Evaluation of upper-extremity function following surgical treatment of displaced proximal humerus fractures in children.

This study aims to assess the functional outcome of children treated with elastic stable intramedullary nailing (ESIN) for displaced proximal humerus fractures using the short version of the disabilities of the arm, shoulder, and hand outcome questionnaire (Quick DASH). Fifty-eight consecutive children with displaced proximal humerus fractures were treated with ESIN. Fifty-two children (89.7%) were available for follow-up and responded to the questionnaire after hardware removal. Average age at time of injury was 11.1 ± 2.8 years (range, 4-15.9). Among the 52 patients available for evaluation, 37 had a Quick DASH score of 0 (71.1%), seven a score of 2.3 (13.5%), four a score of 4.5 (7.7%), and four a score of 6.5 (7.7%). Shoulder and elbow ranges of motion were comparable with the noninjured side. No skin irritation or local infections were observed. There was no radiographic evidence of delayed union, refracture, hardware migration, or secondary displacement. Mean follow-up was 18.3 ± 8.3 months (range, 6-39.5). Our study reports good functional outcomes in children with closed isolated fractures, both physeal and metaphyseal, of the proximal humerus treated with ESIN. The use of a standardized rating scale is recommended to homogeneously compare functional outcome and may facilitate the comparison of clinical outcome in different patient populations.

Gene panel model predictive of outcome in patients with prostate cancer.

In men at high risk for prostate cancer, established clinical and pathological parameters provide only limited prognostic information. Here we analyzed a French cohort of 103 prostate cancer patients and developed a gene panel model predictive of outcome in this group of patients. The model comprised of a 15-gene TaqMan Low-Density Array (TLDA) card, with gene expressions compared to a standardized reference. The RQ value for each gene was calculated, and a scoring system was developed. Summing all the binary scores (0 or 1) corresponding to the 15 genes, a global score is obtained between 0 and 15. This global score can be compared to Gleason score (0 to 10) by recalculating it into a 0-10 scaled score. A scaled score ≥2 suggested that the patient is suffering from a prostate cancer, and a scaled score ≥7 flagged aggressive cancer. Statistical analyses demonstrated a strongly significant linear correlation (p=3.50E-08) between scaled score and Gleason score for this prostate cancer cohort (N=103). These results support the capacity of this designed 15 target gene TLDA card approach to predict outcome in prostate cancer, opening up a new avenue for personalized medicine through future independent replication and applications for rapid identification of aggressive prostate cancer phenotypes for early intervention.

DNA methylation and soy phytoestrogens: quantitative study in DU-145 and PC-3 human prostate cancer cell lines.

AIM: DNA hypermethylation is an epigenetic mechanism which induces silencing of tumor-suppressor genes in prostate cancer. Many studies have reported that specific components of food plants like soy phytoestrogens may have protective effects against prostate carcinogenesis or progression. Genistein and daidzein, the major phytoestrogens, have been reported to have the ability to reverse DNA hypermethylation in cancer cell lines. The aim of this study was to investigate the potential demethylating effects of these two soy compounds on BRCA1, GSTP1, EPHB2 and BRCA2 promoter genes. METHODS & MATERIALS: Prostate cell lines DU-145 and PC-3 were treated with genistein 40 µM, daidzein 110 µM, budesonide (methylating agent) 2 µM and 5-azacytidine (demethylating agent) 2 µM. In these two human prostate cancer cell lines we performed methylation quantification by using Methyl Profiler DNA methylation analysis. This technique is based on a methylation-specific digestion followed by quantitative PCR. We analyzed the corresponding protein expression by western blotting. RESULTS: Soy phytoestrogens induced a demethylation of all promoter regions studied except for BRCA2, which is not methylated in control cell lines. An increase in their protein expression was also demonstrated by western blot analysis and corroborated the potential demethylating effect of soy phytoestrogens. CONCLUSION: This study showed that the soy phytoestrogens, genistein and daidzein, induce a decrease of methylation of BRCA1, GSTP1 and EPHB2 promoters. Therefore, soy phytoestrogens may have a protective effect on prostate cancer. However, more studies are needed in order to understand the mechanism by which genistein and daidzein have an inhibiting action on DNA methylation.

miRNAs differentially expressed in prostate cancer cell lines after soy treatment.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that have aberrant expression in prostate cancer tissues. miRNAs are involved in the initiation and progression of cancer, and several miRNAs have been characterized as tumor suppressors or oncogenes. It has been shown that some miRNAs can be directly regulated from their own promoters by epigenetic alterations in cancer cells. Moreover, phytoestrogens are known to have epigenetic action on gene transcription. Hence, we conducted here an examination of the miRNA expression profile in human prostate cancer cell lines after soy phytoestrogen treatment. MATERIALS AND METHODS: The comparative miRNA expression profiles of prostate cell lines (PC-3, DU145, LNCaP) after a 48-h treatment of 40 µM genistein, 110 µM daidzein, or 2 µM 5-azacytidine (5-AZA, a demethylating agent) were conducted with a Taqman low-density array. RESULTS: We found that out of 377 miRNAs tested, 180, 170 and 150 miRNAs were amplified with 2% of variation in the triplicate in PC-3, DU145 and LNCap cells, respectively, and only 5 miRNAs for PC-3 and DU145 cells and 4 miRNAs for LNCap exhibited a significant change in their expression. Treatment with genistein or daidzein had similar effects on miRNA regulation to those of 5-AZA treatment. CONCLUSION: This work demonstrated a new role of isoflavones on the regulation of miRNAs in prostate cancer.

BRCA1, BRCA2, AR and IGF-I expression in prostate cancer: correlation between RT-qPCR and immunohistochemical detection.

Identification and characterization of biomarkers in prostate cancer are important for improving the diagnosis. The aim of this study was to determine differences in the expression of 4 genes according to the stage of malignancy in prostate cancer. We analyzed BRCA1, BRCA2, androgen receptor (AR) and IGF-I gene expression in a cohort of 98 prostate biopsies. We used TaqMan RT-qPCR for mRNA detection, and correlation with proteins was performed using immunohistochemistry. Among the 98 studied prostate biopsies, high heterogeneity in the expression of the 4 genes was detected among the different histological types. However, down-regulation of BRCA1 and BRCA2 mRNA was detected, particularly in the normal tissues. The expression of AR was dependent on the stage of the tumor. The IGF-I gene was specifically expressed in the tumor tissues. Upon comparison between protein and mRNA expression for BRCA1, BRCA2 and AR, we obtained a trend; however, this did not achieve statistical significance. Regarding IGF-I, a correlation between mRNA expression and staining intensity of the protein was found to be significant (p<0.012). The AR biomarker was found to be slightly correlated with the prostate cancer diagnosis (p=0.013). AR was found to be decreased in the tumors with a 43% sensitivity and 90% specificity. The relative risk of 2.05 (1.13-3.69) indicated a 2­fold higher chance of cancer occurrence when AR was ≤0.206.

Soy phytoestrogens modify DNA methylation of GSTP1, RASSF1A, EPH2 and BRCA1 promoter in prostate cancer cells.

BACKGROUND: The aim of this study was to determine the effects of soy phytoestrogens on the methylation of promoter genes in prostate tumors. The incidence of prostate cancer in Asia is thirty percent lower than in Western countries. Since soy phytoestrogens represent a large portion of the Asian diet, evidence suggests their protective effect against prostate cancer. MATERIALS AND METHODS: In three human prostate cancer cell lines, methylation-specific-PCR was used to determine the effect of soy isoflavones (genistein and daidzein), compared to known demethylating agent 5-azacytidine as control in the promoter regions of glutathione S-transferase P1 (GSTP1), Ras association domain family 1 (RASSF1A), ephrin B2 (EPHB2) and breast cancer 1 (BRCA1) genes. In parallel, immunohistochemistry was used to assess the effects of genistein, daidzein and 5-azacytidine treatment on the corresponding protein expression. RESULTS: All studied promoters, with the exception of that for BRCA1, were strongly methylated without treatment. After treatment by phytoestrogens, demethylation of GSTP1 and EPHB2 promoter regions was observed and an increase in their protein expression was demonstrated by immunohistochemistry. CONCLUSION: Epigenetic modifications of DNA, such as the promoter CpG island demethylation of tumor suppressor genes, might be related to the protective effect of soy on prostate cancer.

Transcriptional profiling of breast cancer cells exposed to soy phytoestrogens after BRCA1 knockdown with a whole human genome microarray approach.

The estrogen-like properties of the soy phytoestrogens could modulate the estrogen-dependent expression of BRCA1 oncosuppressor, which is highly involved in hereditary and sporadic breast cancer. In order to better understand the importance of BRCA1 function and the role of other genes involved around BRCA1 in the phytoestrogen pathways, we have exploited the BRCA1-specific knockdown by RNA interference using double stranded small interfering RNA (siRNA) in breast tumor cell lines (MCF-7, MDA-MB-231) and a fibrokystic breast cell line (MCF-10a) and treated with 18.5 microM genistein or 78.5 microM daidzein for 72 h. We used pangenomic microarrays and subsequently TLDA analysis and demonstrated that cumulated BRCA1 knockdown with soy isoflavone supplementations in breast cell lines seems to modulate apoptosis, MAPK pathway, cell communication, xenobiotic metabolism, and sterol metabolism. Also, transient BRCA1 deficiency in breast cell lines significantly diminished or reversed gene expression after phytoestrogen supplementation. We observed that the significant decrease expression of apoptosis-related genes such as BAX, and the increase expression of BCL2, under BRCA1 knockdown condition, were completely reversed after phytoestrogen treatments. These results underlined the role of BRCA1 expression in breast carcinogenesis and suggested that soy phytoestrogen supplementation could play a role in cancer.

Gene expression profiling of breast cancer cell lines in response to soy isoflavones using a pangenomic microarray approach.

Although the rate of breast cancer differs between women in Asian and Western countries, molecular genetics/genomics basis of this epidemiological observation remains elusive. Moreover, the intake of phytoestrogens is associated with a lower incidence of breast cancer. Genistein and daidzein are the primary soy isoflavones with a chemical structure similar to estrogens. Conceivably, the actions of phytoestrogens on gene expression signatures might mediate their postulated effects on breast cancer pathogenesis. The present study evaluated the transcriptional responsiveness of breast cancer cells to soy phytoestrogens using a whole-genome microarray-based approach. Human breast cancer cell lines and a fibrocystic breast cell line were treated with genistein or daidzein. We identified 278 and 334 differentially expressed genes after genistein or daidzein treatment, respectively, in estrogen-positive (MCF-7) and estrogen-negative (MDA-MB-231, MCF-10a) cells. Hierarchical clustering of this finding revealed a significant modulation, respectively, of 246 or 169 genes after genistein or daidzein exposures. Importantly, the molecular pathways for the differentially expressed genes included those that relate to cell communication, biodegradation of xenobiotics, lipid metabolism, signal transduction, and cell growth/death. These molecular observations collectively contribute to a growing knowledgebase on the putative mechanism(s) of action of phytoestrogens in breast cancer pathogenesis and chemoprevention.

Phytoestrogens regulate the expression of genes involved in different biological processes in BRCA2 knocked down MCF-7, MDA-MB-231 and MCF-10a cell lines.

Breast cancer is a public health problem in the Western countries. Several studies have shown that BRCA2, like BRCA1 oncosuppressors, are strongly involved in hereditary and sporadic mammary carcinogenesis. It has also been suggested that soy has a protective effect against breast cancer in Asia and, more particularly, phytoestrogens such as daidzein and genistein. Thus, phytoestrogens may have an impact on the expression of BRCA2 gene, and there is a possible link between BRCA2 and genes acting around the BRCA2. To focus on these processes, we set up the BRCA2 specific knockdown by RNA interference in two breast tumor cell lines (MCF-7 and MDA-MB-231) and also in a non-tumorigenic breast cell line (MCF-10a). After inhibition of BRCA2 expression, cells were maintained in different conditions and treated with either daidzein or genistein or left untreated. Microarray analysis of mRNAs isolated from the BRCA2 knocked down MCF-7, MDA-MB-231, and MCF-10a cell lines after being treated with phytoestrogens showed 35 differentially expressed genes between positive-ERbeta cells and negative-ERbeta cells. After genistein or daidzein treatments, BRCA1 was found to be up-regulated when knocked down with BRCA2-siRNA MCF-7 and BRCA2 was found to be up-regulated when knocked down with BRCA2-siRNA MDA-MB 231 cells. In MCF-10a, we observed a significant decrease in BAX and BCL2 expressions with a greater effect of daidzein. We also found an increase in BRIP expression between genistein and daidzein treatment knocked down with BRCA2-siRNA MCF-7 and MDA-MB-231 cell lines.

Genistein and daidzein act on a panel of genes implicated in cell cycle and angiogenesis by polymerase chain reaction arrays in human prostate cancer cell lines.

BACKGROUND: The prostate cancer most frequently affects men. The ethnic origin and family antecedents of prostate cancer are established as risk factors. The genetic factors associated with environmental factors such as the nutrition also play a role in the development of the cancer. Epidemiological studies showed that the Asian populations exhibited an incidence of prostate cancer markedly subordinate by comparison with the Western populations. This would be explained partially by their important consumption of soy. Both main phytoestrogens of soy, the genistein and the daidzein, present anti-proliferative properties. METHODS: For that purpose, we used different prostate cancer cell lines (LNCaP, DU 145, PC-3) and, by flow cytometry, we determined the concentration of phytoestrogens inducing a cell cycle arrest and the required time of incubation. RESULTS: Then, the effects of 40microM genistein or 110microM daidzein for 48h were determined and studied on the expression of genes involved in the human cell cycle and angiogenesis and conducted by SYBR green quantitative PCR. CONCLUSION: We demonstrated modulations of cyclin-dependent kinase-related pathway genes, DNA damage-signaling pathway and a down-regulation of EGF and IGF.

Methylation analysis of BRCA1, RASSF1, GSTP1 and EPHB2 promoters in prostate biopsies according to different degrees of malignancy.

Prostate cancer is the most common cancer among men and the second leading cause of cancer-related deaths in the United States. CpG island methylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. Its role was investigated at multiple gene sites during prostate carcinogenesis. Methylation-specific polymerase chain reaction (MS-PCR) was used to analyze 4 interest gene promoter status in 12 patients with adenocarcinoma, 7 patients with prostate intraepithelial neoplasia, 3 patients with peritumor tissues and 15 healthy patients, so a total of 37 prostate biopsy samples constituted the cohort of the study. Despite the biopsy histology, the results have confirmed that BRCA1, RASSF1, GSTP1 and EPHB2 promoter methylation was found in each sample, except two.

Immunohistochemical staining of mucin 1 in prostate tissues.

UNLABELLED: Prostate cancer is a major public health problem in the world. Molecular studies are necessary for the development of prognostic markers in prostate cancer. There is a great interest in mucin studies in treatment development of human malignancies, including prostate cancer. Nevertheless, their expressions in prostate cancer need further investigation. MATERIALS AND METHODS: Mucin 1 (MUC1) expression was examined in 100 prostate biopsies and were compared with prostate carcinoma cell lines (DU-145, PC-3, LNCaP) by immunohistochemistry. RESULTS: Biopsies were healthy, tumor, peritumor or presented an intraepithelial neoplasia. Staining of MUC1 was exihibited in PC-3 cells, was higher in DU-145, and was not expressed by LNCaP. Tumor sections presented more positive expression of MUC1 than non-tumor sections. CONCLUSION: MUC1 expression is correlated with the histological degree of malignancy.

Expression analyses of nuclear receptor genes in breast cancer cell lines exposed to soy phytoestrogens after BRCA2 knockdown by TaqMan Low-Density Array (TLDA).

BACKGROUND: Most of breast cancers are considered sporadic and modulation of the two major genes BRCA1 and BRCA2 expressions caused by tissue-specific somatic mutations lead to this pathology. The nutritional intake of phytoestrogens seems to reduce the risk of breast cancer and investigation of their potential as anticancer agents has increased. However, the possible mechanisms and signalling pathways of phytoestrogen action in breast cancer prevention remains unknown. RESULTS: Using Taqman Low Density Array technology, we investigated the BRCA2 loss of function role in sporadic breast cancers and the links existing with soy isoflavones on a panel of nuclear receptor expression. Human breast cell lines (MCF-7, MDA-MB-231, and MCF-10a) were transfected by BRCA2-siRNA and treated with genistein (18.5 muM) or daidzein (78.5 muM) for 72 h. Generating the transitory knockdown of BRCA2 oncosuppressor, we observed different modulations in several nuclear receptor genes such as ER, RAR and RXR, as well as PPARs and VDR according to the studied breast cell line. Additional isoflavone treatments showed different nuclear receptor gene modulation profiles. CONCLUSION: Our results seemed to implicate the oncosuppressor BRCA2 and the phytoestrogen pathways in different nuclear gene expressions via an ER-independent manner.

Genetic polymorphisms in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 genes and association with bladder cancer risk in a French cohort.

UNLABELLED: Tobacco smoking and environmental exposures are the main known risk factors for bladder cancer (BC) via exposure to chemical carcinogens. Genetic differences in the metabolism of chemicals have been suggested to be associated with individual susceptibility to BC. Polymorphisms in genes coding to metabolising enzymes, resulting in variation of carcinogen detoxification efficiency, may therefore change the response of individuals to chemical carcinogens and be associated with an increased BC risk. PATIENTS AND METHODS: The aim of the study was to investigate the association between functional polymorphisms in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 genes and BC risk, through a hospital-based case-control study. The genotyping of 11 Single Nucleotide Polymorphisms (SNPs) was carried out on DNA of 51 bladder cancer male patients and 45 male controls. The technique of MGB (Minor Groove Binder) probes that utilize allelic discrimination with the Taqman(R) method was used. RESULTS: Individuals with NAT2 slow acetylator genotypes had a significant increase in risk of BC compared to individuals with NAT2 rapid acetylators (OR adjusted for smoking status=2.70; 95% CI, 1.10-6.61). GSTP1 Ile(105)Val variants (deletion of one - Ile/Val- and two -Val/Val-, null genotype- copies) showed a marginal increased risk of BC with OR adjusted for smoking status of 2.27 (95% CI, 0.97-5.31) compared to individuals carrying wild-type genotype (Ile/Ile). No statistically significant effects on BC risk with CYP1A1, CYP11B1 and COMT genotypes were observed. CONCLUSION: The results are consistent with previous literature among Caucasian populations.

Association between genetic polymorphisms and ovarian cancer risk.

BACKGROUND: The etiology of ovarian cancer is not fully understood. Polymorphisms in low penetrance genes involved in carcinogen and estrogen metabolism are hypothesized to play a role in the initiation of carcinogenesis. PATIENTS AND METHODS: A case-control study was conducted to investigate the role of these polymorphisms in ovarian cancer risk. The participants were genotyped for eleven polymorphisms in seven genes involved in estrogen and xenobiotic metabolism (CYP1A1, CYP1B1, COMT, GSTP1, NAT2, estrogen receptor ESR, and progesterone receptor PGR). RESULTS AND CONCLUSION: The odds ratios for ovarian cancer risk were 2.02 (95% confidence interval [CI] = 1.14-3.56) in the NAT2 intermediate acetylators and 4.07 (95% CI = 1.30-12.70) in the slow acetylators. At least three cumulative high-risk genotypes increased ovarian cancer risk, but not significantly. More studies are needed in order to define genetic ovarian risk factors.

DNA repair gene ERCC2, XPC, XRCC1, XRCC3 polymorphisms and associations with bladder cancer risk in a French cohort.

In polygenic diseases, association studies look for genetic variation such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damage have been shown to significantly reduce activity. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study bladder cancer which is viewed as a polygenic disease was investigated. The functional polymorphisms of four DNA repair genes, excision repair cross-complementing group 2 (ERCC2), Xeroderma Pigmentosum group C (XPC), and Xray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3) were analyzed. The studied population included 51 bladder cancer cases and 45 controls. The genotyping of six SNP (single nucleotide polymorphism) was carried out on these populations with the MGB (Minor Groove Binder) probe technique which uses allelic discrimination with the Taqman method. The Gln allele of the XPC 939 polymorphism was found to be associated with an increase in bladder cancer risk.

DNA repair gene ERCC2 polymorphisms and associations with breast and ovarian cancer risk.

Breast and ovarian cancers increased in the last decades. Except rare cases with a genetic predisposition and high penetrance, these pathologies are viewed as a polygenic disease. In this concept, association studies look for genetic variations such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damages have been shown to have significantly reduced. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study, 2 functional polymorphisms in a DNA repair gene ERCC2 were analyzed. The population included 911 breast cancer cases, 51 ovarian cancer cases and 1000 controls. The genotyping of 2 SNP (Single Nucleotide Polymorphism) was carried out on the population with the MGB (Minor Groove Binder) probe technique which consists of the use of the allelic discrimination with the Taqman method. This study enabled us to show an increase in risk of breast cancer with no oral contraceptive users and with women exhibiting a waist-to-hip ratio (WHR) > 0.85 for Asn homozygous for ERCC2 312.