Ribosomal S6 kinase 4 (RSK4) tumor suppressor gene promoter methylation status in ovarian cancer.

BACKGROUND: Previously, we reported lower RSK4 mRNA and protein levels in malignant ovarian tumors compared to normal and benign ovarian tissues. Also, we observed a significant inverse correlation between the advanced ovarian cancer stages and RSK4 mRNA levels. We did not investigate the mechanisms involved in RSK4-reduced expression in ovarian cancer. Thus, this study investigates whether RSK4 promoter methylation in ovarian cancer tissues is responsible for its low expression. Additionally, the reactivation of RSK4 expression and its effect was studied in ovarian cancer cell lines. METHODS AND RESULTS: RSK4 promoter methylation percentage in malignant and benign ovarian tumors and normal ovary tissues was determined by combined bisulfite restriction analysis. The reactivation of RSK4 expression by decitabine treatment was studied in OVCAR3, SKOV3, TOV-112D, and TOV-21G cells by Western blotting. Cell proliferation was determined by XTT. A significantly high methylation percentage of the RSK4 promoter was observed among malignant and benign ovarian tumors but not in normal ovarian tissue. RSK4 promoter methylation was not associated with age, histological subtype, or stages of ovarian cancer. RSK4 promoter methylation correlates weakly but not significantly with RSK4 protein expression. No correlation was shown between RSK4 methylation and RSK4 mRNA expression. Decitabine induces RSK4 reactivation in all cell lines. However, cell proliferation was reduced only in TOV-112D cells. CONCLUSION: These data indicate that although RSK4 promoter methylation is increased in malignant ovarian tumors, this mechanism is unlikely to regulate its expression in ovarian cancer. RSK4 reactivation reduced cell proliferation only in the endometroid histological subtype.

Cadmium exposure reduces invasion of the human trophoblast-derived HTR-8/SVneo cells by inhibiting cell adhesion and matrix metalloproteinase-9 secretion.

Preeclampsia and intrauterine growth restriction, multisystemic disorders characterized by a shallow trophoblast invasion, have been associated with maternal cadmium (Cd) exposure. The molecular mechanisms of this association remain unknown. Cell adhesion and matrix metalloproteinase production are essential for an adequate trophoblast invasion. Thus, the aim of this study was to determine the effect of Cd exposure on invasion, adhesion, and matrix metalloproteinase-9 (MMP-9) production in the trophoblast-derived HTR-8/SVneo cell line. Cultured HTR-8/SVneo trophoblast cells were incubated with different concentrations of CdCl2 for 6 h. Cell invasion was determined by the transwell assay, while cell adhesion was examined on collagen type I. MMP-9 release and activity were measured by ELISA and zymography, respectively. MMP-9 mRNA expression was detected by reverse-transcription polymerase chain reaction, while intracellular MMP-9 protein was assessed by Western blotting. Cd exposure significantly decreased the invasion and adhesion of HTR-8/SVneo cells. Also, MMP-9 levels and activity in the culture medium were significantly reduced after Cd incubation. In contrast, MMP-9 mRNA expression and intracellular protein levels were significantly increased. These data indicate that Cd reduces trophoblast cells invasiveness by inhibiting cell adhesion and MMP-9 secretion.

Progranulin expression induced by follicle-stimulating hormone in ovarian cancer cell lines depends on the histological subtype.

Epithelial ovarian cancer (EOC) is a heterogeneous disease that can be categorized into four major histological subtypes. Its etiology remains poorly understood due mainly to this heterogeneity. Follicle-stimulating hormone (FSH) has been implicated as a risk factor in EOC and has been suggested that may influence the development of specific subtypes. In addition, FSH regulates different aspects of ovarian cancer tumorigenesis. FSH downstream target genes in EOC have not been fully identified. Progranulin (PGRN) overexpression is associated with cell proliferation, invasion, chemoresistance, and shortened overall survival in ovarian cancer. Recently, we demonstrated that PGRN expression is regulated through the PI3K signaling pathway in clear cell ovarian carcinoma (CCOC) cells. In contrast, we also demonstrated that PGRN synthesis in serous ovarian cancer (SOC) cells is regulated via PKC but not by the PI3K signaling pathway. Several studies have demonstrated that FSH induces PKC and PI3K activation. Thus, this study was to investigate the effect of FSH on PGRN production in the CCOC cell line TOV-21G as compared to the SOC cell lines SKOV3 and OVCAR3. Cultured TOV-21G, SKOV3, and OVCAR3 cells were incubated with different concentrations of FSH for 48 h. PGRN mRNA and protein expression were assessed by RT-PCR and Western blotting, while PGRN secretion was measured by ELISA. PGRN mRNA and protein expression, as well as PGRN secretion, significantly increased after FSH stimulation in TOV-21G but not in SKOV3 and OVCAR3 cells. These data indicate that FSH induces PGRN expression and secretion only in CCOC cells. Establishing specific features for CCOC could reveal potential diagnostic and therapeutic targets.

Inhibition of PI3K/AKT/mTOR and MAPK signaling pathways decreases progranulin expression in ovarian clear cell carcinoma (OCCC) cell line: a potential biomarker for therapy response to signaling pathway inhibitors.

Patients with advanced stage ovarian clear cell carcinoma (OCCC) have a poor prognosis due to resistance to conventional platinum chemotherapy. Recent studies have demonstrated that PI3K/AKT/mTOR and ERK1/2 signaling pathways are involved in this chemoresistance. Progranulin (PGRN) overexpression contributes to cisplatin resistance of epithelial ovarian cancer cell lines. Also, PGRN expression is regulated by AKT/mTOR and ERK1/2 signaling pathways in different cell types. Thus, the present study was designed to identify if PGRN expression is regulated by AKT, mTOR, and ERK1/2 signaling pathways in the OCCC cell line TOV-21G. Cultured TOV-21G cells were incubated with different concentrations of pharmacological cell signaling inhibitors. PGRN expression and phosphorylation of ERK1/2, AKT, and mTOR were assessed by Western blotting. Inhibition of AKT, mTOR, and ERK1/2 significantly reduced PGRN expression. Cell viability was not affected, while cell proliferation significantly decreased with all inhibitors used in this study. These observations demonstrated that inhibition of PI3K/AKT/mTOR and ERK1/2 signaling pathways reduces PGRN expression in TOV-21G cells. Thus, PGRN could be considered as a candidate for explaining the high resistance to platinum-based treatment and a potential biomarker for therapy response to cell signaling inhibitors in patients with OCCC.

Cadmium exposure induces interleukin-6 production via ROS-dependent activation of the ERK1/2 but independent of JNK signaling pathway in human placental JEG-3 trophoblast cells.

Maternal exposure to cadmium (Cd) has been associated with preeclampsia (PE), which is a multisystemic disorder characterized by endothelial dysfunction. Elevated interleukin (IL)-6 expression is linked to PE and has been suggested to contribute to maternal endothelial dysfunction. Cd induces IL-6 production in various cell types through different signaling pathways. Thus, this study was designed to investigate the effect of Cd on IL-6 production and the underlying mechanisms in a trophoblast-derived cell line. Cultured JEG-3 trophoblast cells were exposed to non-toxic concentrations of CdCl2 in the presence or absence of various MAPK inhibitors or N-Acetyl-L-cysteine (NAC). IL-6 was measured by ELISA. Phosphorylation of ERK1/2, JNK, and c-Jun was assessed by Western blotting. Cd exposure induced IL-6 production and increased ERK1/2, JNK, and c-Jun phosphorylation. NAC and the inhibition of ERK1/2 significantly reduced Cd-induced IL-6 production. These data indicate that Cd induces IL-6 production in trophoblast cells through a ROS-dependent activation of ERK1/2.

Methods to Study the Role of Progranulin in Preimplantation Mouse Embryo Development.

Progranulin is a 67-88 kDa glycoprotein, also known as acrogranin, PC-cell-derived growth factor, granulin-epithelin precursor, and proepithelin. This protein is present in a variety of mouse, rat, and human tissues. Progranulin, which is a growth factor, mediates cell cycle progression and cell migration in normal and pathological conditions. In several types of cancers, progranulin expression is upregulated, whereas function-interfering mutations in the granulin gene in humans have been linked to a subset of heritable cases of frontotemporal lobar degeneration. Also, progranulin has important effects on mouse preimplantation embryo development in vitro, including regulation of the appearance of the epithelium in the developing mouse blastocyst and growth of trophectoderm. Furthermore, progranulin promotes mouse blastocyst hatching, adhesion, and outgrowth in vitro. In this chapter, we describe some of the techniques that may be useful in the study of progranulin in embryo development.

The follicle-stimulating hormone receptor Asn680Ser polymorphism is associated with preterm birth in Hispanic women.

OBJECTIVE: Recently, a study based on the analysis of accelerated evolution of related genes at birth identified the follicle-stimulating hormone receptor (FSHR) as a possible candidate for the development of preterm delivery. Additionally, FSHR expression has been described in extragonadal tissue including the placenta. Therefore, the aim of the present study was to determine the association between the N680S polymorphism of the follicle-stimulating hormone receptor and preterm birth in a population of Hispanic women. METHODS: Placenta samples were obtained from 64 women who had preterm births and 54 control cases. DNA was extracted and genotyped for the N680S FSHR gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The χ2 test and t-test were used to calculate statistical significance. RESULTS: Statistically significant differences in genotype frequencies for the N680S polymorphism were observed between preterm and term groups (p = .04). Based on the Akaike information criterion values, the dominant model showed that the NN genotype had a significantly increased risk of preterm birth compared with the SS + NS genotype (OR 2.52, 95% CI 1.20-5.33, p = .02). CONCLUSIONS: The results herein suggest that the FSHR polymorphism N680S is significantly associated with preterm birth in the Hispanic population.

Progranulin and its biological effects in cancer.

Cancer cells have defects in regulatory mechanisms that usually control cell proliferation and homeostasis. Different cancer cells share crucial alterations in cell physiology, which lead to malignant growth. Tumorigenesis or tumor growth requires a series of events that include constant cell proliferation, promotion of metastasis and invasion, stimulation of angiogenesis, evasion of tumor suppressor factors, and avoidance of cell death pathways. All these events in tumor progression may be regulated by growth factors produced by normal or malignant cells. The growth factor progranulin has significant biological effects in different types of cancer. This protein is a regulator of tumorigenesis because it stimulates cell proliferation, migration, invasion, angiogenesis, malignant transformation, resistance to anticancer drugs, and immune evasion. This review focuses on the biological effects of progranulin in several cancer models and provides evidence that this growth factor should be considered as a potential biomarker and target in cancer treatment.

Synergistic effect of DDT and its metabolites in lipopolysaccharide-mediated TNF-α production is inhibited by progesterone in peripheral blood mononuclear cells.

Increased TNF-α levels have been associated with adverse pregnancy outcomes. Lipopolysaccharide (LPS), 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) induce TNF-α release in peripheral blood mononuclear cells (PBMC). Conversely, progesterone (P4) inhibits TNF-α secretion. Pregnant women in malaria endemic areas may be co-exposure to these compounds. Thus, this study was to investigate the synergistic effect of LPS and these pesticides in PBMC and to assess P4 influence on this synergy. Cultured PBMC were exposed to each pesticide in the presence of LPS, P4, or their combination. TNF-α was measured by ELISA. All pesticides enhanced TNF-α synthesis in PBMC. Co-exposure with LPS synergizes TNF-α production, which is blocked by progesterone. These results indicate that these organochlorines act synergistically with LPS to induce TNF-α secretion in PBMC. This effect is blocked by P4.

Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines.

Survival rate in ovarian cancer depends on the stage of the disease. RSK4, which has been considered as a tumor suppressor factor, controls cells invasion due to its antiinvasive and antimetastatic properties. Modulation of RSK4 expression could be an important event to increase the survival rate in ovarian cancer patients. Thus, the goal of the present study was to establish the differences in RSK4 expression among normal, benign and malignant ovarian tissues and to determine whether antineoplastic drugs regulate its expression in SKOV3 and TOV-112D cells. RSK4 levels in 30 malignant ovarian tumors, 64 benign tumors and 36 normal ovary tissues were determined by reverse transcription polymerase chain reaction and Western blot. Modulation of RSK4 expression by two antineoplastic drugs (cisplatin and vorinostat) was also studied in the SKOV3 and TOV-112D ovarian cancer cell lines using the same techniques. RSK4 mRNA and protein levels were decreased in malignant ovarian tumors as compared to benign tumors and normal tissue. These low-RSK4 levels were significantly associated with advanced stages of ovarian cancer. RSK4 expression was increased after incubation of SKOV3 and TOV-112D cell lines with cisplatin and vorinostat for 24 h. The combination of these antineoplastic drugs did not produce a synergistic or additive effect. These results suggest that RSK4 is expressed at low levels in malignant ovarian tumors, which correlates with advanced stages of the disease. Additionally, RSK4 expression is regulated by cisplatin and vorinostat in two ovarian cancer cell lines.

Matrix metalloproteinase-8 promoter gene polymorphisms in Mexican women with ovarian cancer.

Increased levels of matrix metalloproteinase-8 (MMP-8) have been associated with tumor grade and stage in ovarian cancer. Also, it has been reported that higher concentrations of this enzyme in fluid from malignant ovarian cysts compared with benign ovarian cysts. However, no genetic analysis has been conducted yet to assess the contribution of MMP-8 polymorphisms in ovarian cancer. Thus, this study was performed to investigate the frequencies of MMP-8 genotypes in Mexican women with ovarian cancer. MMP-8 promoter genotypes were examined in 35 malignant ovarian tumors, 51 benign tumors, and 37 normal ovary tissues. Two single nucleotide polymorphisms were selected and characterized using polymerase chain reaction-restriction fragment length polymorphism analysis. The chi-square test was used to calculate statistical significance. Haplotype analysis was performed using the SNPstats web tool. Of the two polymorphisms, only the MMP-8 -799 T/T genotype was significantly associated with an increased risk of ovarian cancer (OR 3.78, 95 % CI 1.18-12.13). The Kaplan-Meier analysis for this polymorphism showed that patients with the T/T genetic variant had a tendency toward significant worse overall survival compared with patients with the C/C + C/T genotypes. Haplotype analysis revealed no significant differences in haplotype distribution between benign ovarian tumors, malignant ovarian cancer, and controls. This study suggests that MMP-8 promoter gene polymorphism -799 T/T is significantly associated with an increased risk of ovarian cancer in Mexican women.

Differential effect of DDT, DDE, and DDD on COX-2 expression in the human trophoblast derived HTR-8/SVneo cells.

The purpose of this study was to investigate the effect of 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) isomers on COX-2 expression in a human trophoblast-derived cell line. Cultured HTR-8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX-2 mRNA and protein expression were assessed by RT-PCR, Western blotting, and ELISA. Prostaglandin E2 production was also measured by ELISA. Both COX-2 mRNA and protein were detected under control (unexposed) conditions in the HTR-8/SVneo cell line. COX-2 protein expression and prostaglandin E2 production but not COX-2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX-2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX-2 by these organochlorines pesticides appears to be at the translational level.

PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

OBJECTIVE: Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. METHODS: We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. RESULTS: Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. CONCLUSIONS: These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

Effect of clindamycin treatment on vaginal inflammatory markers in pregnant women with bacterial vaginosis and a positive fetal fibronectin test.

OBJECTIVE: To compare the levels of interleukin (IL)-1beta, IL-6, and matrix metalloproteinase (MMP)-8 in the vaginal secretions of pregnant women with a positive fetal fibronectin (fFN) test result with or without asymptomatic bacterial vaginosis (BV) before and after treatment with oral clindamycin. METHODS: A prospective cohort study was conducted among 43 pregnant women with a positive fFN test result. All patients were treated with clindamycin, and the pre- and post-treatment levels of IL-1beta, IL-6, and MMP-8 were compared. RESULTS: Before treatment, levels of IL-1beta and MMP-8 were significantly higher in women with BV compared with women without BV (P<0.05). Vaginal levels of IL-1beta and IL-6, but not MMP-8, decreased after treatment in pregnant women with BV. CONCLUSIONS: The inability of clindamycin to decrease MMP-8 vaginal levels may explain why it is ineffective in reducing preterm birth in pregnant women with positive fFN and BV.

No evidence of the human chorionic gonadotropin-beta gene 5 betaV79M polymorphism in Mexican women.

Human chorionic gonadotropin (hCG) is a placental hormone essential for the maintenance of pregnancy. Previous studies have shown a G to A transition in exon 3 of the hCGbeta gene 5, which changes the naturally occurring valine to methionine in codon 79. The frequency of this transition varies among different ethnic groups, being high in USA women, and less common, or absent, in various European populations. The purpose of the present study was to determine the frequency of the betaV79M allelic variant of the beta-subunit of hCG in a Mexican population, and to compare this frequency with those found in other ethnic groups. Placental DNA from 161 pregnant Mexican women was genotyped for the betaV79M by polymerase chain reaction (PCR)-restriction fragments length polymorphism analysis. No polymorphic betaV79M alleles were identified in the population studied. The allele and genotypic frequencies of betaV79M polymorphism in Mexican Mestizo women were significantly different from those reported for the US population, but not from five different European populations. In contrast to what has been found in women from the USA, it seems that the hCGbeta V79M polymorphism is absent or extremely rare in Mexican Mestizo women.

Progesterone receptor gene polymorphisms are not associated with preterm birth in a Hispanic population.

OBJECTIVE: To determine whether increased frequency of mutant alleles of the progesterone receptor gene (PGR) was associated with preterm birth in a population of Hispanic women. METHODS: Placental DNA from 64 patients who had preterm births and 54 control patients was genotyped for 4 progesterone receptor gene polymorphisms by polymerase chain reaction (PCR) restriction fragment length polymorphism. The chi(2) test and t test were used to calculate statistical significance. Linkage disequilibrium was calculated using the Linkage Disequilibrium Analyzer program. RESULTS: The genotypic frequencies of the 4 polymorphisms were not significantly different between the study and control groups. In addition, there was complete linkage disequilibrium between V660L, H770H, and PROGINS polymorphisms, but not with +331G/A polymorphism. CONCLUSIONS: The present study suggests that polymorphisms in the progesterone receptor gene are unlikely to be associated with an increased risk of preterm birth in a Hispanic population.

Vaginal matrix metalloproteinase levels in pregnant women with bacterial vaginosis.

OBJECTIVE: To compare matrix metalloproteinase (MMP)-8 and MMP-9 levels in the vaginal secretions of pregnant women with or without asymptomatic bacterial vaginosis (BV). METHODS: In this study, vaginal levels and molecular forms of MMP-8 and MMP-9 were studied in 36 pregnant women between 28 and 34 weeks of gestation with asymptomatic BV and 41 pregnant women, matched for gestational age, without BV. RESULTS: Vaginal MMP-8 concentrations were significantly higher (P = .023) in BV-positive women. There was no significant difference in MMP-9 levels between healthy pregnant controls and BV-positive pregnant women. The presence of MMP-8 was confirmed by a 38-kd band on Western blots. CONCLUSIONS: Our findings show that BV is associated with increased levels of MMP-8 in vaginal fluid. Increased production of collagen-degrading enzymes such as MMP-8 is a possible cause of spontaneous preterm delivery in pregnant women with asymptomatic BV.

Genetic variation in the interleukin-8 gene promoter and vaginal concentrations of interleukin-8 are not associated with bacterial vaginosis during pregnancy.

Interleukin-8 (IL-8) may play a role in the activation of the vaginal immune system during bacterial vaginosis. However, contradictory results were obtained regarding the involvement of IL-8 in the immunological response during bacteria vaginosis. These apparently contradictory results could be due to different genetic variations of the study groups. Since some gene polymorphisms may affect the level of IL-8 production, the aim of this study was to determine whether the frequency IL-8 promoter alleles and levels of IL-8 in vaginal fluid are associated with bacterial vaginosis during pregnancy. Genotyping for IL-8 polymorphisms in the promoter region of the gene was performed in 34 pregnant women with asymptomatic bacterial vaginosis matched for gestational age with 38 pregnant women without vaginosis. Additionally, vaginal IL-8 levels were assayed by the dual monoclonal antibody sandwich enzyme-linked immunosorbent assay technique. The frequencies of the three polymorphisms were not significantly different between control women and women with bacterial vaginosis. In addition, there was no linkage disequilibrium between the polymorphisms. Furthermore, there was no statistical difference in median vaginal levels of IL-8 between both groups. Neither the frequencies of IL-8 polymorphic alleles nor levels of IL-8 in vaginal fluid were associated with bacterial vaginosis.

Effects of progranulin on blastocyst hatching and subsequent adhesion and outgrowth in the mouse.

Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn, also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of Grn mRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.