Determination of In Vitro and In Vivo Effects of Taxifolin and Epirubicin on Epithelial-Mesenchymal Transition in Mouse Breast Cancer Cells.

Background: One of the most significant characteristics of cancer is epithelial-mesenchymal transition and research on the relationship between phenolic compounds and anticancer medications and epithelial-mesenchymal transition is widespread. Methods: In order to investigate the potential effects of Taxifolin on enhancing the effectiveness of Epirubicin in treating breast cancer, specifically in 4T1 cells and an allograft BALB/c model, the effects of Taxifolin and Epirubicin, both individually and in combination, were examined. Cell viability assays and cytotoxicity assays in 4T1 cells were performed. In addition, 4T1 cells were implanted into female BALB/c mice to conduct in vivo studies and evaluate the therapeutic efficacy of Taxifolin and Epirubicin alone or in combination. Tumor volumes and histological analysis were also assessed in mice. To further understand the mechanisms involved, we examined the messenger RNA and protein levels of epithelial-mesenchymal transition-related genes, as well as active Caspase-3/7 levels, using quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays, respectively. Results: In vitro results demonstrated that the coadministration of Taxifolin and Epirubicin reduced cell viability and cytotoxicity in 4T1 cell lines. In vivo, coadministration of Taxifolin and Epirubicin suppressed tumor growth in BALB/c mice with 4T1 breast cancer cells. Additionally, this combination treatment significantly increased the levels of active caspase-3/7 and downregulated the messenger RNA and protein levels of N-cadherin, ß-catenin, vimentin, snail, and slug, but upregulated the E-cadherin gene. It significantly decreased the messenger RNA levels of the Zeb1 and Zeb2 genes. Conclusion: The in vitro and in vivo results of our study indicate that the concurrent use of Epirubicin with Taxifolin has supportive effects on breast cancer treatment.

Silencing effects of FOXD1 inhibit metastatic potentials of the PCa via N-cadherin - Wnt/ß-catenin crosstalk.

The elucidation of the mechanisms controlling the metastatic processes is important for the development of new treatment methods to prevent the progression of localized disease to metastasis. Forkhead box D1 (FOXD1) is a member of the FOX transcription factor family and has been reported to play an important role in the development and progression of various cancers. However, its role in prostate cancer (PCa) remains only partially understood. Therefore, we aimed to explore the effects on the associated regulatory signal pathway of FOXD1 in prostate cancer. To clarify the roles of FOXD1 in prostate cancer, we used siRNA to suppress its expression in 22Rv1 cells with relatively higher expression of FOXD1. The effects of FOXD1 silencing on cell proliferation, migration and invasion were determined. WST-1 assays were used to determine cell proliferation. Cell migration and invasion were evaluated through wound healing and transwell assays. The possible underlying mechanism of FOXD1 silencing on 22Rv1 was evaluated by determining the expression of proteins related to EMT and Wnt/ß-catenin signaling pathway. Our results showed that FOXD1 was highly expressed in prostate cancer cell lines -PC-3, DU145, LNCaP and 22Rv1- compared to normal prostate epithelial cell line RWPE-1. Additionally, silencing of FOXD1 significantly reduced proliferation, migration and invasion of 22Rv1 cells. Furthermore, silencing of FOXD1 decreased the expression of ß-catenin and cyclin D1, which are involved in the Wnt/ß-catenin signaling pathway. However, it did not appear to affect the expression of EMT-related proteins other than N-cadherin. Our results suggest that silencing of FOXD1 suppresses metastatic potentials of the PCa via N-cadherin - Wnt/ß-catenin crosstalk. Therefore, the expression status of FOXD1 may be a new prognostic factor as well as a potential therapeutic target in prostate cancer treatment.

miR-148a, miR-152 and miR-200b promote prostate cancer metastasis by targeting DNMT1 and PTEN expression.

MicroRNAs (miRs) modulate the expression of target genes in the signal pathway on transcriptome level. The present study investigated the 'epigenetic-based miRNA (epi-miRNA)-mRNA' regulatory network of miR-34b, miR-34c, miR-148a, miR-152, miR-200a and miR-200b epi-miRNAs and their target genes, DNA methyltransferase (DNMT1, 3a and 3b), phosphate and tensin homolog (PTEN) and NK3 Homeobox 1 (NKX3.1), in prostate cancer (PCa) using reverse transcription-quantitative PCR. The expression level of NKX3.1 were not significantly different between the PCa, Met-PCa and control groups. However, in the PCa and Met-PCa groups, the expression level of DNMT1 was upregulated, while DNMT3a, DNMT3b and PTEN were downregulated. Overexpression of DNMT1 (~5 and ~6-fold increase in the PCa and Met-PCa groups respectively) was accompanied by a decreased expression in PTEN, indicating a potential negative association. Both groups indicated that a high level of DNMT1 is associated with the aggressiveness of cancer, and there is a a directly proportional relationship between this gene and PSA, GS and TNM staging. A significant ~2 to ~5-fold decrease in the expression levels of DNMT3a and DNMT3b was found in both groups. In the PCa group, significant associations were identified between miR-34b and DNMT1/DNMT3b; between miR-34c/miR-148a and all target genes; between miR-152 and DNMT1/DNMT3b and PTEN; and between miR-200a/b and DNMT1. In the Met-PCa group, miR-148a, miR-152 and miR-200b exhibited a significant association with all target genes. A significant negative association was identified between PTEN and DNMT1 in the Met-PCa group. It was also revealed that that miR-148a, miR-152 and miR-200b increased the expression of DNMT1 and suppressed PTEN. Furthermore, the 'epi-miRNA-mRNA' bidirectional feedback loop was emphasised and the methylation pattern in PCa anti-cancer therapeutics was highlighted.

Contrast effects of autophagy in the treatment of bladder cancer.

Bladder cancer is a disease that negatively affects patients' quality of life, but treatment options have remained unchanged for a long time. Although promising results have been achieved with current bladder cancer treatments, cancer recurrence, progression, and therapy resistance are the most severe problems preventing the efficiency of bladder cancer treatments. Autophagy refers to an evolutionarily conserved catabolic process in which proteins, damaged organelles, and cytoplasmic components are degraded by lysosomal enzymes. Autophagy regulates the therapeutic response to the chemotherapy drugs, thus determining the effect of therapy on cancer cells. Autophagy is a stress-induced cell survival mechanism and its excessive stimulation can cause resistance of tumor cells to therapeutic agents. Depending on the conditions, an increase in autophagy may cause treatment resistance or autophagic cell death, and it is related to important anti-cancer mechanisms, such as apoptosis. Therefore, understanding the roles of autophagy under different conditions is important for designing effective anti-cancer agents. The dual role of autophagy in cancer has attracted considerable attention in respect of bladder cancer treatment. In this review, we summarize the basic characteristics of autophagy, including its mechanisms, regulation, and functions, and we present examples from current studies concerning the dual role of autophagy in bladder cancer progression and therapy.

Targeting Cancer Cell Metabolism with Metformin, Dichloroacetate and Memantine in Glioblastoma (GBM).

AIM: To investigate the effects of metformin, dichloroacetate (DCA), and memantine on T98G and U87-MG human glioblastoma (GBM) cells to target tumor cell metabolism in a multi-directional manner. MATERIAL AND METHODS: IC50 levels for metformin, DCA, metformin+DCA and memantine were determined by MTT assay in T98G and U87-MG cells in vitro. Casp3, Bcl-2, Bax, c-Myc and GSK-3B protein expressions were investigated post treatments. Fifteen GBM+ tumor tissues were assessed for Casp-3, Bcl-2, Bad, Bax for apoptotic protein expression patterns. RESULTS: Cancer cell metabolism targeting drugs metformin, DCA, metformin+DCA and memantine induced cytotoxicity in a dose-dependent manner in T98G and U87-MG cells. IC50 for memantine is found as 0.5 mM (p < 0.01) which is nearly 10 times lower concentration than that of metformin. Fifteen GBM+ tumor tissues had differential apoptotic protein expressions. CONCLUSION: Memantine exerted anti-cancer mechanism of action in T98G and U87-MG cells, however, such a mechanism requires deeper investigation for GBM treatment.

Upregulation of potential regulatory signaling molecules correlate with androgen receptor splice variants AR-V7 and AR-V567es in prostate cancer metastasis.

AIM: Androgen receptor splice variants (AR-Vs) produced by alternative splicing of the AR play an important role in the treatment resistance and progression of prostate cancer (PCa). In this study, two most common AR variants and how they associate with the inflammatory response (NF-Kß) and regulatory transcriptional activity (HSP-27) genes were investigated in patients with PCa and metastatic PCa (Met-PCa). METHODS: Our study was carried out with the whole blood obtained from 25 healthy control subjects, 25 PCa patients and 39 Met-PCa patients. We examined the expression levels of AR, AR-V7 and AR-V567es genes via Real-time PCR and those of HSP-27 and NF-Kß via ELISA method. RESULTS: AR, AR-V7 and AR-V567es expressions were observed in 84.61%, 64.1%, 23.07% of Met-PCa patients respectively. The expression levels of full-length AR and variants (AR-V7 and AR-V567es) were associated with the prostate cancer stage. In the Met-PCa, the expression levels of AR, AR-V7 and AR-V567es were associated with the Gleason Scores but not with the PSA levels. AR-V7 expression levels in stage T4 patients significantly increased. NF-Kß and HSP-27 protein levels were significantly higher in Met-PCa patients. DISCUSSION: Our findings highlight the targeting of the proteostasis and inflammation pathways through inhibiting HSP-27 and NF-Kß. This might be a valuable strategy to overcome anti-androgen resistance and improve drug therapy in Met-PCa patients whose gene expression levels of AR-V7 and AR-V567es variants are high.

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 (miR-34b/c, miR-148a, miR-152), GS's (miR-34a-5p, miR-34b/c, miR-101-2, miR-126, miR-148a, miR- 152, miR-185-5p) 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 onco-miR 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.

Cucurbitacin B and cisplatin induce the cell death pathways in MB49 mouse bladder cancer model.

IMPACT STATEMENT: Alternative agents that will increase the effectiveness of cisplatin, which are widely used in the advanced stage and metastatic bladder cancer, are being investigated. In previous studies, Cucurbitacin B (CuB), which is a natural compound from the Cucurbitaceae family has been shown to inhibit the proliferation of tumor cells and create synergistic effects with cisplatin. In this study, we investigated the synergistic effect of CuB with cisplatin for the first time in bladder cancer in vitro and in vivo models. Our findings showed that CuB treatment with cisplatin reduced cell proliferation, and reduced tumor development through activating apoptosis and autophagy via PI3K/AKT/mTOR signaling pathway. Our results showed that CuB may be a new agent that can support conventional treatment in bladder cancer. Our study is important in terms of enlightening new pathways and developing new treatment methods in the treatment of bladder cancer.

Hsp-27 and NF-κB pathway is associated with AR/AR-V7 expression in prostate cancer cells.

In the present study, NF-κB inhibitor BAY 11-7082 and/or Hsp-27 inhibitor KRIBB-3 agents were used to investigate the molecular mechanisms mediating androgen receptor expression on prostate cancer cell lines. The decrease observed in androgen receptor and p65 expressions, particularly at 48 h, in parallel with the decrease in the phosphorylation of the p-IKK α/ß and p-Hsp-27 proteins in the LNCaP cells, indicated that androgen receptor inactivation occurred after the inhibition of the NF-κB and Hsp-27. In 22Rv1 cells, androgen receptor variant-7 was also observed to be decreased in the combined dose of 48 h. The association of this decrease with the decrease in androgen receptor and p65 expressions is a supportive result for the role of NF-κB signaling in the formation of androgen receptor variant. In androgen receptor variant-7 siRNA treatment in 22Rv1 cell lines, decrease of expression of androgen receptor variant-7 as well as decrease of expression of androgen receptor and p65 were observed. The decrease statistically significant in androgen receptor and p65 expressions was even greater when siRNA treatment was followed with low dose and time (6 h) combined treatment after transfection. We also showed that increased Noxa and decreased Bcl-2 protein level, indicated that apoptotic induction after this combination. In conclusion, inhibition of NF-κB and Hsp-27 is also important, along with therapies for androgen receptor variant-7 inhibition.

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: ∼40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, ß-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 effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines.

CONTEXT: Thyroid cancers (TCs) are the most common endocrine malignancies. There were two problems with the current cancer chemotherapy: the ineffectiveness of treatment due to resistance to cancer cell, and the toxic effect on normal cells. AIMS: This study was aimed to determine the effects of thymoquinone (TQ) and genistein (Gen) phytotherapeutics on telomerase activity, angiogenesis, and apoptosis in follicular and anaplastic thyroid cancer cells (TCCs). MATERIALS AND METHODS: Cell viability, caspase-3 (CASP-3) activity, and messenger RNA (mRNA) expression levels of human telomerase reverse transcriptase (hTERT), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-kB), cyclin-dependent kinase inhibitor 1 (p21), and vascular endothelial growth factor-A (VEGF-A) genes were analyzed. RESULTS: It was found that TQ and Gen treatment on TCCs caused a statistically significant decrease of cell viability, and mRNA expression levels of hTERT, VEGF-A, and NF-kB genes, but a statistically significant increase of PTEN and p21 mRNA expression levels. In addition, TQ and Gen treatment also caused a statistically significant increase active CASP-3 protein level in TCCs. Moreover, our results demonstrated that, when compared with follicular TCCs, anaplastic TCCs were more sensitive to the treatment of TQ and Gen. CONCLUSIONS: Based on these results, two agents can be good options as potential phytochemotherapeutics against TCCs.

Do the expressions of epithelial-mesenchymal transition proteins, periostin, integrin-α4 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-α4, 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-α4, 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-α4 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. Impact statement Prostate cancer is the third most common cancer in the world and the most common cancer among men. Development of metastatic castration-resistant prostate cancer (mCRPC) 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 (EMT). The present study analyzes for the first time the expressions of EMT marker proteins - periostin, integrin α4, fibronectin - in mCRPC and in benign prostatic hyperplasia (BPH) with the aim to determine the clinical relevance of changes in these three proteins vis-a-vis the PCa aggressive phenotype. In doing so, it sheds light on the molecular mechanism underlying the disease. We concluded that elevated periostin and fibronectin levels in mCRPC patients may be appropriate targets of therapeutic intervention in the future; hence, adopting methods that target these proteins may help treat prostate cancer effectively.

Expression profiles of CD11b, galectin-1, beclin-1, and caspase-3 in nasal polyposis

Background/aim: Nasal polyposis is a chronic inflammatory disease affecting the paranasal sinuses and nasal mucosae. It is thought that genetic and molecular mechanisms in inflammatory and apoptotic pathways are the main factors in the etiopathogenesis of nasal polyposis. The aim of this study was to investigate the expression patterns of CD11b, galectin-1, beclin-1, and caspase-3 in nasal polyps.Materials and methods: The mRNA expression levels of CD11b, galectin-1, beclin-1, and caspase-3 protein and western blot analysis of caspase-3 protein were evaluated in inferior turbinate mucosae and nasal polyp tissues.Results: CD11b expression was markedly higher in nasal polyp tissues when compared to turbinate mucosae (5.5 times higher, P < 0.05). Expression of galectin-1 was not statistically higher in nasal polyp tissues when compared to the controls. Beclin-1 expression in nasal polyp tissues was lower than in controls (17 times lower, P < 0.05). Caspase-3 expression was significantly lower in nasal polyp tissues than in controls (5.5 times lower, P < 0.05).Conclusion: Inflammation, apoptosis, and hyperproliferation are the major cellular processes in nasal polyposis and these proteins may take part and play some important roles in formation of this disease and the targeting of new treatment protocols.

Synergistic effects of cisplatin and proteasome inhibitor bortezomib on human bladder cancer cells.

The proteasome inhibitor bortezomib is a promising novel agent in bladder cancer therapy; however, inducible cytoprotective mechanisms may limit its potential efficacy. To date, the cellular and molecular effects of proteasome inhibitors on bladder cancer cells have been poorly characterized. Despite the consistent rate of initial responses, cisplatin treatment typically results in the development of chemoresistance, leading to therapeutic failure. Therefore, the present study aimed to characterize the molecular mechanisms underlying the anti-proliferative effects of cisplatin and bortezomib combination therapy on the human T24 bladder cancer cell line, by analyzing the protein expression levels of apoptotic genes. Cytotoxic effects were measured using a water-soluble tetrazolium salt-1 assay, and the apoptosis-associated molecules were examined using western blot analysis and ELISA. It was observed that combined administration of cisplatin and bortezomib induced upregulation of caspase-3, -8 and -9, B-cell lymphoma-2 (Bcl-2)-like 11 and Bcl-2-interacting killer, but downregulated Bcl-2 and Bcl-extra large protein expression levels in T24 cells in a dose-dependent manner. Furthermore, enhanced protein expression of caspase-8 and -9, in line with the significantly increased caspase-3 activation, was detected when the cells were treated with a combination of cisplatin and bortezomib, compared with that of either agent alone. Bortezomib appeared to synergize with cisplatin to promote apoptosis via the extrinsic and intrinsic apoptotic pathways. Taken together, the results of the current study provide the preclinical framework for additional evaluation of the effects of combining bortezomib with other agents to induce apoptosis in bladder cancer cells.

The Inhibitory Effect of Platelet-Rich Plasma on Botulinum Toxin Type-A: An Experimental Study in Rabbits.

BACKGROUND: Combination treatments of botulinum toxin type-A and other rejuvenation agents or instruments are gradually becoming more popular. After observing a high incidence of therapy failure following simultaneous applications of botulinum toxin type-A and platelet-rich plasma mesotherapy, we aimed to investigate whether PRP has an inhibitory effect on botulinum toxin type-A. METHODS: Twenty-four New Zealand white rabbits were divided into 4 groups, and the anterior auricular muscle and overlying skin were used for injections. Groups I and II both received onabotulinumtoxinA intramuscular injections. In addition, autologous platelet-rich plasma mesotherapy was performed in Group I while Group II received saline mesotherapy. Group III was designed as the in vitro mixture group in which onabotulinumtoxinA and platelet-rich plasma were mixed and then administered intramuscularly. Group IV received saline within the mixture instead of platelet-rich plasma. The contralateral ears of all the rabbits served as control and were only treated with onabotulinumtoxinA. Visual evaluation of ear positions and electroneuromyographic studies were done prior to all procedures and at day 14. Anterior auricular muscles were harvested at day 14 and were evaluated with quantitative real-time PCR. RESULTS: Visual and electroneuromyographic studies revealed less onabotulinumtoxinA activity in Groups I and III. When platelet-rich plasma was administered through skin mesotherapy, onabotulinumtoxinA activity failure was more severe in comparison with direct contact. No significant difference in SNAP-25 mRNA expression through quantitative real-time PCR was observed between groups. CONCLUSION: Although we could not explain the exact mechanism underlying this interaction, platelet-rich plasma applications result in less onabotulinumtoxinA muscle paralysis activity.

Does Wnt/ß-catenin pathway contribute to the stability of DNMT1 expression in urological cancer cell lines?

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/ß-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/ß-catenin signaling pathway was activated by using SB216763 which is a glycogen synthase kinase-3 (GSK3) ß inhibitor. Cell proliferation levels in bladder cancer cells, renal cell carcinoma, and prostate cancer cells treated with GSK3ß 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 ß-catenin and WIF-1 by real-time PCR; and protein levels of ß-catenin, DNMT1, pGSK3ß(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 ß-catenin increased after treatment with SB216273 and protein levels of pGSK3ß(Ser9), ß-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, ß-catenin, HAUSP, and UHRF1 decreased after re-expression of WIF-1 following treatment with DAC. In Caki-2 cells, ß-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/ß-catenin signal pathway on DNMT1. This may allow us to identify new molecular targets for the treatment of urogenital cancers.

Does Pattern Scan Laser (PASCAL) photocoagulation really induce less VEGF expression in murine retina than conventional laser treatment?

To investigate the differences in the mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in murine retina between mice subjected to conventional laser (AG) and those subjected to Pattern Scan Laser (PASCAL) system. Male C57BL/6 mice were randomly assigned to one of three groups: Group 1 (G1) receiving retinal scatter laser photocoagulation using with AG photocoagulator (n=16), Group 2 (G2) receiving retinal scatter laser photocoagulation using with PASCAL (n=16) and Group 3 (G3) served as an untreated control group (n=6). Molecular and morphological analyses of VEGF were performed on days 1, 2 and 5 by ELISA, real-time PCR and immuno-histochemical analysis. In samples which underwent AG (G1), when compared with the control group (G3), VEGF mRNA level increased 2.4 folds on day 2, whereas it decreased on day 5 (p□0.001). In samples which underwent PASCAL (G2), on the other hand, VEGF mRNA level increased 1.8 folds on day 1 and 2.2 folds on day 5 when compared with the control group (G3). In samples which underwent AG (G1), when compared with the control group (G3), VEGF protein level increased significantly on day 2, whereas it decreased on day 5 (p□0.001). In group G2, the VEGF levels in the sensory retina significantly increased as compared to control groups at both 2 and 5 days after laser photocoagulation using PASCAL laser (p=0.012, both time points). The peak expressions of VEGF protein in samples which underwent PASCAL and conventional laser were found on day 5 and day 2 respectively. In retinas of PASCAL-treated mice, VEGF immunoreactivity gradually increased during the 5-day follow-up. However, in argon laser group, the strongest VEGF immunoreactivity was detected on day 2, then started to decrease on day 5. In summary, the expression of VEGF protein and mRNA gradually increase during a 5-day follow-up period in PASCAL-treated mouse eyes, whereas in AG group they reach their peak levels on the second day of follow-up and started decreasing after then. These results may also explain why the PASCAL system is less effective in regressing neovascularization in the clinic.

The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24).

The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both ß-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 µM), DAC (10 µM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3ß, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of ß-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/ß-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3ß mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/ß-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.

Effects of AG490 and S3I-201 on regulation of the JAK/STAT3 signaling pathway in relation to angiogenesis in TRAIL-resistant prostate cancer cells in vitro.

The aim of the present study was to analyze the molecular mechanisms involved in blocking the signaling pathway and the effects of this on the progression of prostate cancer (CaP) cells in vitro. LNCaP human CaP cell line was stimulated with interleukin-6 (IL-6) in the presence/absence of Janus kinase (JAK) 2 (AG490), signal transducer and activator of transcription 3 [(STAT3) S3I-201] inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cytotoxic activity, the activation of phosphorylated (p)-STAT3 protein, caspase (CASP) 3 activity at protein level, vascular endothelial growth factor (VEGF) A, VEGFC, vascular endothelial growth factor receptor 2, STAT3, matrix metalloproteinase-2, myeloid cell leukemia sequence 1 (MCL-1), CASP8 and CASP9 messenger RNA (mRNA) levels were determined. Morphology and apoptosis were confirmed by DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. IL-6 rapidly induced the phosphorylation of STAT3 in a dose- and time-dependent manner with a peak expression at 3 h at a concentration of 25 ng/ml. In addition, AG490 (50 µM) and S3I-201 (300 µM) inhibited STAT3 activation. Western blotting results revealed that p-STAT3 protein expression decreased significantly with AG490 and S3I-201 treatment in LNCaP cells. AG490 and S3I-201 induced the downregulation of VEGFA, MCL-1 and STAT3 and the upregulation of CASP8 and CASP9 mRNA transcription levels. In addition, the inhibitors increased the level of CASP3 protein. Combinations of AG490- and S3I-201-TRAIL did not result in an increase in this effect. Parallel results were found by DAPI staining and TUNEL assay. To the best of our knowledge, this is the first study to investigate the possible clinical use of AG490 or S3I-201, together with the reduced use of chemotherapeutic agents with high cytotoxicity, for their ability to exert an apoptotic effect, targeting the JAK/STAT3 pathway.

DNA methyltransferase inhibitor-mediated apoptosis in the Wnt/ß-catenin signal pathway in a renal cell carcinoma cell line.

The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3ß) is an important regulator of the Wnt/ß-catenin signaling pathway. The mechanisms underlying GSK3ß regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2'-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3ß (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of ß-catenin (CTNNB1) expression decreased. The p-GSK3ß (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 µM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice.