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.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines.

INTRODUCTION: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. METHODS: The cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. RESULTS: Treatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. CONCLUSION: DHA might be a potential anti-cancer therapeutic for CLL.

Platinum nanoparticles Protect Against Lipopolysaccharide-Induced Inflammation in Microglial BV-2 Cells via Decreased Oxidative Damage and Increased Phagocytosis.

Neuroinflammation and oxidative stress cooperate to compromise the function of the central nervous system (CNS). Colloidal platinum nanoparticles (Pt NPs) are ideal candidates for reducing the deleterious effects of neuroinflammation since they act as free radical scavengers. Here we evaluated the effects of Pt NPs on several markers of lipopolysaccharide (LPS)-induced inflammation in cultured BV-2 microglial cells. BV-2 cells were treated with increased dilutions (1-100 ppm) of Colloidal Pt and/or LPS (1-10 µg/mL) at different exposure times. Three different protocols of exposure were used combining Pt NPs and LPS: (a) conditioning-protective effect (pre-post-treat), (b) therapeutic effect (co-treat) and (c) conditioning-therapeutic effect (pre-co-treat). After exposure to LPS for 24 h, cells were used for assessment of cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, apoptosis and caspase-3 levels, cell proliferation, mitochondrial membrane potential, inducible nitric oxide (iNOS) activity, pro-inflammatory cytokine (IL-1ß, TNF-α and IL-6) levels, and phagocytic activity. Low concentrations (below or equal to 10 ppm) of Colloidal Pt prevented or ameliorated the LPS-induced increase in ROS formation, loss of mitochondrial membrane potential, induction of apoptosis, increase in LDH release, increase in pro-inflammatory cytokines and iNOS, inhibition of phagocytosis linked to microglial persistence in the M1 phase phenotype, loss of cell adhesion, differentiation and/or proliferation, as well as loss of cell viability. These protective effects were evident when cells were preconditioned with Pt NPs prior to LPS treatment. Collectively, the findings demonstrate that at low concentrations, Pt NPs can regulate the function and phenotype of BV-2 cells, activating protective mechanisms to maintain the microglial homeostasis and reduce inflammatory events triggered by the inflammatory insults induced by LPS. These preventive/protective effects on the LPS pro-inflammatory model are linked to the antioxidant properties and phagocytic activity of these NPs.

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.

Oleuropein and rutin protect against 6-OHDA-induced neurotoxicity in PC12 cells through modulation of mitochondrial function and unfolded protein response.

Parkinson's disease (PD) is a highly prevalent neurodegenerative disorder, often associated with oxidative stress-induced transcriptional changes in dopaminergic neurons. Phenolic antioxidants, oleuropein (OLE) and rutin (RUT) have attracted a great interest due to their potential to counteract oxidative protein aggregation and toxicity. This study aimed at examining the effects of OLE and RUT against 6-OHDA-induced stress response in rat pheochromocytoma cells. When differentiated PC12 cells were exposed to oxidative stress composer 6-OHDA (100 µM, 8 h), a decreased mitochondrial membrane potential (ΔΨm) was observed along with a significant loss of cell viability and apoptotic nuclear changes. Exposure to 6-OHDA resulted in unfolded protein response (UPR) in differentiated PC12 cells as evidenced by an increased level of endoplasmic reticulum (ER)-localized transmembrane signal transducer IRE1α, adaptive response proteins ATF-4 and proapoptotic transcription factor CHOP. OLE or RUT pretreatment (24 h) at low doses (1-50 µM) protected the differentiated PC12 cells from 6-OHDA-induced cytotoxicity as assessed by increased viability, improved ΔΨm and inhibited apoptosis, whereas relatively high doses of OLE or RUT (>50 µM) inhibited cell growth and proliferation, indicating a typical hormetic effect. In hormetic doses, OLE and RUT up-regulated 6-OHDA-induced increase in IRE1α, ATF-4 and inhibited CHOP, PERK, BIP and PDI. 6-OHDA-activated XBP1 splicing was also inhibited by OLE or RUT. The presented results suggest that neuroprotection against 6-OHDA-induced oxidative toxicity may be attributable to neurohormetic effects of OLE or RUT at low doses through regulating mitochondrial functions, controlling persistent protein misfolding, activating and/or amplificating the adaptive response-related signaling pathways, leading to UPR prosurvival output.

Effects of Electroporation on Tamoxifen Delivery in Estrogen Receptor Positive (ER+) Human Breast Carcinoma Cells.

Estrogen receptor positive breast cancer is the most common type of breast cancer and in most cases, hormone therapy is considered complementary to surgery. Tamoxifen is one of the most common drugs used in hormone therapy for treating estrogen receptor positive breast cancer cells. However, it has severe side-effects depending on the duration of treating breast cancer and amount of tamoxifen used. In this study, we examined the effects of electroporation on the tamoxifen uptake in estrogen receptor positive MCF-7 breast cancer cells. The survival rate of MCF-7 cells had a negative relationship with energy dissipation in cells. Similarly, the electrical charge delivered to cells during electroporation was inversely proportional to survival rate. The combined application of electroporation and tamoxifen is much more effective than the usage of tamoxifen alone in the treatment of estrogen receptor positive breast cancer. The application of electroporation increased the uptake of tamoxifen into MCF-7 cells and reduced the minimal tamoxifen dosage which, is needed for the treatment of estrogen receptor positive breast cancer.

Mitochondrial hyperpolarization and cytochrome-c release in microwave-exposed MCF-7 cells.

This study examines the effects of a 2.1-GHz WCDMA-modulated microwave (MW) radiation on apoptotic activity and mitochondrial membrane potential (ΔΨm) in MCF-7 cells. The cells were exposed to the MW at a specific absorption rate (SAR) of 0.528 W/kg for 4 or 24 h. The antiproliferative effect of MW exposure was determined by the MTT test. Cytochrome-c and p53 levels were determined by an ELISA method. The relative ΔΨm was analysed by JC-1 staining using flow cytometer. Apoptotic rate of the cells was measured by Annexin-V-FITC staining. All assays were performed after certain time of incubations (15 min-4 h) following MW exposure. MW-exposed cells showed a significant decrease in viability when compared to unexposed cells. A significantly larger decrease was observed after longer exposure. The percentage of apoptotic cells, amount of cytochrome-c, and relative ΔΨm were significantly higher in MW-exposed cells. The percent of apoptotic cells and relative ΔΨm in 24 h MW-exposed group was significantly higher than those in 4 h MW-exposed group. However, no significant change was observed in p53 levels. These results demonstrated that exposure to 2.1-GHz WCDMA-modulated MW radiation caused hyperpolarization of mitochondria that in turn induced apoptosis in MCF-7 cells.

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.

Does MW Radiation Affect Gene Expression, Apoptotic Level, and Cell Cycle Progression of Human SH-SY5Y Neuroblastoma Cells?

Neuroblastoma (NB) is a cancer that occurs in sympathetic nervous system arising from neuroblasts and nerve tissue of the adrenal gland, neck, chest, or spinal cord. It is an embryonal malignancy and affects infants and children. In this study, we investigated the effects of microwave (MW) radiation on apoptotic activity, cell viability, and cell cycle progression in human SH-SY5Y NB cells which can give information about MW radiation effects on neural cells covering the period from the embryonic stages to infants. SH-SY5Y NB cells were exposed to 2.1 GHz W-CDMA modulated MW radiation for 24 h at a specific absorption rate of 0.491 W/kg. Control samples were in the same conditions with MW-exposed samples but they were not exposed to MW radiation. The apoptotic activity of cells was measured by Annexin-V-FITC and propidium iodide staining. Moreover, mRNA levels of proliferative and cell cycle proteins were determined by real-time RT-PCR. The change in cell cycle progression was observed by using CycleTest-Plus DNA reagent. No significant change was observed in apoptotic activity of MW-exposed cells compared to control cells. The mRNA levels of c-myc and cyclin D1 were significantly reduced in MW group (p < 0.05). The percentage of MW-exposed cells in G1 phase was significantly higher than the percentage of control cells in G1 phase. MW radiation caused cell cycle arrest in G1 phase. These results showed that 2.1 GHz W-CDMA modulated MW radiation did not cause apoptotic cell death but changed cell cycle progression.

Effects of Rat Bone Marrow-Derived Mesenchymal Stem Cells and Demineralized Bone Matrix on Cranial Bone Healing.

BACKGROUND: Studies in tissue engineering about mesenchymal stem cells (MSCs) provide promising results for bone regeneration. The aim of this study was to evaluate the effects of rat bone marrow-derived MSCs (rMSCs) alone and when combined with demineralized bone matrix (DBM) on critical-sized cranial defects of rats. METHODS: Ten rats were used to obtain allogeneic rMSCs. Forty rats were separated equally into 4 groups. A full-thickness circular bone defect was created in the frontal bone of the rats. Group 1 was an operative control group. In group 2 DBM, in group 3 rMSCs, and in group 4 DBM combined with rMSCs were applied into the defects. Bone regeneration was evaluated by computed tomographic analysis and immunohistochemistry. RESULTS: In radiological evaluation, the percentage of area healed in group 3 at the 12th week was statistically significantly greater than in group 1. In group 3 and group 4, distributed healing patterns were observed more than in group 2 and in group 1. Immunohistochemical evaluation revealed that group 4 had the best osteoinductive potential. Osteoinductive potential of group 3 was similar to group 2 and was better than group 1. CONCLUSIONS: Allogeneic rMSC applications have created a statistically significant radiologic reduction of the bone defect areas at the end of the 12 weeks. The MSC applications have also increased the bone density and changed the healing patterns. Combined use of the DBM and rMSCs has created more osteoinductive responses. This combination can provide better results in craniofacial bone reconstruction.

Effects of Cisplatin Electrochemotherapy on Human Neuroblastoma Cells.

Electrochemotherapy is the usage of electroporation to introduce chemotherapeutic drugs through membrane pores into target cells for cancer treatment. The effectiveness of chemotherapeutic drugs would be increased dramatically when they are used in electrochemotherapy than standard chemotherapy. In the present study, we investigated the effects of cisplatin treatment with electroporation on human SH-SY5Y neuroblastoma cells. SH-SY5Y cells were treated with different concentrations (0.15-24 µg/mL) of cisplatin and then exposed to 1500 volts per centimeter (V/cm), 100 microseconds (µs) pulse duration, and 1 Hertz (Hz) electric pulses. Cisplatin alone showed a dose-dependent effect on cell viability. On the other hand, cisplatin + electroporation treatment was more effective than cisplatin treatment alone. Lower doses of cisplatin treatment with electroporation was as effective as higher doses of cisplatin treatment without electroporation. These results indicated that cisplatin cytotoxicity was potentiated after exposure of cells to high intensity electric pulses and low doses of cisplatin can be used with electroporation in the treatment of neuroblastoma.

Virus-like nanostructures for tuning immune response.

Synthetic vaccines utilize viral signatures to trigger immune responses. Although the immune responses raised against the biochemical signatures of viruses are well characterized, the mechanism of how they affect immune response in the context of physical signatures is not well studied. In this work, we investigated the ability of zero- and one-dimensional self-assembled peptide nanostructures carrying unmethylated CpG motifs (signature of viral DNA) for tuning immune response. These nanostructures represent the two most common viral shapes, spheres and rods. The nanofibrous structures were found to direct immune response towards Th1 phenotype, which is responsible for acting against intracellular pathogens such as viruses, to a greater extent than nanospheres and CpG ODN alone. In addition, nanofibers exhibited enhanced uptake into dendritic cells compared to nanospheres or the ODN itself. The chemical stability of the ODN against nuclease-mediated degradation was also observed to be enhanced when complexed with the peptide nanostructures. In vivo studies showed that nanofibers promoted antigen-specific IgG production over 10-fold better than CpG ODN alone. To the best of our knowledge, this is the first report showing the modulation of the nature of an immune response through the shape of the carrier system.

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt's lymphoma (Raji) cells.

We investigated the effects of 1.8 MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt's lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8 GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350 W/kg in a CO2 incubator. The duration of the exposure was 24 h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW + Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.

Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.

TCTP/HRF pathway and angiogenesis: a feasible intercourse in chronic lymphocytic leukemia.

This prospective study was planned to determine the intercourse between translationally controlled tumor protein (TCTP)/histamine releasing factor (HRF)/histamine pathway and angiogenesis in chronic lymphocytic leukemia (CLL). A total of 153 CLL patients were included. Serum histamine levels were higher in CLL patients. A positive correlation was found between microvessel density (MVD)-mast cell (MC) count; MVD-TCTP/HRF and MC count-TCTP/HRF. Microvessel density, MC and ZAP 70 were significantly higher in TCTP/HRF-positive group. Time to first treatment was shorter in patients with increased MVD and TCTP/HRF. Further data is essential to ascertain the role of TCTP/HRF pathway in tumor angiogenesis and CLL prognosis.

A novel mutation pattern emerging during lamivudine treatment shows cross-resistance to adefovir dipivoxil treatment.

AIMS: This study was conducted to clarify the resistance profile of a novel mutation pattern emerging during lamivudine (3TC) therapy and showing cross-resistance to adefovir dipivoxil (ADV) in a patient with chronic hepatitis B. METHODS AND RESULTS: Successful suppression of hepatitis B virus (HBV) replication by sequential therapy of 9 MU thrice weekly interferon (IFN) and 3TC was followed by genotypical resistance detected at month 28 of therapy (month 19 of lamivudine treatment). ADV was added to 3TC therapy on month 44 of antiviral treatment. Neither alanine aminotransferase normalization nor a stable decrease in HBV viral load was observed, although ADV was used for more than 40 months. The HBV pol region was amplified from serum samples obtained before and after ADV treatment. The complete genome was cloned into a TA vector. PCR products and 7-10 clones from each cloned vector were sequenced. A novel mutation, A181S, in the reverse transcriptase gene leading to a conversion of W172C in the overlapping surface antigen gene was detected along with a M2041 mutation. The complete genome comprising the A181S+M2041 pattern was cloned into an expression vector and its in vitro susceptibility to 3TC, ADV, tenofovir (PMPA), clevudine (L-FMAU) and emtricitabine (FTC) were determined in transiently transfected Huh7 cells. This mutation pattern displayed more than 1000-fold resistance to the nucleoside analogues 3TC and FTC and approximately sixfold resistance to L-FMAU, while it confers 28.23- and 5.57-fold resistance for the nucleotide analogues ADV and PMPA, respectively. CONCLUSION: A new mutation pattern, A181S+M2041, arising under lamivudine treatment confers cross-resistance to ADV both in vivo and in vitro.