[Effects of Diallyl Disulfide on the Proliferation and Apoptosis of Epithelial Ovarian Cancer Cells by Inducing G2/M Arrest].

Objective To explore the effects of diallyl disulfide(DADS)-induced G2/M phase arrest on proliferation and apoptosis of ovarian cancer cells and its possible molecular mechanism.Methods DADS was used to incubate SK-OV-3 and OVCAR-3 cells,respectively,in different concentrations. Cell proliferation was measured by MTT assay and cell apoptosis rate was detected by flow cytometry assay. Xenograft model assay were performed to analyze the antitumor effect in vivo. Cell cycle phase distribution was detected by flow cytometry. Expressions of cell cycle G2/M phase as well as proliferation- and apoptosis-related proteins were measured by Western blotting.Results MTT assay showed that,after treatment of SK-OV-3(F=247.86,P=0.000)and OVCAR-3 cells(F=302.54,P=0.000)with different concentrations of DADS,the cell proliferation inhibition rate was significantly elevated with the increase of DADS concentrations in a concentration-dependent manner. The inhibition rate of SK-OV-3(F=335.12,P=0.000)and OVCAR-3 cells(F=347.43,P=0.000)at 24 h was significantly higher than that at 12 h and 48 h,showing a significant time-dependence manner. Flow cytometry showed that,after SK-OV-3 and OVCAR-3 cells were treated with different concentrations of DADS,the apoptosis rates increased significantly with the increase of DADS concentration in a concentration-dependent manner(P<0.05). The apoptotic rates of SK-OV-3 and OVCAR-3 cells treated with DADS at 24 h was significantly higher than that at 12 h and 48 h in a significant time-dependence manner(P<0.05). Compared with the blank treatment group,intraperitoneal injection of DADS solution significantly inhibited the xenograft volume of ovarian cancer cells in nude mice(F=548.23,P=0.000;F=311.84,P=0.000). After 30 mg/L of DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the percentage of cells in G2 phase of SK-OV-3 and OVCAR-3 cells increased significantly(F=375.11,P=0.000;F=256.48,P=0.000),compared with the blank cells. After 30 mg/L DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the expressions of p-Chk1(ser345)(F=108.89,P=0.013;F=97.58,P=0.018),p-CDC25C(ser216)(F=87.25,P=0.025;F=114.25,P=0.009),p-P53(ser15)(F=112.41,P=0.011;F=255.87,P=0.000),P21WAF1(F=246.38,P=0.001;F=141.36,P=0.005)and p-CDK1(Thr14/Tyr15)protein(F=298.12,P=0.000;F=233.15,P=0.000)were significantly increased,whereas the expressions of CDK1(F=308.24,P=0.000;F=257.55,P=0.000)and CyclinB1 protein(F=223.15,P=0.001;F=241.28,P=0.000)were significantly reduced.The expressions of proliferation and apoptosis-related proteins PCNA(F=77.36,P=0.031;F=157.28,P=0.001),Ki-67(F=205.64,P=0.007;F=315.22,P=0.000)and Survivin(F=122.13,P=0.013;F=188.24,P=0.000)were significantly decreased and Cleaved-caspase3 protein was significantly increased(F=86.46,P=0.023;F=99.11,P=0.009).Conclusion DADS can inhibit the proliferation of ovarian cancer cells and induce their apoptosis,which may be related to the activation of Chk1-CDC25C and P53-P21WAF1 signaling pathways in G2/M checkpoint,decreased kinase activity of CDK1,down-regulated expressions of CDK1 and CyclinB1 proteins,and ultimately cell cycle arrest at G2/M phase.

Inhibition of the IGF signaling pathway reverses cisplatin resistance in ovarian cancer cells.

BACKGROUND: This study was aimed at investigating whether metformin can reverse the resistance of ovarian cancer cells to cisplatin and exploring the underlying mechanism. METHODS: Ovarian cancer cell proliferation in vitro was evaluated using a CCK-8 assay. The resistance index of platinum-resistant ovarian cancer cells was determined and cell cycle and apoptosis rate determined by annexin V/propidium iodide double-staining in CP70 cells. Western blotting was used to determine IGF1, IGF1R, AKT, p-IGF1, p-IGF1R, p-AKT, and MRP2 levels in cells treated with different concentrations of metformin and LY29400, an inhibitor of the insulin-like growth factor pathway. Changes in gene expression levels of MRP2, IGF1, IGF1R, and AKT were determined by fluorescence real-time quantitative PCR assay of CP70 cells treated with metformin. Tumors of human ovarian cancer cell lines CP70 and A2780 were established by subcutaneous transplantation of cells in nude mice and the effect of metformin on MRP2 expression and tumor inhibition assessed. RESULTS: The IC50 value of cisplatin in CP70 cells decreased significantly as metformin concentration increased (P < 0.05). The cell cycle distribution in CP70 cells changed with metformin treatment; the percentage of cells in the G0/G1 phase, as well as the natural apoptosis rate was significantly increased with metformin treatment (P < 0.05). IGF1, IGF1R, AKT p-IGF1, p-IGF1R, and p-Akt protein expression was enhanced dose-dependently with metformin, and was also significantly changed by treatment of CP70 cells with 0 mM metformin +10 mM LY294002. Moreover, changes in the expression of MRP2, IGF1, IGF1R, and AKT was metformin-concentration dependent, and was significantly different from that in the untreated control group (P < 0.05). In nude mice, the tumor volumes of the cisplatin-treated groups were significantly less than in the control group, and was further suppressed by co-treatment with cisplatin and metformin (P < 0.05), indicating that these 2 drugs had a synergistic effect on tumor inhibition. CONCLUSION: Metformin can improve the sensitivity of ovarian cancer CP70 cells to cisplatin in a concentration-dependent manner by activating the AKT signaling pathway, inhibiting the IGF1R signaling pathway, and reducing MRP2 expression.

Management of polycystic ovarian syndrome with Diane-35 or Diane-35 plus metformin.

In this study, we assessed the efficacy and safe usage of the oral contraceptive, Diane-35, in the treatment of polycystic ovary syndrome (PCOS) when combined with the drug metformin. Eighty-two patients with PCOS were randomly divided into two equal groups: Diane-35 treatment group and Diane-35 plus metformin group. Three treatment cycles were administered. Patients' biomedical data such as height, weight, waist circumference, hip circumference, body fat percentage, acne score, hirsutism score and serum hormone levels were selected, which were tested between the second and the fifth day of the menstrual cycle and follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), blood glucose, blood lipids and insulin levels(IR) were analyzed. Significant reduction in body mass index (BMI), acne score, LH and T levels were observed in both groups after three months of treatment; on the other hand, high-density lipoprotein cholesterol (HDL) concentration elevated (p < 0.05). Combined treatment group had a significant change in BMI index and fasting blood glucose levels compared to Diane-35 alone treatment group (p < 0.05). With personalized nutrition and exercise program, Diane-35 only group or Diane-35 plus metformin group had both significantly lowered their serum testosterone levels and had improved acne symptoms. Diane-35 plus metformin combination had shown reduced fat percentage levels in patients with PCOS, and had shown improved glucose and lipid metabolism.

Mutation analysis and prenatal diagnosis in a Chinese family with succinic semialdehyde dehydrogenase and a systematic review of the literature of reported ALDH5A1 mutations.

AIMS: Succinic semialdehyde dehydrogenase (SSADH) deficiency is a neurometabolic disease in which the degradation of γ-aminobutyric acid (GABA) is impaired. The purpose of this study was to report two novel ALDH5A1 mutations responsible for SSADH deficiency in a Chinese family and the prenatal diagnosis of an at-risk fetus with DNA sequencing. RESULTS: Genetic analysis of ALDH5A1, in a child with SSADH deficiency, parents, and 10 weeks' gestation at-risk fetus and 100 healthy unrelated volunteers, was performed. The coding sequence and the intron/exon junctions of ALDH5A1 were analyzed by bidirectional DNA sequencing. The proband was identified to have a compound heterozygous mutations with c.496T>C (p.W166R) and c.589G>A (p.V197M). Each of his parents carried a deleterious mutation. DNA sequencing of chorionic villus revealed the fetus was a carrier, but not affected, and this was confirmed after birth by genetic analysis of umbilical cord blood and urine organic acid analysis. A study in 2003 described 35 mutations of ALDH5A1 in 54 unrelated families, and the current study and systematic literature review identified nine additional novel mutations in eight unrelated families bringing the total number of unique mutations of ALDH5A1 resulting in SSADH deficiency to 44, and the 44 mutations occur from exon 1 to exon 10. No mutational hotspots or prevalent mutations were observed, and all mutations appeared vital for the function of SSADH. CONCLUSIONS: Two novel ALDH5A1 mutations likely responsible for SSADH deficiency were identified, and DNA sequencing provided an accurate diagnosis for an at-risk fetus whose sibling had SSADH deficiency.

[Sucrose gel for treatment of bacterial vaginosis: a randomized, double-blind, multi-center, parallel-group, phase III clinical trial].

OBJECTIVE: To evaluate the cure effectiveness and safety of sucrose gel in the treatment of bacterial vaginosis through a multi-center, randomized, double-blind, parallel controlled clinical study. METHODS: A clinical research method of multi-center, randomly double-blind, and dose group parallel comparison was adopted. In the study, 533 patients with bacterial vaginosis were randomly divided into two groups, which included 214 cases in the control group (5.0 g metronidazole gel) and 319 cases in the trial group (5.0 g sucrose gel ). The patients were treated with different medication according to the group where they were. All the cases in these two groups were treated with drugs vaginally twice in a day, morning and evening separately, for 5 days. The curative effect and safety evaluation were assessed from 7 to 10 days and 21 to 30 days after treatment respectively. RESULTS: The efficacy of the comprehensive clinical treatment showed that the cure rate of metronidazole gel group and sucrose gel group were 70.53% and 80.83% respectively 7 to 10 days after treatment. The recovery rate of Nugent score for vaginal smear were 71.50% and 81.15% respectively. The differences in the efficacy between these two groups were significant statistically (P<0.05). However, the cure rates of metronidazole gel group and sucrose gel group were 63.29% and 61.98% respectively 21 to 30 days after treatment. No statistically significant difference (P>0.05) could be found in the cure rates of the two groups. CONCLUSION: The clinical comprehensive efficacy and recovery of vaginal bacteria of sucrose gel group in the treatment of bacterial vaginosis were obviously superior to those of metronidazole gel 7 to 10 days after treatment. The susucrose gel could improve the clinical efficacy index and laboratory index of bacterial vaginosis. Other effects included the release of clinical symptoms, and the recovery of the normal micro-environment in the vagina according to the Nugent score. The curative efficacy of sucrose gel was equal to that of metronidazole gel 21 to 30 days after treatment. In the future, sucrose gel treatment can be a new strategy for the treatment of bacterial vaginosis. Various advantages can be taken to improve the cure rate of bacterial vaginosis and reduce the shortcomings produced by this disease.

Tyrosinase gene mutations in the Chinese Han population with OCA1.

Oculocutaneous albinism (OCA) is a heterogeneous autosomal recessive genetic disorder that affects melanin synthesis. OCA results in reduced or absent pigmentation in the hair, skin and eyes. Type 1 OCA (OCA1) is the result of tyrosinase (TYR) gene mutations and is a severe disease type. This study investigated TYR mutations in a Chinese cohort with OCA1. This study included two parts: patient genetic study and prenatal genetic diagnosis. A total of 30 OCA1 patients were subjected to TYR gene mutation analysis. Ten pedigrees were included for prenatal genetic diagnosis. A total of 100 unrelated healthy Chinese individuals were genotyped for controls. The coding sequence and the intron/exon junctions of TYR were analysed by bidirectional DNA sequencing. In this study, 20 mutations were identified, four of which were novel. Of these 30 OCA1 patients, 25 patients were TYR compound heterozygous; two patients carried homozygous TYR mutations; and three were heterozygous. Among the ten prenatally genotyped fetuses, three fetuses carried compound heterozygous mutations and seven carried no mutation or only one mutant allele of TYR and appeared normal at birth. In conclusion, we identified four novel TYR mutations and showed that molecular-based prenatal screening to detect TYR mutations in a fetus at risk for OCA1 provided essential information for genetic counselling of couples at risk.

[Expression of metastasis-associated in colon cancer-1 in different stages of epithelial ovarian cancer].

OBJECTIVE: To investigate the expression of metastasis-associated in colon cancer-1 (MACC1) in different International Federation of Gynecology and Obstetrics(FIGO)stages of epithelial ovarian cancer and its relationship with prognosis. METHODS: Between May 2008 and August 2010, 52 epithelial ovarian cancer patients were selected from the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Zhengzhou University. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of MACC1 mRNA and protein in the primary lesions of epithelial ovarian cancer patients, the levels of MACC1 in different stage patients were compared, and the relationship between expression of MACC1 and prognosis of ovarian cancer patients was analyzed by Kaplan-Meier analysis. RESULTS: The relative expression levels of MACC1 mRNA in epithelial ovarian cancer from 1 stage to 4 stage were 0.72±0.01, 0.75±0.01, 0.78±0.01, and 0.81±0.02, respectively (F=51.305, P=0.000). The expression levels of MACC1 protein from 1 stage to 4 stage were 0.71±0.04, 0.73±0.02, 0.76±0.01, and 0.84±0.05, respectively (F=65.142, P=0.000). At the end of the follow-up, the expression of MACC1 protein in recurrence and dead patients of 3-4 stages was obviously higher than that in the patients with stable disease (0.85±0.03 vs.0.74±0.05, F=72.324, P=0.000). Compared to 1-2 stage patients with lower MACC1 expression, the survival time of 3-4 stage patients with higher MACCC1 expression was significantly shorter (χ(2)=29.804, P=0.000). CONCLUSIONS: Increased expression of MACC1 may indicate poor prognosis of ovarian cancer patients. Therefore, MACC1 may be a potential biomarker for advanced ovarian cancer.

MiR-106a targets Mcl-1 to suppress cisplatin resistance of ovarian cancer A2780 cells.

Resistance to chemotherapy is a major obstacle for the effective treatment of advanced ovarian cancer. The mechanism of chemoresistance is still poorly understood. Recently, more and more evidence showed microRNAs (miRNAs) modulated many key molecules and pathways involved in chemotherapy. microRNA-106a (miR-106a) has been implicated in many cancers, but its role in ovarian cancer and drug resistance still remains unexplored. This study was to investigate whether miR-106a mediated resistance of the ovarian cancer cell line A2780 to the chemotherapeutic agent cisplatin (DDP). The different levels of miR-106a in A2780 cells and their resistant variant A2780/DDP cells were identified by using real-time PCR. MTT assay and flow cytometry were used to analyze the effect of miR-106a on cisplatin resistance of these paired cells. Real-time PCR, Western blotting and luciferase reporter assay were applied to explore whether Mcl-1 was a target of miR-106a. As compared to A2780 cells, the expression of miR-106a was down-regulated in the cisplatin resistant cell line A2780/DDP. Moreover, knockdown of miR-106a dramatically decreased antiproliferative effects and apoptosis induced by cisplatin in A2780 cells, while overexpression of miR-106a significantly increased antiproliferative effects and apoptosis induced by cisplatin in A2780/DDP cells. Furthermore, miR-106a inhibited cell survival and cisplatin resistance through downregulating the expression of Mcl-1. Mcl-1 was a direct target of miR-106a. These results suggest that miR-106a may provide a novel mechanism for understanding cisplatin resistance in ovarian cancer by modulating Mcl-1.

Effect of siRNA targeting MTA1 on metastasis malignant phenotype of ovarian cancer A2780 cells.

Ovarian cancer is the fifth lethal gynecologic malignancy. Metastasis-associated gene 1 (MTA1) is overexpressed in many malignant tumors with high metastatic potential. This study investigated whether down-regulation of MTA1 expression by RNAi in A2780 ovarian cancer cells could affect proliferation, anoikis, migration, invasion and adhesion of the cells and to research the potential for MTA1 gene therapy of ovarian cancer. After transfection with effective Mta1 gene siRNA, the effects on proliferation, anoikis, migration, invasion and adhesion of A2780 cells were tested by MTT assay, flow cytometry, wound-healing assay, Transwell assay and adhesion assay. Expression levels of PTEN, beta 1 integrin, MMP-9, phosphor-AKT (Ser473), and total AKT activity were evaluated in control and transfected cells. The results showed that inhibition of MTA1 mediated by Mta1-siRNA transfection decreased the cell invasion, migration and adhesion, and induced the increased cell anoikis, but no significant difference was found in proliferation of A2780 cancer cells. In addition, beta 1 integrin, MMP-9, and phosphor-AKT protein levels were significantly down-regulated, while PTEN was significantly up-regulated. These results demonstrated that MTA1 played an important role in the cell metastasis in ovarian cancer. MTA1 could serve as another novel potential therapeutic target in ovarian cancer.

[Mutation analysis and first-trimester prenatal diagnosis for a Chinese family with hidrotic ectodermal dysplasia].

OBJECTIVE: To analyze GJB6 gene mutations in a Chinese family with hidrotic ectodermal dysplasia and to provide first-trimester prenatal diagnosis for a fetus. METHODS: Mutation scanning was carried out with PCR and bilateral direct sequencing in 2 affected and 6 unaffected individuals from the family. After the mutation was confirmed, prenatal diagnosis was performed on chorionic villi samples obtained at 11th gestational week. RESULTS: A heterozygous missense mutation c.31G>A of the GJB6 gene was discovered in all of the patients, which has led to substitution of glycine by arginine at codon 11 (p.G11R) at the N-terminal of the GJB6 protein. Prenatal diagnosis indicated that the fetus had also carried the same p.G11R mutation. Following termination of the pregnancy, analysis of the aborted tissues was consistent with prenatal diagnosis. CONCLUSION: The missense mutation c.31G>A(p.G11R) of the GJB6 gene probably underlies the disease in this family. Prenatal diagnosis with DNA sequencing can facilitate genetic counseling of this family.

Outcomes of day 3 embryo transfer with vitrification using Cryoleaf: a 3-year follow-up study.

OBJECTIVE: To compare success rates of vitrified-warmed with fresh and frozen-thawed ETs DESIGN: Retrospective. SETTING: Public fertility center. PATIENT(S): Cryopreserved- thawed/warmed ETs were included in this study. Fresh cycles, in which supernumerary embryos were cryopreserved, were set as the fresh control group. INTERVENTION(S): Supernumerary day 3 embryos were cryopreserved by slow-freezing or vitrification and transferred after thawing or warming. MAIN OUTCOME MEASURE(S): Comparison of two cryopreservation techniques with respect to post-thaw survival of embryos, implantation and pregnancy rates, neonatal outcome, and congenital birth defects. RESULTS: A total of 962 fresh, 151 freezing-thawed and 300 vitrified-warmed cycles were included in this study. The survival and intact cell rates in the vitrification group were significantly higher compared with those in the slow freezing group (88.5 % vs 74.5 % and 86.6 % vs 64.0 %). The implantation, clinical pregnancy and live birth rates of the vitrification group were similar to the fresh and significant higher than slow freezing group. There were no significant differences in mean gestational age, birth weight, stillbirth, birth defects and the prevalence of neonatal diseases among three groups. CONCLUSION: Vitrified-warmed ETs yield comparable outcomes with fresh ETs and is superior to frozen-thawed ETs regarding the survival rate and clinical outcomes.

[Early prenatal genetic diagnosis of oculocutaneous albinism type I in seven families].

OBJECTIVE: To evaluate the feasibility of genetic analysis of tyrosinase gene (TYR) in oculocutaneous albinism type I (OCA1). Mutation analysis and prenatal genetic diagnosis of TYR gene for seven pedigrees with OCA1 were performed. METHODS: PCR was used to amplify the exons, exon-intron boundaries and promoter of the TYR gene in the probands and/or their parents. The products were further analyzed by direct sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of the probands or their parents were determined. RESULTS: Compound heterozygous mutations were detected in all pedigrees, which included 9 mutations, namely R76Q, c.232insGGG, R116X, R278X, R299H, c.929-930insC, IVS2-11delTT, Q399X and W400L. Among these, R76Q and Q399X were identified for the first time. Seven families have requested prenatal diagnoses. One fetus was detected with double mutations of TYR gene, and the parents have decided to have therapeutic abortion. Two fetuses did not carry the mutations identified in the probands, whilst other four fetuses were carriers of heterozygous mutations. Six families decided to carry on with the pregnancies. And the neonates did not show any symptoms of OCA after birth. CONCLUSION: Direct sequencing of the TYR gene is helpful for genetic counseling, prenatal diagnosis and carriers screening of OCA1.

[Detection of ED1 gene mutations in six pedigrees with hypohidrotic ectodermal dysplasia].

OBJECTIVE: To identify potential mutations of ED1 gene in six pedigrees with hypohidrotic ectodermal dysplasia (HED), and to provide genetic counseling and prenatal diagnosis. METHODS: Eight coding exons of ED1 gene of patients with clinically diagnosed HED and their relatives were amplified by polymerase chain reaction (PCR). The products were further analyzed by direct sequencing. RESULTS: Various mutations of ED1 gene were detected, which included R153C, A349T, G299S, A349T and X392Q. Heterozygous double peaks at the same position were found in female carriers. Deletion of exon 9 was detected in one pedigree. R153C, X392Q and deletion of exon 9 were first identified in ethnic Han Chinese. CONCLUSION: The identified mutations of ED1 gene may be responsible for the disease. Genetic counseling, prenatal diagnosis and carrier screening are now available for these families.

Pilot study: alteration of deleted in liver cancer1 and phosphorylated focal adhesion kinase Y397 cytoplasmic expression and the prognostic value in advanced epithelial ovarian carcinoma.

BACKGROUND: Deletion in liver cancer gene (DLC1) and phosphorylated focal adhesion kinase (p-FAK) have recently been reported as metastasis-related genes. However, the roles and prognostic values of their expression in epithelial ovarian carcinomas (EOCs) remain unclear. METHODS: The expression and prognostic value of DLC1 and p-FAK Y397 in EOC were evaluated by immunohistochemistry and multivariate analysis. RESULTS: Low expression of DLC1 and high expression of p-FAK Y397 were found in the 76 cases of EOC. The expression of DLC1 and p-FAK Y397 were negatively correlated. Multivariate analysis showed that the combination of them was an independent prognostic marker of EOC (P = 0.0319). CONCLUSIONS: DLC1 and pFAK Y397 had an association with the clinicopathologic characteristics of EOC. Expression of neither of these genes was a prognostic factor alone, but the combination revealed a significant prognostic value in the 60 cases of advanced stage EOC.

[Expression of hSef and FGF-2 in epithelial ovarian tumor].

OBJECTIVE: To detect the expression of human similar expression to FGF gene(hSef) and fibroblast growth factor-2(FGF-2) and their correlation with epithelial ovarian tumor. METHODS: Immunohistochemical SP staining was used to detect the expression of hSef and FGF-2 proteins in 31 cases of epithelial ovarian carcinoma (EOC), 18 cases of benign epithelial tumor (BET), 10 cases of normal ovarian (NO) tissues collected from July 2007 to May 2008. The expression of hSef mRNA in 24 cases of EOC, BET and NO collected from July 2008 to May 2009 were analyzed by RT-PCR. RESULTS: The results of immunohistochemical study showed that the expression of hSef in the EOC tissues were significantly lower than that in the NO and BET (P < 0.001). However, the expression of FGF-2 was higher (P = 0.002). The expression of hSef had a negative correlation with FGF-2 (r(s) = -0.324, P = 0.012). The RT-PCR results showed that there was a gradually declined trend of expression of hSef in NO, BET to EOC (P < 0.001), but the expression of FGF-2 in NO, BET to EOC was gradually increased (P < 0.001), with a significant negative correlation (NO: r(s) = -0.910, P < 0.001; BET: r(s) = -0.859, P < 0.001; EOC: r(s) = -0.888, P < 0.001). CONCLUSIONS: The expression of hSef is decreased in epithelial ovarian carcinoma tissue, but the expression of FGF-2 is increased. It is likely that low hSef expression is related to the the carcinogenesis and development of epithelial ovarian carcinoma by suppressing the promoting effects of FGF-2 to cell proliferation.

[Mutation analysis of phenylalanine hydroxylase gene in patients w ith phenylketonuria in Henan province].

OBJECTIVE: To study the characteristics of the phenylalanine hydroxylase gene (PAH) mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. METHODS: Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. RESULTS: A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88.3%). Among them, E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C to G mutation was reported in Chinese PKU population. The mutations p.R243Q, EX6-96A to G, p.Y356X, IVS401G to A, p.R111X, p.V399V and p.R413P, were the prevalent mutations with relative frequencies of 20.5%, 12.0%, 9.6%, 9.6%, 8.4%, 8.4% and 7.2% respectively. CONCLUSION: The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.

[Effects of PD98059 and LY294002 on subcutaneous xenograft of human endometrial carcinoma in nude mice].

OBJECTIVE: To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) in endometrial carcinoma xenografts. METHODS: Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium (MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups (n = 6), normal saline group, PD98059 group (PD group), LY294002 group (LY group) or PD98059 + LY294002 group (PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. RESULTS: (1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and(or) Y294002, in which A(570) values of cells decreased showing both time-dependent and concentration-dependent manner (LY294002: F(group) = 9.801, P = 0.002; F(time) = 10.398, P = 0.001. PD98059: F(group) = 8.213, P = 0.015; F(time) = 6.839, P = 0.036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G(0)/G(1) phase (F(time) = 35.049, P = 0.004; F(group) = 32.024, P < 0.01) increased and percentage of S phase cells (F(time) = 7.789, P = 0.049; F(group) = 30.132, P < 0.01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63.3 ± 0.5)% vs (30.7 ± 20.1)% vs (40.8 ± 1.3)%; F = 621.059, P < 0.01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously (F = 23.545, P < 0.01), and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9)% vs (32 ± 16)% or (38 ± 17)%; F = 10.283, P < 0.05]. (3) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13.7 ± 1.5)%, (14.1 ± 1.2)%, (29.0 ± 1.8)%; F = 320.344, P < 0.01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. CONCLUSION: The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.

UCHL3 promotes ovarian cancer progression by stabilizing TRAF2 to activate the NF-κB pathway.

The inflammatory response plays an important role in carcinogenesis. However, the functional role and mechanism of the UCHL3-associated inflammatory response in ovarian cancer remain to be characterized. Here, we report that increased expression of UCHL3 facilitates tumourigenesis by targeting TRAF2 protein, thereby enhancing the inflammatory response. The expression of UCHL3 is elevated in ovarian cancer patients and is associated with an unfavourable prognosis. Genetic ablation of UCHL3 was found to markedly block ovarian cancer cell proliferation, viability and migration both in vitro and in vivo. Mechanistically, luciferase pathway screening results show that NF-κB signalling is clearly activated compared with other pathways. UCHL3 was found to activate NF-κB signalling by deubiquitinating and stabilizing TRAF2, leading to tumourigenesis. Our results indicate that highly expressed UCHL3 enhances inflammation by stabilizing TRAF2, which in turn facilitates tumourigenesis in ovarian cancer, and that UCHL3 is a potential target for ovarian cancer patients with increased inflammation.

Increased staining for phosphorylated AKT and nuclear factor-kappaB p65 and their relationship with prognosis in epithelial ovarian cancer.

AKT plays an important role in malignant behavior of tumors. The purpose of the present study was to determine the expression of phosphorylated AKT (P-AKT) and nuclear factor-kappaB (NF-kappaB) p65 and their association with clinicopathological parameters and prognosis in epithelial ovarian tumor. On immunohistochemistry 115 samples of ovarian tissue that included 68 specimens of epithelial ovarian cancer, 12 of borderline tumor, 24 of epithelial benign tumor and 11 of normal ovary, were evaluated. Sixty-three patients with ovarian cancer were followed up from 7 to 68 months. The positive expression rate of P-AKT and NF-kappaB p65 were higher in epithelial ovarian cancer than in normal ovarian tissue (P<0.01). Elevated P-AKT or NF-kappaB p65 expression was significantly correlated with late clinical stage (P<0.05 and P<0.01) and poor histological differentiation (both P<0.01). P-AKT expression was significantly correlated with NF-kappaB p65 immunostaining (phi=0.272, P<0.05). Elevated expression of P-AKT was negatively correlated with the survival of ovarian cancer patients, but it was not an independent prognostic factor after multivariate analysis. Overexpression of P-AKT and NF-kappaB p65 were involved in the carcinogenesis and metastasis of ovarian cancer. P-AKT might contribute to the malignant transformation through NF-kappaBp65 upregulation.

Association of p53 and mitochondrial gene with chemosensitization by metformin in ovarian cancer.

OBJECTIVE: This study aims to investigate the association of p53 and D-loop gene with drug resistance and sensitization induced by metformin in ovarian cancer. RESULTS: Metformin suppresses cells in a time-dependent manner, but the inhibition does not change with the dose of metformin. This suggests that within a certain range of concentration in the body, metformin has a constant inhibitory effect on cells; and the long-term use of metformin yields a better effect. CONCLUSIONS: Metformin enhances the sensitivity of drug-resistant ovarian cancer cells to chemotherapy. METHODS: The third passage cells of 17 cancerous ovarian tissues, which were successfully passaged five times, were used as the study objects; and the SKOV3 cell line was used as the positive control. After three adaptations, cells were cultured for 72 hours in an orthogonal experiment using drugs that were used for adaptation. Then, the inhibitory rate on cells in the experimental group was observed by CCK8 assay, in order to study the sensitization effect of metformin in different chemotherapies of ovarian cancer.