonography consistently revealed a heterogeneous mass in the cervix. Repeated UAE followed by immediate curettage was performed and complete resolution was achieved. Case 2 was also successfully managed by UAE followed by immediate curettage after failure of medical treatment. This report suggests that UAE followed by immediate curettage is a safe and efficient procedure for controlling heavy bleeding and avoiding recurrent bleeding when fertility capacity is desired in cases of cervical pregnancy with fetal cardiac activity and high beta-hCG concentration.

[Construction and package of the expression plasmid pAdEasy-1 system encoding the human papillomavirus 16 E7 gene and the gene's influence on HeLa cells].

OBJECTIVE: To investigate the effect of human papillomavirus 16 E7 gene on cell cycle of cervical cancer HeLa cell, through construction and expression of human papillomavirus 16 E7 gene with adenovirus vector. METHODS: Recombinant adenovirus which expressed E7 gene was constructed and packed. Flow cytometry (FCM) was used to detect the changes of cell cycle phase and cyclin D1 between cells infected and uninfected by recombinant adenovirus. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphennyltetrazolium bromide (MTT) colorimetric assay was used in the detection of the alteration of cell growth. RESULTS: The recombinant adenovirus had stable efficiency of infection and E7 genes could be expressed stably. The result of MTT showed the multiplication of HeLa cells was accelerated from 0.27 +/- 0.03 before infection to 0.38 +/- 0.02 after infection (P < 0.01). FCM showed the number of cells in S phase increased from (26.0 +/- 0.4)% to (36.0 +/- 2.0)% at 12 hours and (49.9 +/- 4.2)% at 24 hours after infection (P < 0.05). Cyclin D1 expression was 22.4% before infection, and decreased to 55.2% after infection (P < 0.01). CONCLUSIONS: The recombinant adenovirus expressing E7 gene could infect target cells. E7 gene can influence cell-cycle of HeLa cells, which can be used to restrain cervical cancer.

[Effect of pirenzepine on form deprivation myopia in chicks and its possible mechanism].

OBJECTIVE: To observe the effect of M1-selective muscarinic antagonist, pirenzepine, on form deprivation myopia and investigate the expression of MMP-2 and its inhibitor TIMP-2 in the fibrous sclera in order to better understand the mechanism by which pirenzepine inhibits myopia. METHODS: 40 chicks after birth one day were divided into 4 groups randomly: I. Control group; II. Form deprivation group; III. Vehicle application group; IV. Pirenzepine injected group. Form deprivation myopia was established in right eyes of group II, III, IV by placement of a translucent occluder. The deprived eyes of group III and IV received daily subconjunctival administration of vehicle PBS and pirenzepine respectively. Optical measures such as refraction, axial length, equatorial diameter were made at the end of the experiment. Total RNA and protein were extracted from the posterior fibrous sclera chicks. The expression of MMP-2 and TIMP-2 mRNA and protein were investigated with RT-PCR and Western blot analysis respectively. RESULTS: Refraction status, axial length, equatorial diameter of the eyes in pirenzepine injected group were significantly lower when compared with form deprivation group (P < 0.01), but the parameters were higher when compared with normal control group therefore relatively myopic changes were detected. There were no significant difference between drug control and pirenzepine injected group when optical measures and the expression of MMP-2, TIMP-2 were concerned (P > 0.05). The expressions (mRNA and protein) of both MMP-2 and TIMP-2 were significantly different in form deprivation group when compared with normal control group (MMP-2 mRNA increased by 143.51%, P < 0.01; protein increased by 114.60%, P < 0.01; TIMP-2 mRNA decreased by 55.05%, P < 0.01; protein decreased by 53.73%, P < 0.01). In pirenzepine injected group the relative expression of MMP-2 mRNA and protein were decreased obviously by 41.95% (P < 0.01) and by 36.16% (P < 0.01), while TIMP-2 mRNA and protein expression was increased significantly by 72.46% (P < 0.01) and by 53.05% (P < 0.01) respectively compared with the form deprived group. CONCLUSION: Subconjunctivally administration of the M1 selective muscarinic antagonist, pirenzepine, partly prevents or restrains form deprivation induced myopia. It may exert its inhibitory effect by modulating the expression of MMP-2 and TIMP-2 in fibrous sclera.

[Study on the changes of T-lymphocyte subsets in the patients with endometriosis].

OBJECTIVE: To investigate the function of T-lymphocyte subsets in patients with endometriosis. METHODS: The levels of interleukin-2 (IL-2) and interleukin-6 (IL-6) in the serum, and peritoneal fluid of 30 cases with endometriosis were detected using enzyme linked immunoabsorbent assay and compared with those of 20 non-endometriosis cases. The expression of IL-2 and IL-6 in ectopic endometrial tissue from the patients with endometriosis and the endometrial tissues of 10 normal women was investigated by immunohistochemistry method. RESULTS: Significantly elevated levels of IL-6 (cytokines of T help cell 2) were found in the serum and peritoneal fluid of patients with endometriosis (Median: 5.3 ng/L, 2.1 ng/L, P < 0.05) compared with that of non-endometriosis patients median: 2.5 ng/L, 0.9 ng/L). The level of IL-6 in the serum and peritoneal fluid of endometriosis patients in early stages (stages I, II) was 3.7 ng/L, 1.6 ng/L, significantly lower than those in advanced stages (stages III, IV) (13.6 ng/L, 4.1 ng/L) (P < 0.05). The ratio of IL-2/IL-6 in the serum and peritoneal fluid in patients with endometriosis (0.7, 1.1) was significantly lower than those in the control non-endometriosis group (0.8, 6.2, P < 0.05). The levels of IL-6 detected in the peritoneal fluid of patients with endometriosis had a positive correlation with those in the serum (r = 0.745, P < 0.01). The levels of IL-6 in serum and peritoneal fluid in the patients with endometriosis had a negative correlation with the IL-2/IL-6 ratios in the serum and peritoneal fluid respectively (r = -0.406, r = -0.480, P < 0.05). IL-2 and IL-6 were expressed in the interstitial cells of ectopic endometrial tissue, with an expression rate of 56.7%, and 60.0% respectively. There was significant difference in the expression of IL-2 and IL-6 between the ectopic endometrial tissue and normal endometrial tissue. CONCLUSIONS: The levels of IL-6 in the serum and peritoneal fluid of patients with endometriosis are increased, implying that IL-6 might play a role in the pathophysiology of endometriosis. The ratio of IL-2/IL-6 in the serum and peritoneal fluid was decreased in patients with endometriosis compared with the control group, suggesting shift of Th1 cell toward Th2 cell in patients with endometriosis. Stronger expression of IL-2 and IL-6 in the ectopic endometrial tissues may contribute to the disturbed immune regulation in patients with endometriosis.

[Detection of mutation and protein expression of PTEN gene in endometrial carcinoma].

BACKGROUND & OBJECTIVE: PTEN (phosphatase and tensin homolog deleted on chromosome ten), a novel tumor suppressor gene identified recently, is called the house-keeping gene of endometrium. However, little is known about its precise role in genesis and development of endometrial carcinoma. In the present study, the mutation and protein expression of PTEN gene were investigated to seek the clinical significance. METHODS: Fifty-two endometrial carcinoma samples and 10 normal endometrial tissues were collected. The mutations of exon 5 and exon 8 of PTEN gene were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing analysis. The expression of PTEN protein was evaluated by immunohistochemistry method. The results associated with clinical pathological features were analyzed. RESULTS: In endometrial carcinomas, the rates of mutation and protein expression deletion of PTEN were 25% and 60%, respectively, which were significantly higher than that of normal endometrium (0%) (P< 0.05). The samples at pathological G(1) and G(2) and depth of myometrial invasion less than 1/2 demonstrated higher mutation rate than that of G3 and depth of myometrial invasion more than or equal to 1/2 (P< 0.05). In contrast, the rate of protein expression deletion of G(1) and G(2) was significantly lower than that of G3 (P< 0.05). Both mutation and protein expression deletion showed statistical differences between endometrioid adenocarcinoma and other types of endometrial carcinomas (P< 0.05), but no significant difference was found at different surgical-pathological stage (P >0.05). CONCLUSION: Mutation and positive protein expression of PTEN occurred more frequently in the endometrial carcinoma cases with low pathological stages.