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.

Investigation Covering the Effect of Boron plus Taurine Application on Protein Carbonyl and Advanced Oxidation Protein Products Levels in Experimental Alzheimer Model.

Alzheimer's disease (AD) is the most common form of dementia that occurs in the brain. This is a chronic neurodegenerative disease which is valid in 60-70% of all dementia patients. Boron, regarded as a potential antioxidant, has the effect of reducing oxidative stress. Taurine, as one of the thiol-containing amino acids, exists at different concentrations in both the neurons and glial cells of the central nervous system. It plays an important role in the protective and adjuvant therapies as an antioxidant due to its characteristics of maintaining the oxidant-antioxidant balance of the body as well as cell integrity and increasing body resistance. Based on this information, our objective was to reveal the effect of boron alone, taurine alone plus co-administration of taurine and boron application on brain tissue protein carbonyls (PC) and serum advanced oxidation protein products (AOPP) levels in the experimental Alzheimer's model. For this purpose, 5 groups were formed in our study which consisted of 30 Wistar albino male rats. The rats were given a single dose of STZ stereotaxically. At the end of this period, the rats were decapitated, plus their brain tissues and blood were removed. Our findings suggested that taurine alone and co-administration of boron and taurine had a decreasing effect on AOPP and PC levels of the experimental Alzheimer model of the rats.

Efficiency of resveratrol in the prevention and treatment of age-related hearing loss.

Age-related hearing loss (ARHL) is a major public health concern, which is characterized by gradual, progressive sensorineural hearing loss and deterioration of sound localization, with no effective treatment available to date. The aim of the present study was to evaluate the efficacy of resveratrol to prevent and treat ARHL. For this purpose, 32 male C57BL/6 mice were assigned to four groups: Early treatment, late treatment, control and sham control. The experiment lasted for 15 months. Treatment was started at three months of age in the early treatment group and at sixth months in the late treatment group. The auditory brainstem response test was performed once every three months. At the end of the study period, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, NF-κB, Bcl-2, Bcl-xL, Bax, Bcl-2 homologous antagonist/killer (Bak), caspase-3 and caspase-9 levels in the cochlear tissues of the animals were analyzed by reverse transcription-quantitative PCR. Hearing thresholds of the mice in the early treatment group were better than those in the other groups (P<0.001) at the end of the study. However, hearing levels in the late treatment group were not significantly different from those in the control groups (P>0.05), although mean thresholds were lower. The threshold shift in the early treatment group was significantly lower at all frequencies when compared with those in the control groups (P<0.001). The mRNA expression levels of pro-apoptotic genes Bax and Bak were lower (P<0.05), anti-apoptotic genes Bcl-2 and Bcl-xL were higher (P<0.05), NF-κB, COX-2 and iNOS as genes that have a role in inflammation and caspase-3 and caspase-9 as genes with a vital role in apoptosis were lower (P<0.05) in the early treatment group when compared with the late treatment and control groups. These results suggested that resveratrol is effective in the prevention of ARHL, particularly when started prior to the beginning of hearing loss.

Involvement of endometrial IGF-1R/IGF-1/Bcl-2 pathways in experimental polycystic ovary syndrome: Identification of the regulatory effect of melatonin.

The involvement of endometrial IGF-1R/IGF-1/Bcl-2 pathways and the potential regulatory effects of exogenously administrated melatonin on this expression is investigated in the experimental PCOS model in the present study. Thirty-two 6-8 week old Sprague Dawley rats were divided into four groups: the Sham Control Group (1% CMC/day by oral gavage [o.g.]); the Melatonin Group (2 mg/kg/day melatonin by subcutaneous administration [s.c.]); the Experimental PCOS Group (1 mg/kg/day Letrozole by o.g.); and the Experimental PCOS + Melatonin Group (1 mg/kg/day Letrozole by o.g. and 2 mg/kg/day melatonin by s.c. administration). Vaginal smear samples were taken from the 14th day to the end of the experiment for colpocytological measurements. At the end of the 21 day experimental period, uterine tissues were taken; Hematoxylin-Eosin histochemical, IGF-1R/IGF-1/Bcl-2, PCNA immuno-histochemical stainings and western blot analyses were performed for related antibodies. All of the data was supported statistically. The epithelium of endometrium lost its single-layer structure in some parts, separation was observed between the epithelium and the basal membrane junction, intracellular edema was found in the uterine glands by the polycystic ovary-induction. Also this induction increased the expression of IGF-1R/IGF-1, Bcl-2, and PCNA proteins. Morphological degenerations returned to its normal appearance generally by the melatonin administrations and melatonin also regulated the increased expression of endometrial IGF-1R/IGF-1/Bcl-2 and PCNA pathways. It is concluded that additional studies are needed, using melatonin as a supporting agent may be appropriate in cases of PCOS.

The effect of intracerebroventricular amyloid beta 1-42 application on cognitive functions in aged rats supplemented with taurine and the change of peroxisomal proteins in this process.

OBJECTIVE: The aim of our study is to investigate the change of peroxisomal proteins in the neurodegenerative and oxidative process caused by the neurotoxicity of Aß 1-42 in aged rats supplemented with taurine and to show the possible positive effects of taurine in this process. METHODS: 30 Wistar albino rats were randomly divided into 5 groups as control, sham, Aß 1-42, taurine, and Aß 1-42+taurine. Taurine administration continued for 6 weeks (1000 mg/kg/day with drinking water). Stereotaxic surgery was applied to all groups (intracerebroventricular per lateral ventricle needle only or 5 µl, PBS, or Aß 1-42). Spatial learning and memory performances of the animals were evaluated with Morris water maze and elevated plus maze. The levels of MDA and GSH were measured as oxidative stress parameters in the cerebral cortex and hippocampus. Expressions of CAT, PEX14, PMP70 of peroxisomal membrane proteins were indicated by Western blot analysis. RESULTS: Our results showed that injection of Aß 1-42 decreased the spatial learning and memory performance, cortex CAT and hippocampus PEX14, PMP70 and GSH levels, and increased cortex and hippocampus MDA levels (p < 0.05). Although the administration of taurine partially ameliorated the adverse effects of Aß 1-42 injection, a significant difference was found only at the hippocampus GSH levels (p < 0.05). Also, taurine caused anxiety at this dose (p < 0.05). DISCUSSION: In conclusion, decreased peroxisomal proteins and antioxidant capacity in neurodegenerative and oxidative processes induced by intracerebroventricular Aß 1-42 injection showed that peroxisomes may play a role in this process and taurine supplementation may have positive effects especially in increasing antioxidant capacity.

Resveratrol enhances the sensitivity of FL118 in triple-negative breast cancer cell lines via suppressing epithelial to mesenchymal transition.

We aimed to investigate whether resveratrol (RSV) could sensitize human triple-negative breast cancer (TNBC) cells to FL118-induced cell death, epithelial to mesenchymal transition (EMT), invasion, and migration. The effects of sequential administration of RSV and FL118 on MDA-MB-436 and MDA-MB-468 cells were evaluated in terms of cell viability, cytotoxicity, apoptosis, cell cycle distribution, active caspase-3/7 levels, migration and invasion. Furthermore, mRNA and protein levels of EMT associated genes and proteins were also evaluated. Sequential administration of RSV and FL118 inhibited the cell viability in both TNBC cell lines. Meanwhile sequential administration of RSV and FL118 also dramatically reduced the migratory and invasive capabilities, it also reversed the EMT process in both TNBC cells. Moreover, sequential administration of RSV and FL118 led to a significant increase of apoptotic cells, as well as active Caspase-3/7 levels. Sequential administration of RSV and FL118 caused TNBC cells accumulating in the G1 phase, and markedly suppressed the mRNA and protein levels of N-cadherin, ß-catenin, Vimentin, Snail, and Slug, and also significantly downregulated mRNA levels of Fibronectin, Twist1, Twist2, Zeb1, and Zeb2 genes, while enhanced the mRNA and protein levels of E-cadherin genes. RSV sensitized TNBC cells to FL118 via facilitating apoptosis, migration, invasion, and EMT and enhancing intracellular entrapment of FL118. Thus, our results suggest that since RSV enhanced the in vitro anticancer activity of FL118 in BC, it may be a potential therapeutic agent in advanced BC.

Phenoxodiol sensitizes metastatic colorectal cancer cells to 5-fluorouracil- and oxaliplatin-induced apoptosis through intrinsic pathway.

Colorectal cancer (CRC) is one of the most common types of cancer seen in the world. 5-Fluorouracil (5-Fu) plus Oxaliplatin (1-OHP) remains the backbone of CRC chemotherapeutics, but with limited success. Phenoxodiol (Pxd) is an isoflavone analog with antitumor activity against various types of cancers, and sensitizes chemoresistant cancer cells to chemotherapeutics including platinum and taxanes. This study was, therefore, undertaken to examine whether Pxd pre-treatment with conventional chemotherapeutic agent(s) 5-Fu and 1-OHP co-administration be a therapeutic strategy for CRC. Cell viability and cytotoxicity were evaluated using dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase assays. The percentage of apoptotic and necrotic cells were determined by fluorescence microscopy analysis. Besides, active Caspase-3 levels by ELISA and relative mRNA levels of Caspase 3 (CASP3), CASP8 and CASP9 genes were determined by quantitative real-time PCR (qPCR) analysis. The pre-treatment of Pxd followed by 5-Fu and 1-OHP co-administration was more effective at inhibiting cell viability than either chemotherapeutic agents treatment alone. When compared to 5-Fu with 1-OHP alone treatment, Pxd pre-treatment overwhelmingly increased apoptotic Caspase-3 activity levels in CRC cells. Moreover, qPCR analyses showed that CASP3 and CASP9 mRNA levels significantly increased after pre-treatment with Pxd followed by 5-Fu and 1-OHP treatments, compared to 5-Fu with 1-OHP alone. Our results suggested that Pxd enhanced the in vitro antitumor activity of 5-Fu and 1-OHP. Our study also suggested that Pxd may be a potential candidate agent in advanced CRC and inclusion of Pxd to the conventional chemotherapeutic agent(s) could be an effective therapeutic strategy for CRC.

Luteolin protects microglia against rotenone-induced toxicity in a hormetic manner through targeting oxidative stress response, genes associated with Parkinson's disease and inflammatory pathways.

Rotenone, an environmental toxin, triggers Parkinson's disease (PD)-like pathology through microglia-mediated neuronal death. The effects and molecular mechanisms of flavonoid luteolin against rotenone-induced toxicity was assessed in microglial BV2 cells. Cells were pretreated with luteolin (1-50 µM) for 12 h and then was co-treated with 20 µM of rotenone for an additional 12 h in the presence of luteolin. The viability (MTT), IL-1ß and TNF-α levels and lactate dehydrogenase (LDH) release (ELISA), and Park2, Lrrk2, Pink1, Nrf2 and Trx1 mRNA levels (qRT-PCR) were measured. In rotenone exposed microglia, luteolin increased viability significantly at lower concentrations (1-5 µM) compared to higher concentrations (25-50 µM). Rotenone increased LDH release and IL-1ß levels in a dose-dependent manner (1-20 µM). Luteolin inhibited rotenone-induced LDH release, however the activity decreased in concentration-dependent manner Neither rotenone nor luteolin altered TNF-α levels, but luteolin reduced IL-1ß levels in a concentration dependent manner in rotenone exposed cells. The mRNA levels of Nrf2 and Trx1, which are the master regulators of redox state, were increased by rotenone, as well as by luteolin, which exhibited an inverse relationship between its concentration and effect (1-20 µM). Park2 mRNA levels increased by luteolin, but decreased by rotenone. Pink1 mRNA levels was not altered by rotenone or luteolin. Lrrk2 mRNA levels reduced by luteolin, while it was increased by rotenone. Results suggest that luteolin have favorable effects on regulation of oxidative stress response, genes associated with PD and inflammatory pathways, hence protects microglia against rotenone toxicity in a hormetic manner.

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.

Corneal Cross-Linking Has No Effect on Matrix Metalloproteinase 9 and 13 Levels During Fungal Keratitis on the Early Stage.

The aim of our study was to investigate matrix metalloproteinases, MMP-9 and MMP-13 levels, in the rabbit model of Fusarium and Candida keratitis treated by corneal cross-linking (PACK-CXL). Rabbit corneas were inoculated with fungal inoculum for keratitis. Each group divided into four subgroups, including un-treated group, PACK-CXL group, voriconazole group and PACK-CXL plus voriconazole group. PACK-CXL was applied with 0.25% riboflavin in accelerated Dresden protocol, and 0.1% voriconazole drops were administered. All corneal buttons excised at tenth day after ophthalmological examination. Fungal cell counts and Scheiber scores were determined in all groups. Corneal tissue MMP mRNA levels were evaluated quantitative reverse transcriptase PCR. The difference in MMP-9 and MMP-13 levels at all groups was not statistically significant (p > 0.05). PACK-CXL with 0.25% riboflavin either alone or combined with antifungal drops was unable to provide decline in inflammatory findings in both macroscopic and microscopic levels similar to medical antifungal treatment.

Expression of iNOS, COX-2 and VEGF in canine mammary tumours and non-neoplastic mammary glands: Association with clinicopathological features and tumour grade.

The aim of the present study was to investigate the relationship between the expression of iNOS, COX-2 and VEGF mRNA levels and malignancy degree in canine malignant mammary tumours. Thirty-five bitches presented with the complaint of mammary masses, aged 6-10 years and representing different breeds, were used. The expressions of iNOS, COX-2 and VEGF mRNA levels were significantly higher in both benign and malignant tumours than in the adjacent nonneoplastic mammary glands (P < 0.05). The iNOS, COX-2 and VEGF mRNA expression levels of grade 2 tumours were higher than those of grade 1 tumours; however, the highest expression levels were detected in grade 3 tumours. Thus, increased iNOS, COX-2 and VEGF gene mRNA levels were found to be related with the histological grade of malignancy in dogs with mammary tumours.

Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells.

Cranial bone repair and regeneration via tissue engineering principles has attracted a great deal of interest from researchers during last decade. Here, within this study, 6 mm critical-sized bone defect regeneration via genetically modified mesenchymal stem cells (MSC) were monitored up to 4 months. Cranial bone repair and new bone formations were evaluated by histological staining and real time PCR analysis in five different groups including autograft and bone morphogenetic protein-2 (BMP-2) transfected MSC groups. Results presented here indicate a proper cranial regeneration in autograft groups and a prospering regeneration for hBMP-2 encoding mesenchymal stem cells.

Association of rs10757274 and rs2383206 Polymorphisms on 9p21 locus with Coronary Artery Disease in Turkish Population.

BACKGROUND AND OBJECTIVES: Genetic predisposition is an important risk factor for coronary artery disease (CAD). In this study, we aimed to evaluate the impact of rs10757274 and rs2383206 polymorphisms in chromosome 9p21 on presence and severity of CAD in a Turkish population. SUBJECTS AND METHODS: A total of 646 patients who underwent coronary angiography were included in this study. Coronary vessel score and Gensini score were calculated to assess the angiographic severity of CAD. Alleles of AA, AG, and GG were determined for rs10757274 (polymorphism-1) and rs2383206 (polymorphism-2) polymorphisms located in chromosome 9p21 from the blood samples. RESULTS: There was a significant difference between the alleles in polymorphism-1 in the presence of coronary artery disease (38.9% in AA, 48.0% in GG and 56.4% in AG, p=0.017). However, there was no difference between the alleles in polymorphism-2. According to vessel scores, there was a significant difference between the alleles in polymorphism-1 (AA 0.71±1.04, GG 0.88±1.07, AG 1.06±1.12, p=0.018). In polymorphism-2, vessel scores did not show a difference between the alleles. In polymorphism-1, there was a significant difference in Gensini score (p=0.041). Gensini scores did not differ between the alleles in polymorphism-2 (p>0.05 for all). In multivariate analyses, none of the alleles was an independent factor for presence of CAD. CONCLUSION: The presence of rs10757274 polymorphism including AG allele in chromosome 9p21 was related to CAD. However, this relationship was not independent of other cardiovascular risk factors.

HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells.

Histone deacetylases (HDACs) play a major role in the regulation of chromatin structure and gene expression by changing acetylation status of histone and non-histone proteins. MS-275 (entinostat, MS) is a well-known benzamide-based HDACI and Salermide (SAL), a reverse amide compound HDACI, have antiproliferative effects on several human cancer cells. In this study, we aimed to investigate the effects of HDACIs (MS and SAL) alone and/or combined use with EF24 (EF), a novel synthetic curcumin analog, on human pancreatic cancer cell line (BxPC-3). In vitro, BxPC-3 cells were exposed to varying concentrations of MS, SAL with or without EF, and their effects on cell viability, acetylated Histone H3 and H4 levels, cytotoxicity, and cleaved caspase 3 levels, and cell cycle distribution were measured. The viability of BxPC-3 cells decreased significantly after treatment with EF, MS and SAL treatments. MS and SAL treatment increased the acetylation of histone H3 and H4 in a dose dependent manner. MS and SAL alone or combined with EF were increased the number of cells in G1 phase. In addition, treatment with agents significantly decreased the ratio of cell in G2/M phase. There were significant dose-dependent increases at cleaved Caspase 3 levels after MS treatment but not after SAL treatment. Our results showed that HDAC inhibitors (MS and SAL), when combined with EF, may effectively reduce pancreatic cancer cell (BxPC-3) progression and stop the cell cycle at G1 phase. Further molecular analyses are needed to understand the fundamental molecular consequences of HDAC inhibition in pancreas cancer cells.