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DNA methylation is considered as one of the most important epigenetic mechanisms and it is catalyzed by DNA methyltransferases (DNMTs). DNMT1 abundance has been frequently seen in urogenital system tumors but the reasons for this abundance are not well understood. We aimed to look into the effects of Wnt/β-catenin signaling pathway on overexpression of DNMT1 and aberrant expression of UHRF1 and HAUSP which are responsible for stability of DNMT1 at transcriptional and protein levels in urogenital cancers. In this context, firstly, Wnt/β-catenin signaling pathway was activated by using SB216763 which is a glycogen synthase kinase-3 (GSK3) β inhibitor. Cell proliferation levels in bladder cancer cells, renal cell carcinoma, and prostate cancer cells treated with GSK3β inhibitor (SB216763) were detected by WST-1 reagent. WIF-1 gene methylation profile was determined by methylation-specific PCR (MSP); expression levels of target genes β-catenin and WIF-1 by real-time PCR; and protein levels of β-catenin, DNMT1, pGSK3β(Ser9), HAUSP, and UHRF1 by Western Blot. Our results indicated that treatment with SB216763 caused an increased cell proliferation at low dose. mRNA levels of β-catenin increased after treatment with SB216273 and protein levels of pGSK3β(Ser9), β-catenin, and DNMT1 increased in comparison to control. HAUSP and UHRF1 were either up-regulated or down-regulated at the same doses depending on the type of cancer. Also, we showed that protein levels of DNMT1, β-catenin, HAUSP, and UHRF1 decreased after re-expression of WIF-1 following treatment with DAC. In Caki-2 cells, β-catenin pathway might have accounted for the stability of DNMT1 expression, whereas such relation is not valid for T24 and PC3 cells. Our findings may offer a new approach for determination of molecular effects of Wnt/β-catenin signal pathway on DNMT1. This may allow us to identify new molecular targets for the treatment of urogenital cancers

Foxa1 Knock-Out Via Crispr/Cas9 Altered Casp-9, Bax, Ccnd1, Cdk4, And Fibronectin Expressions In Lncap Cells

Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P < 0.05, fold change: similar to 40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, beta-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment.Wo

Might E-Cadherin Promoter Polymorphisms Of Rs16260 And Rs5030625 Associate With The Risk Of Nephrolithiasis?

Purpose To study whether −160 C > A (rs16260) and −347 G > GA (rs5030625) single nucleotide polymorphisms of the regulatory region (rSNPs) of CDH1 gene modulate the risk of nephrolithiasis. Methods Genomic DNA of 101 patients with calcium oxalate nephrolithiasis and 114 healthy controls were screened for both polymorphisms, using polymerase chain reaction-restriction fragments length polymorphism method (PCR-RLFP). Haplotype frequencies were also analyzed. To determine the association of rSNPs of CDH1 gene with the clinicopathological features of nephrolithiasis, nearly all possible etiological factors were documented. These factors were family history, gender, age, body mass index, liquid consumption, eating habits, tea–coffee and meat (oxalate rich) consumption, adequate physical activity, and all serum and urine levels—the serum levels of Na, K, Cl, phosphate, Ca, Mg, uric acid, albumin, blood urea nitrogen (BUN), creatinine and serum parathyroid hormone (PTH) as well as 24 h urine excretions of creatinine, Na, K, Cl, phosphate, Ca, Mg, citrate, oxalate, uric acid, albumin and BUN. Results Significant differences were found between rs16260 and the risk of nephrolithiasis. Patients having CA genotype of rs16260 CDH1 polymorphism were associated with an almost trifold increased risk for developing kidney stone than those with the AA genotype (95 % CI 1.08–7.28, OR 2.8, P = 0.033). We also found that non-A allele carriers (CC) had significantly higher nephrolithiasis risk associated with the clinicopathological characteristics including serum calcium (P = 0.027) and 24 h urinary magnesium level (P = 0.042). Moreover, we did find a directly proportional relationship between the CA genotype and serum calcium levels (P = 0.041). There was no significant difference between patients and controls in terms of the distribution of rs5030625 genotypes and alleles (P > 0.05). Likewise, no associations between the rs16260 and rs5030625 haplotypes and susceptibility to kidney stone were observed (P > 0.05). Conclusion Regulatory variants of rs16260 of the CDH1 gene may confer susceptibility to nephrolithiasis. This may have important implications for understanding the pathophysiological mechanisms of the disease and suggesting novel targets for drug treatment.PubMedWoSScopu

Do The Expressions of Epithelial-Mesenchymal Transition Proteins, Periostin, Integrin-Alpha 4 and Fibronectin Correlate With Clinico-Pathological Features and Prognosis of Metastatic Castration-Resistant Prostate Cancer?

Development of metastatic castration-resistant prostate cancer is a result of the lack of an apoptotic response by the tumor cells and loss of the ability to stick to adjacent cells through epithelial-mesenchymal transition. Although there are several strongly recommended biomarkers for determining prognosis of metastatic castration-resistant prostate cancer, only few of them may help decide the selection of the optimal treatment option. The mode of treatment sequencing in metastatic castration-resistant prostate cancer will be based on the individual characteristics of the patient. In this study, we aimed to explain the correlation between the expression characteristics of periostin, integrin-alpha 4, and fibronectin in metastatic castration-resistant prostate cancer patients and their clinico-pathological data comprising Gleason score, PSA levels, and metastatic sites in the process of epithelial-mesenchymal transition. We evaluated by using Western blotting, periostin, integrin-alpha 4, and fibronectin expressions in peripheral blood samples of metastatic castration-resistant prostate cancer patients (n=40), benign prostatic hyperplasia patients (n=20), and the healthy control group (n=20). Associations between changes in the protein expressions and clinico-pathological parameters were also analyzed in the metastatic castration-resistant prostate cancer group. When comparing BPH and healthy groups with the metastatic castration-resistant prostate cancer group, a reduced expression of integrin-alpha 4 was found in metastatic patients, albeit being statistically insignificant (P>0.05). Protein expressions of periostin and fibronectin in the metastatic castration-resistant prostate cancer group were higher than those in the BPH and heathy groups (P<0.001). Increased periostin expression in metastatic patients was significantly associated with bone metastasis (P<0.05). Elevated periostin and fibronectin levels in metastatic castration-resistant prostate cancer patients may be appropriate targets of therapeutic intervention in the future.Wo

Might E-cadherin promoter polymorphisms of rs16260 and rs5030625 associate with the risk of nephrolithiasis?

Purpose: To study whether - 160 C > A (rs16260) and -347 G > GA (rs5030625) single nucleotide polymorphisms of the regulatory region (rSNPs) of CDH1 gene modulate the risk of nephrolithiasis. Methods: Genomic DNA of 101 patients with calcium oxalate nephrolithiasis and 114 healthy controls were screened for both polymorphisms, using polymerase chain reaction-restriction fragments length polymorphism method (PCR-RLFP). Haplotype frequencies were also analyzed. To determine the association of rSNPs of CDH1 gene with the clinicopathological features of nephrolithiasis, nearly all possible etiological factors were documented. These factors were family history, gender, age, body mass index, liquid consumption, eating habits, tea-coffee and meat (oxalate rich) consumption, adequate physical activity, and all serum and urine levels-the serum levels of Na, K, Cl, phosphate, Ca, Mg, uric acid, albumin, blood urea nitrogen (BUN), creatinine and serum parathyroid hormone (PTH) as well as 24 h urine excretions of creatinine, Na, K, Cl, phosphate, Ca, Mg, citrate, oxalate, uric acid, albumin and BUN. Results: Significant differences were found between rs16260 and the risk of nephrolithiasis. Patients having CA genotype of rs16260 CDH1 polymorphism were associated with an almost trifold increased risk for developing kidney stone than those with the AA genotype (95 \% CI 1.08-7.28, OR 2.8, P = 0.033). We also found that non-A allele carriers (CC) had significantly higher nephrolithiasis risk associated with the clinicopathological characteristics including serum calcium (P = 0.027) and 24 h urinary magnesium level (P = 0.042). Moreover, we did find a directly proportional relationship between the CA genotype and serum calcium levels (P = 0.041). There was no significant difference between patients and controls in terms of the distribution of rs5030625 genotypes and alleles (P > 0.05). Likewise, no associations between the rs16260 and rs5030625 haplotypes and susceptibility to kidney stone were observed (P > 0.05). Conclusion: Regulatory variants of rs16260 of the CDH1 gene may confer susceptibility to nephrolithiasis. This may have important implications for understanding the pathophysiological mechanisms of the disease and suggesting novel targets for drug treatment

Effects of Meloxicam, Alone and in Combination With Chemotherapeutic Agents on the Raf/Mek/Erk Pathway in Raji Cells

WOS: 000344634100027Background: Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to induce apoptosis and inhibit cell proliferation in tumor cells when combined with conventional chemotherapeutic agents. We aimed to investigate the effect of NSAIDs (meloxicam and lornoxicam) plus conventional chemotherapeutic agents on RAF/MEK/ERK pathway in Burkitt's lymphoma cell line (Raji). Methods: Raji cells were treated with meloxicam, lornoxicam, carboplatin, gemcitabine and 5-fluorourasil (5-FU) for 24h. Then, the cells were treated with selected low and high doses of meloxicam alone or in combination with two different gemcitabine concentrations. Presence of apoptotic cells were assessed using fluorescence microscopy. mRNA expression levels of RAF1, MEK1, ERK1 and ERK2 genes were analyzed with quantitative real time PCR. Results: Apoptotic and anti-proliferative effects were not observed by carboplatin, 5-FU and lornoxicam. However, meloxicam and gemcitabine treatment resulted in a decrease in viability. Therefore, we examined the effects of low and high doses of meloxicam, gemcitabine and their combinations on this pathway. The antiproliferative effects of gemcitabine were not significantly enhanced in the presence of meloxicam. Furthermore, mRNA expression levels of RAF1, MEK1 and ERK1/2 genes were downregulated in gemcitabine alone and upregulated in meloxicam alone. Interestingly, the decrease shown in low and high doses of gemcitabine alone reversed in low dose meloxicam plus gemcitabine treatment. Conversely, downregulation of these genes by high dose gemcitabine did not change with meloxicam treatment. Conclusion: We believe that it may be fundamental to further evaluate this inhibitory drug interaction

FOXA1 knock-out via CRISPR/Cas9 altered Casp-9, Bax, CCND1, CDK4, and fibronectin expressions in LNCaP cells

Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P < 0.05, fold change: similar to 40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, beta-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment

The Impact of Gene Polymorphisms on the Success of Anticholinergic Treatment in Children with Overactive Bladder

Aim. To determine the impact of gene polymorphisms on detrusor contraction-relaxation harmony in children with lower urinary tract symptoms (LUTS). Materials and Methods. Toilet trained children older than 5 years of age with LUTS and normal neurological examination underwent videourodynamic study. The control group was composed of age matched children with no voiding complaints. The study group who filled out the voiding dysfunction symptom score before and after the treatment received standard oxybutynin treatment and was reevaluated 1 year after treatment. Genomic DNA was isolated from all patients and subjected to PCR for amplification. Genotyping of ARGHEF10, ROCK2, ADRB3, and CYP3A4 was carried out with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Results. 34 (45%) and 42 (55%) patients were enrolled in the study and control group, respectively. ARGEF10 GG, ADRB3 TC, and CYP3A4 AG genotype patients displayed insignificant difference between pre-and posttreatment voiding dysfunction symptom score and bladder volumes. Conclusions. The polymorphism of genes in the cholinergic pathway did not significantly differ clinical parameters. On the other hand, polymorphic patients in the adrenergic pathway seemed to suffer from clinical disappointment. For this reason, we think that the neglected adrenergic pathway could be a new therapeutic target for the treatment of anticholinergic resistant LUTS in children

Comparative analysis of epi-miRNA expression levels in local/locally advanced and metastatic prostate cancer patients

Abnormal expression of enzymes involved in epigenetic mechanisms, such as DNA methyl transferases, can trigger large chaos in cellular gene expression networks and eventually lead to cancer progression. In our study, which is a pioneer in the literature that clinicopathologically evaluates the expression of 30 epi-miRNAs in prostate cancer (PCa), we investigated which of the new miRNA class epi-miRNAs could be an effective biomarker in the diagnosis and progression of PCa. In this study, the expression levels of 30 epi-miRNAs in whole blood samples from 25 control, 25 PCa and 40 metastatic PCa patients were investigated by the Quantitative Real-Time PCR method. Then, promoter methylation levels of 11 epi-miRNAs, whose expression levels were found to be significantly higher, were examined by methylation-specific qPCR method. The correlations between miRNA expression levels and clinicopathological parameters (Gleason Score (GS), PSA levels, TNM Staging) in different stages of PCa groups as well as disease-specific expression levels were examined. We found a hypomethylation in the promoter regions of miRNAs that showed a direct proportional increase with PSA levels (miR34b/c, miR-148a, miR-152), GS's (miR-34a-5p, miR-34b/c, miR-101-2, miR-126, miR-148a, miR-152, miR-1855p) and T staging (miR-34a-5p, miR-34b/c, miR-101-2, miR-126, miR-140, miR-148a, miR-152, miR-185-5p) (p < 0.05). When miR-200a/b was evaluated according to clinicopathological parameters, it acted as an oncomiR in local/local advanced PCa and as a tumor-suppressor-miR in metastatic stage. This study is novel in the sense that our findings draw attention to the important role of miRNAs as diagnostic and prognostic biomarkers in PCa.Gazi University Research FundGazi University [01/2019-11]This study was conducted with financial support from the Gazi University Research Fund [the project code number 01/2019-11].WOS:0005646940000012-s2.0-85088216243PubMed: 3268307