A Slug-dependent mechanism is responsible for tumor suppression of p53-stabilizing compound CP-31398 in p53-mutated endometrial carcinoma.

Mutation in the tumor suppressor gene p53 is the most frequent molecular defect in endometrial carcinoma (EC). Recently, CP-31398, a p53-stabilizing compound, has been indicated to possess the ability to alter the expression of non-p53 target genes in addition to p53 downstream genes in tumor cells. Herein, we explore the alternative mechanisms underlying the restoration of EC tumor suppressor function in mutant p53 by CP-31398. A p53-mutated EC cell was constructed in AN3CA cells with restored or partial loss of Slug using lentiviral vectors, followed by treatment with 25 µM CP-31398. A p53-independent mechanism of CP-31398 was confirmed by the interaction between mouse double minute 2 homolog (MDM2) and Slug AN3CA cells treated with IWR-1 (inhibitor of Wnt response 1). Furthermore, the AN3CA cells were treated with short hairpin RNA against Slug, Wnt-specific activators (LiCl) or inhibitors (XAV-939) followed by CP-31398 treatment. Moreover, AN3CA cell proliferation and apoptosis were examined. A tumorigenicity assay was conducted in nude mice. CP-31398 could promote the apoptosis of p53-mutated EC cells, while Slug reversed this effect. Slug ubiquitination was found to occur via binding of Slug to MDM2 in AN3CA cells. We found that CP-31398 increased the GSK-3ß, p-Slug, Puma, Wtp53, and Bax expressions whereas Wnt, Mtp-53, Slug, Bcl-2, and Ki-67 expressions were decreased. However, these findings were reversed following the activation of the Wnt pathway and overexpression of Slug. Finally, the in vivo experimental evidence confirmed that CP-31398 with depleted Slug suppressed tumor growth by downregulating the Slug. Collectively, CP-31398-regulated Slug downregulation represses the p53-mutated EC via the p53/Wnt/Puma pathway.

CP-31398 inhibits the progression of cervical cancer through reversing the epithelial mesenchymal transition via the downregulation of PAX2s.

CP-31398, a styrylquinazoline, emerges from a screen for therapeutic agents that restore the wild-type DNA-binding conformation of mutant p53 to suppress tumors in vivo, but its effects on cervical cancer (CC) remain unknown. Hence, this study aimed to explore the effects CP-31398 has on the CC cells and to investigate whether it is associated with paired box 2 (PAX2) expression. CC cells were treated with different concentrations of CP-31398 (1, 2, 4, 6, 8, and 10 µg/ml) to determine the optimum concentration using fluorometric microculture cytotoxicity assay. After constructing the sh-PAX2 vector, CC cells were transfected with sh-PAX2 or treated with CP-31398. The effects of CP-31398 or PAX2 silencing on CC cell proliferation, apoptosis, invasion, and migration were evaluated. Epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, N-cadherin, snail, and twist in CC cells were detected. Tumor formation experiment in nude mice was performed to observe tumor growth. The optimum concentration of CP-31398 was 2 µg/ml. PAX2 was overexpressed in CC cells. CC cells treated with CP-31398 or treated with sh-PAX2 inhibited proliferation, invasion, and migration but promoted apoptosis with decreased PAX2 expression. The EMT process in CC cells was also reversed after treatment with CP-31398 or sh-PAX2. Moreover, the tumor formation experiment in nude mice revealed the inhibitory activity of CP-31398 in CC tumor in nude mice by suppressing PAX2. Our results provide evidence that CP-31398 could inhibit EMT and promote apoptosis of CC cells to curb CC tumor growth by downregulating PAX2.

RNAi­mediated downregulation of asparaginase­like protein 1 inhibits growth and promotes apoptosis of human cervical cancer line SiHa.

Asparaginase like 1 (ASRGL1) protein belongs to the N­terminal nucleophile group, cleaving the isoaspartyl­dipeptides and L­asparagine by adding water. It tends to be overexpressed in cancerous tumors including ovarian cancer and breast tumors. The present study assessed the potential ability of ASRGL1 as a molecular target in gene­based cervical cancer treatment. The protein expression level of ASRGL1 was determined in paraffin­embedded tumor specimen by immunohistochemistry. Additionally, in order to assess the activity of ASRGL1 during the process of cervical cancer cell multiplication, ASRGL1­short hairpin (sh) RNA­expressing lentivirus was established, which was used to infect SiHa cells. The Cellomics ArrayScan VT1 Reader identified the influence of downregulation on SiHa caused by RNA interference­intervened ASRGL1. Flow cytometric analysis was also performed to evaluate the influence. The cyclin dependent kinase (CDK2), cyclin A2, B­cell lymphoma 2 (Bcl­2) and Bcl­2­associated X protein (Bax) expression levels were assessed by western blot analysis. ASRGL1 was observed to be overexpressed in cervical cancer tissues when compared with the adjacent normal tissues. The knockdown of ASRGL1 in SiHa by ASRGL1­shRNA lentivirus infection significantly inhibited cell growth and enhanced cellular apoptosis; the cells were also captured during the S phase. The knockdown of ASRGL1 expression led to the increased expression of Bax and decreased expression of Bcl­2, CDK2 and cyclin A2. In conclusion, ASRGL1 was closely associated with growth and apoptosis in cervical cancer. Therefore, ASRGL1 may be a novel, potentially effective anti­cervical cancer therapy.

Clinical outcomes of frozen-thawed single blastocyst transfer in patients requiring whole embryo freezing.

We compared clinical outcomes amongst frozen-thawed cleavage-stage embryo, double and single blastocyst transfers in patients requiring whole embryo freezing. Data of infertile patients undergoing in-vitro fertilization and embryo transfer (IVF-ET) in our Reproductive Medicine Center from January 2010 to December 2012 were retrospectively analyzed. According to patients' wishes, patients were divided into cleavage-stage embryo transfer groups (group A, n = 456), double blastocyst transfer group (group B, n = 106), and single blastocyst transfer group (group C, n = 402). We found that the number of frozen embryos was significantly less in groups B and C than in group A (all p < 0.05), but the implantation rate was significantly higher in groups B and C as compared to group A (all p < 0.05). The clinical pregnancy rate and pregnancy rate per included cycle were significantly higher in group B than in groups A and C (all p < 0.05). The multiple pregnancy rate was significantly lower in group C than in groups A and B (all p < 0.05). The rate of early abortion was significantly lower in group C as compared to group A (p < 0.05). The data support the view that it may be the best clinical strategy for patients who require whole embryo freezing and have four or more Day 3 embryos available, to incubate Day 3 embryos into blastocysts, which are then vitrified for elective single blastocyst transfer.

[Role of collagen III, CTGF and TNF-alpha in premature rupture of human fetal membranes].

OBJECTIVE: To detect the expression of collagen III, CTGF and TNF-alpha in human fetal membranes with premature rupture of fetal membranes (PROM) and non-PROM, and explore their roles in the pathogenesis of PROM. METHODS: Fifty women with PROM were enrolled in this study, and divided into preterm premature rupture of membranes (pPROM, n=24) and term premature rupture of membranes (tPROM, n=26) according to gestational age. Fifty non-PROM pregnancies matching the same gestational age were recruited as control group. Collagen III CTGF and TNF-alpha in fetal membranes were detected immunohistochemically and analyzed by computer image analysis system. Linear correlation analysis was used to determine the relationship between collagen III, CTGF and TNF-alpha. RESULTS: (1) The expression of collagen III and CTGF in pPROM group (88.81+/-5.25, 85.45+/-6.91) and tPROM group (94.53+/-6.43, 88.15+/-4.93) were decreased significantly (P<0.05, P<0.01) when compared to their control groups (preterm labor 95.99+/-8.41, 90.30+/-5.74, normal term delivery 100.80+/-9.77, 93.20+/-5.33); The expression of collagen III in pPROM group appeared significantly decrease when compared to that in tPROM group (P<0.01). The expression of TNF-alpha in pPROM group (98. 23+/-8.35) and tPROM group (107.85+/-8.68) were increased significantly (P<0.01) when compared to their control groups (preterm labor 83.82+/-6.03, normal term delivery84.50+/-6.14); The expression of TNF-alpha in pPROM group was decreased but not significantly when compared to that in tPROM group (P>0.05). (2) The expression level of Collagen III was positively correlated with that of CTGF in pPROM group (r=0.838, P=0.001), but no correlation was found in tPROM group. Collagen III was negatively correlated with TNF-alpha in both pPROM (r=-0.689, P=0.002) and tPROM group (r=-0. 655, P=0.001). CONCLUSION: Abnormal collagen development caused by CTGF and excess apoptosis of membrane cell caused by TNF-alpha are related to the occurrence of pPROM. However, the basic reason of abnormal collagen resulted in tPROM group is not from CTGF, it may be related with the degeneration of fetal membrane and the TNF-alpha-induced excess cell apoptosis.

[Correlation of toll-like receptor 3 and tumor necrosis factor-alpha with idiopathic fetal growth restriction.].

OBJECTIVE: To investigate the expression and the significance of toll-like receptor 3 (TLR-3) in placenta, tumor necrosis factor-alpha(TNF-alpha) in maternal and cord blood of idiopathic fetal growth restriction (IFGR), and their correlation with the pathogenesis of symmetric and asymmetric IFGR. METHODS: From April 2008 to April 2009, 42 primiparae of singleton pregnancy and their IFGR babies, who delivered at term through cesarean section, in the Third Affiliated Hospital of Zhengzhou University were enrolled. All subjectects were divided into symmetric IFGR group (n = 20) and asymmetric IFGR group (n = 22). Another 42 non-IFGR pairs were randomly selected as the control group. The polink-2 plus polymerized horseradish peroxidase (HRP) immunohistochemical method and the enzyme linked immunosorbent assay (ELISA) were applied to detect TLR-3 and TNF-alpha levels. RESULTS: (1) The expression of TLR-3 protein were observed in all maternal placenta of the three groups. TLR-3 essentially expressed in syncytiotrophoblasts and hofbouer cells in the symmetric IFGR and control group, but expressed mostly in hofbouer cells and less in syncytiotrophoblasts in the asymmetric IFGR group. (2) The expression of TLR-3 in the syncytiotrophoblasts of the symmetric and asymmetric IFGR group was significantly lower than in the control group (111 +/- 14 and 118 +/- 11 vs. 156 +/- 9, P < 0.01). The number of TLR-3 positive in Hofbourer cell in the symmetric IFGR group was lower than the control group (8.9 +/- 2.8 vs 17.5 +/- 2.8, P < 0.01), but the number in the asymmetric IFGR group was higher (23.8 +/- 3.7) compared with the control group (P < 0.01). (3) The TNF-alpha levels in the maternal and cord blood of the symmetric and the asymmetric group were higher than that of the control group [maternal: (90 +/- 10) microg/L and (86 +/- 11) microg/L vs. (73 +/- 9) microg/L; cord blood: (92 +/- 12) microg/L and (96 +/- 8) microg/L vs. (79 +/- 9) microg/L; P < 0.01]. (4) Neither symmetric nor the asymmetric IFGR group showed any correlations between the maternal and cord blood levels of TNF-alpha (P > 0.05). (5) Significant correlation was found between the TNF-alpha level of the cord blood and TLR-3 expression in the placenta in both the symmetric and asymmetric IFGR group (P < 0.05), but no relationship was found between the maternal blood TNF-alpha level and TLR-3 expression in the placenta (P > 0.05). CONCLUSIONS: The variantions of TLR-3 expression in placenta and the increased expression of TNF-alpha in cord blood are associated with the genesis IFGR. The reduced expression of TLR-3 may related to symmetric IFGR, while the increased TLR-3 level in hofbouer cells may lead to asymmetric IFGR.

[Expression of Fas antigen and ligand, placental growth factor in placenta of pregnant women with pre-eclampsia].

OBJECTIVE: To investigate the expression of Fas antigen (Fas) and ligand (FasL), placental growth factor (PlGF) in placenta of pregnant women with pre-eclampsia. METHODS: Expressions of Fas, FasL and PlGF of placenta were determined by immunohistological streptavidin-peroxidase-biotin (SP) method and compared between normal late pregnancy (24 cases) and mild pre-eclampsia (24 cases) and severe pre-eclampsia (24 cases) groups. RESULTS: Different expression of levels Fas, FasL and PlGF were observed in the trophoblasts of most placentae. Positive immunostain of Fas, FasL and PlGF was mainly located in the cytoplasm and membrane of syncytiotrophoblast. FasL and PlGF expressions (63 +/- 4, 81 +/- 6 and 42 +/- 6, 65 +/- 6) were significantly less (P < 0.01), and Fas expression (51 +/- 4, 67 +/- 6) significantly greater in the study subjects compared with controls (P < 0.01). CONCLUSION: Altered expression of Fas and FasL in trophoblasts of placenta may influence pathogenesis or sequelate of pre-eclampsia.