Daily Consumption of High-Polyphenol Olive Oil Enhances Hippocampal Neurogenesis in Old Female Rats.

OBJECTIVE: The aim of this study is to evaluate the effect of daily consumption of high-polyphenol (HP) olive oil on neurogenesis by investigating neuronal cell proliferation and maturation in the hippocampus of old rats, and to evaluate the relationship between neurogenesis, spatial memory, and anxiety-like behavior. METHODS: A total of 34 female, 20-22-month-old Sprague Dawley rats were divided into three groups: control group, low-polyphenol (LP) group, and high-polyphenol (HP) group. The animals were fed distilled water, LP olive oil and HP-extra virgin olive oil, respectively for 6 weeks using an oral gavage. At 43 days, animals were tested using the Morris Water Maze to evaluate spatial memory, and the Open-field test to evaluate anxiety-like behavior. Neural cell proliferation in the dentate gyrus (DG) was determined by BrdU labeling and Nestin protein expression. Neuronal maturation was determined by NeuN labeling. Synaptic density in the hippocampus and prefrontal cortex was examined by measuring Synaptophysin (SYN) levels. Hippocampal Calbindin levels were measured to assess cellular calcium metabolism. RESULTS: Daily consumption of HP olive oil significantly improved cell proliferation and neuronal maturation in the DG of old rats. HP-olive oil significantly increased SYN levels in the prefrontal cortex, and nestin and calbindin levels in the hippocampus (p < 0.05). LP olive oil diet has shown no effect on any parameter (p > 0.05). We also did not find any statistically significant difference between the groups in terms of spatial memory and anxiety-like behavior (p > 0.05). CONCLUSION: Our study is first to show that daily consumption of HP-olive oil enhances hippocampal neurogenesis in old rats, which has been confirmed by proliferation and maturation biomarkers. In addition, increased SYN and calbindin levels showed that the generated cells were also functionally developed in the HP group. We suggest that daily consumption of HP olive oil may have beneficial effects on brain aging by triggering neurogenesis.

Melatonin Prevents UVB-Induced Skin Photoaging by Inhibiting Oxidative Damage and MMP Expression through JNK/AP-1 Signaling Pathway in Human Dermal Fibroblasts.

Exposure to ultraviolet (UV) irradiation causes damage to the skin and induces photoaging. UV irradiation stimulates production of reactive oxygen/nitrogen species, which results in activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) in fibroblasts. MAPKs are responsible for activation of activator protein-1 (AP-1), which subsequently upregulates expression of matrix metalloproteinases (MMPs). Melatonin is a potent free radical scavenger which is known to have photoprotective effects. The aim of this study is to investigate the underlying molecular mechanisms for the photoprotective effects of melatonin in UVB-irradiated primary human dermal fibroblasts (HDFs) in terms of EGFR activation, oxidative/nitrosative damage, JNK/AP-1 activation, MMP activities, and the levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) and type I procollagen (PIP-C). In this study, HDFs were pretreated with 1 µM of melatonin and then irradiated with 0.1 J/cm2 of UVB. Changes in the molecules were analyzed at different time points. Melatonin inhibited UVB-induced oxidative/nitrosative stress damage by reducing malondialdehyde, the ratio of oxidized/reduced glutathione, and nitrotyrosine. Melatonin downregulated UV-induced activation of EGFR and the JNK/AP-1 signaling pathway. UVB-induced activities of MMP-1 and MMP-3 were decreased and levels of TIMP-1 and PIP-C were increased by melatonin. These findings suggest that melatonin can protect against the adverse effects of UVB radiation by inhibiting MMP-1 and MMP-3 activity and increasing TIMP-1 and PIP-C levels, probably through the suppression of oxidative/nitrosative damage, EGFR, and JNK/AP-1 activation in HDFs.

Borax Pentahydrate and Disodium Pentaborate Decahydrate Are Candidates as Anti-leukemic Drug Components by Inducing Apoptosis and Changing Bax/Bcl-2 Ratio in HL-60 Cell Line.

Acute myeloid leukemia (AML) is the most common form of acute leukemia and has the lowest 5-year survival rates. Current treatment strategies do not meet the expectations also. Therefore, there is a need to improve therapeutic approaches still. Boron, which is a natural trace element in human diet, is gaining attention with its important roles in cellular processes for the development of new anti-cancer drug candidates. For instance, bortezomib, a dipeptidyl boronic acid, has encouraging results in the treatment of multiple myeloma and mantle cell lymphoma. However, severe toxic effects and resistance development are the limitations to its application for AML treatment. Hence, the development of alternative boron-derived anti-AML agents is unmet need. Therefore, we aimed to evaluate anti-leukemic effect of two promising boron compounds, borax pentahydrate (BP) and disodium pentaborate decahydrate (DPD), and comparison of each other in terms of the capacity to trigger apoptosis on acute promyelocytic leukemia cells (HL-60). Cell viability was assessed by MTT assay. Apoptotic effects of the boron compounds on HL-60 cells were evaluated by annexin V/propidium iodide dyes and caspase 3/7 activity assay by flow cytometry. In addition, Bax/Bcl-2 and cleaved PARP levels were detected by western blotting. Although BP showed greater apoptosis-inducing capacity, we observed that both DPD (6 mM) and BP (24 mM) treatment showed anti-leukemic effect by triggering apoptotic pathway through increasing Bax/Bcl-2 ratio for the first time. Our study suggests that BP and DPD are the promising candidates for anti-AML drug development research, which may be confirmed by further wide-spectrum studies.

Development of biological meniscus scaffold: Decellularization method and recellularization with meniscal cell population derived from mesenchymal stem cells.

Tissue engineering approaches which include a combination of cells and scaffold materials provide an alternative treatment for meniscus regeneration. Decellularization and recellularization techniques are potential treatment options for transplantation. Maintenance of the ultrastructure composition of the extracellular matrix and repopulation with cells are important factors in constructing a biological scaffold and eliminating immunological reactions.The aim of the study is to develop a method to obtain biological functional meniscus scaffolds for meniscus regeneration. For this purpose, meniscus tissue was decellularized by our modified method, a combination of physical, chemical, and enzymatic methods and then recellularized with a meniscal cell population composed of fibroblasts, chondrocytes and fibrochondrocytes that obtained from mesenchymal stem cells. Decellularized and recellularized meniscus scaffolds were analysed biochemically, biomechanically and histologically. Our results revealed that cellular components of the meniscus were successfully removed by preserving collagen and GAG structures without any significant loss in biomechanical properties. Recellularization results showed that the meniscal cells were localized in the empty lacuna on the decellularized meniscus, and also well distributed and proliferated consistently during the cell culture period (p < 0.05). Furthermore, a high amount of DNA, collagen, and GAG contents (p < 0.05) were obtained with the meniscal cell population in recellularized meniscus tissue.The study demonstrates that our decellularization and recellularization methods were effective to develop a biological functional meniscus scaffold and can mimic the meniscus tissue with structural and biochemical features. We predict that the obtained biological meniscus scaffolds may provide avoidance of adverse immune reactions and an appropriate microenvironment for allogeneic or xenogeneic recipients in the transplantation process. Therefore, as a promising candidate, the obtained biological meniscus scaffolds might be verified with a transplantation experiment.

Synergistic Induction of Apoptosis by Quercetin and Curcumin in Chronic Myeloid Leukemia (K562) Cells: II. Signal Transduction Pathways Involved.

Flavonoids are phenolic substances with chemo-preventive and chemotherapeutic properties. They are widely found in fruits and vegetables. The polyphenols quercetin and curcumin have antioxidant, anti-inflammatory, anti-carcinogenic, and pro-apoptotic properties. They were successfully used against different human cancers, especially chronic myeloid leukemia cancer cells. We have previously investigated anti-proliferative and apoptotic effects of quercetin and curcumin combination in K562 cells. Our data showed that they had beneficial synergistic effects. Based on these findings, we aimed to clarify signaling pathways involved in synergistic combination treatment with quercetin and curcumin in these cells. Proteins were investigated by Western blotting and by confocal microscopy. Changes in several genes in 10 different pathways related to cell proliferation, apoptosis, cell cycle, inflammation, hypoxia and oxidative stress were observed. Combination of quercetin and curcumin was effective on genes that were particularly related to p53, NF-κB and TGF-α pathways. Down-regulatory (CDKN1B, AKT1, IFN-γ) and up-regulatory (BTG2, CDKN1A, FAS) effects on genes and related protein expressions may provide a multi-targeted therapy potential for chronic myeloid leukemia cancer cells without affecting healthy cells.

Combinations of polyphenols disaggregate Aß1­42 by passing through in vitro blood brain barrier developed by endothelium, astrocyte, and differentiated SH­SY5Y cells.

Disaggregation of amyloid ßeta (Aß) is considered as one of the promising therapeutic strategies for Alzheimer's disease. Polyphenols are promising molecules for the disaggregation of Aß. However, in order to find a potential therapeutic candidate, the in vitro analyses need to be performed on a model that mimics the blood­brain barrier (BBB) as much as possible. Therefore, we aimed to establish an in vitro BBB representative transwell system by using differentiated human neuroblastoma (SH­SY5Y), cerebral microvascular endothelial, and astrocyte cells to investigate transition and Aß disaggregation capacity of punicalagin (PU), ellagic acid (EA), epigallocatechin gallate (EGCG), gastrodin, and their combinations on the established system. The efficiency of the established transwell systems was evaluated by measuring the transendothelial electrical resistance (TEER) and paracellular permeability coefficients (Pe) values. The transition and Aß disaggregation capacities of the polyphenols were evaluated in the established tri­culture transwell system based on obtained TEER (50,07 Ω.cm2) and Pe (65x10­6 cm/s) values. Our results revealed that all polyphenols can successfully pass across the BBB system and disaggregate Aß. While Aß disaggregation capacities of the polyphenols were in the range of 30.52-45.01%, the percentages of their combinations were higher (75% for EGCG­PU (Com 1) and 64% for EGCG­EA (Com 2)). Consequently, this study provides the first evidence that Com 1 and Com 2 are promising polyphenol combinations in terms of Aß disaggregation. Besides, the developed tri­culture transwell system, containing differentiated SH­SY5Y cells, may provide a new tool that closely mimics the BBB for basic research and testing of candidate agents.

Melatonin attenuates the detrimental effects of UVA irradiation in human dermal fibroblasts by suppressing oxidative damage and MAPK/AP-1 signal pathway in vitro.

BACKGROUND: People living in Mediterranean countries are mostly exposed to solar ultraviolet (UV) radiation that damages skin and results in photoaging which involves activation of epidermal growth factor receptor (EGFR) and downstream signal transduction through mitogen-activated protein kinases (MAPKs) in fibroblasts. Generation of reactive oxygen/nitrogen species by UV radiation is also critical for EGFR and MAPKs activation. MAPKs are responsible for activation of AP-1 subunits in the nucleus which induce matrix metalloproteinases. Melatonin, along with its metabolites, are known to be the most effective free radical scavenger and protective agent due to its ability to react with various radicals, lipophilic/hydrophilic structures. OBJECTIVES: In this study, we investigated the effects of melatonin on UVA-irradiated primary human dermal fibroblasts (HDFs) by following the alteration of molecules from cell membrane to the nucleus and oxidative/nitrosative damage status of the cells in a time-dependent manner which have not been clearly elucidated yet. METHODS: To mimic UVA dosage in Mediterranean countries, HDFs were exposed to UVA with sub-cytotoxic dosage (20 J/cm2 ) after pretreatment with melatonin (1 µmol/L) for 1 hour. Changes in the activation of the molecules and oxidative/nitrosative stress damage were analyzed at different time points. RESULTS: Our results clearly show that melatonin decreases UVA-induced oxidative/nitrosative stress damage in HDFs. It also suppresses phosphorylation of EGFR, activation of MAPK/AP-1 signal transduction pathway and production of matrix metalloproteinases in a time-dependent manner. CONCLUSION: Melatonin can be used as a protective agent for skin damage against intracellular detrimental effects of relatively high dosage of UVA irradiation.

Synergistic Induction of Apoptosis by Quercetin and Curcumin in Chronic Myeloid Leukemia (K562) Cells.

Chronic myeloid leukemia is a major hematopoietic malignancy characterized by expansion of myeloid cells. In this study, we have investigated whether quercetin, curcumin and their combination induce apoptosis and inhibit growth of K562 cells. We have observed that quercetin and curcumin combination induced apoptosis accompanied by increased ROS and decreased GSH levels as well as loss of mitochondrial membrane potential. Our mRNA and protein expression results suggested that cytochrome c was released from mitochondria causing PARP and caspase-9 cleavages, the hallmarks of mitochondrial apoptotic pathway. We believe that triggering of apoptosis is mostly via mitochondrial pathway and ROS generation may induce impairment of mitochondrial membrane potential. The use of quercetin and curcumin combination potentiates individual apoptotic effects of the polyphenols and reduces their effective dose thereby preventing potential toxic effects on normal cells. Additional preclinical studies and clinical trials are certainly required to further validate their usefulness as potent anticancer agents.

Quercetin-Induced Cell Death in Human Papillary Thyroid Cancer (B-CPAP) Cells.

In this study, we have investigated the antiproliferative effect of quercetin on human papillary thyroid cancer cells and determined the apoptotic mechanisms underlying its actions. We have used different concentrations of quercetin to induce apoptosis and measured cell viability. Apoptosis and cell cycle analysis was determined by flow cytometry using Annexin V and propidium iodide. Finally, we have measured changes in caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels as hallmarks of apoptosis and Hsp90 protein expression level as a marker of proteasome activity in treated and control cells. Quercetin treatment of human papillary thyroid cancer cells resulted in decreased cell proliferation and increased rate of apoptosis by caspase activation. Furthermore, it was demonstrated that quercetin induces cancer cell apoptosis by downregulating the levels of Hsp90. In conclusion, we have shown that quercetin induces downregulation of Hsp90 expression that may be involved in the decrease of chymotrypsin-like proteasome activity which, in order, induces inhibition of growth and causes cell death in thyroid cancer cells. Thus, quercetin appears to be a promising candidate drug for Hsp90 downregulation and apoptosis of thyroid cancer cells.

Amyloid Beta Peptides Affect Pregnenolone and Pregnenolone Sulfate Levels in PC-12 and SH-SY5Y Cells Depending on Cholesterol.

Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25-35, AB 1-40 and AB 1-42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose-and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25-35 were higher than those in AB 1-40 and AB 1-42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9 mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase might be playing a key role in the cell death mechanism of AB toxicity depending on cellular cholesterol levels.

Diagnostic and prognostic values of preoperative serum levels of YKL-40, HE-4 and DKK-3 in endometrial cancer.

OBJECTIVE: The objective of this study is to determine the efficiency of YKL-40, HE-4 and DKK-3 levels in early diagnosis of patients with endometrial cancer and in the pre-operative estimation of the prognostic parameters such as stage, grade and the extension of the disease. METHODS: In this prospective study, 50 patients diagnosed with endometrial cancer and 50 women as a control group, who applied to Dokuz Eylul University and Ege University Faculties of Medicine, Obstetrics and Gynecology Clinics between May 2011-May 2012 were included. CA125, HE-4, YKL-40 and DKK-3 serum levels were measured by ELISA and compared between two groups. The relation between serum levels and histopathological results, extension of disease and prognostic factors were analyzed. RESULTS: Preoperative serum CA125, HE-4 and YKL-40 levels were significantly higher in endometrial cancer group (p<0.001). Serum HE-4 levels were significantly higher in advanced stages (p=0.004). When we examined early stage patients, YKL-40 levels were significantly higher in non-endometrioid histology compared with endometrioid adenocarcinoma (p=0.022). We also examined the relation between the markers and prognostic factors. Different from other markers, HE-4 levels were significantly higher in endometrial cancer patients who had lymphovascular space involvement, lower uterine segment involvement, endocervical stromal involvement, and deep myometrial invasion. CONCLUSION: YKL-40 and HE-4 were significantly higher in patients with endometrial cancer. HE-4 seems to be superior to YKL-40 in discriminating early and advanced stages. Additionally, HE4 is significantly correlated with prognostic factors. HE-4 and YKL-40 may be successful in early determination of endometrial cancer and in detection of high risk subsets before surgery.

The effect of resveratrol on signal transduction pathways and the role of pro-apoptotic Bax protein on apoptosis in HCT- 116 colon carcinoma cell lines.

Colon carcinoma is the third among the cancer related deaths. The role of pro-apoptotic Bax protein on the resveratrol related apoptosis, mitochondrial membrane potential and signal pathways has not been identified in colon carcinoma cells. In this direction, HCT-116 bax positive and HCT-116 negative cell lines were utilized to detect the apoptotic effect and I?B, MEK1 and STAT 3 signal transduction pathways of resveratrol. The impact on the cell viability and IC50 value of resveratrol has been determined via WST-1 viability assay. The ratio of apoptosis has been evaluated via flow cytometry following Annexin V/Propidium iodide (PI) double staining. Changes in the mitochondrial membrane potential have been analyzed by flow cytometry and JC-1 fluorometric staining. IkB, MEK1 and STAT3 molecules were measured by Enspire device. Data showed the IC50 value for resveratrol as 50M. According to the flow cytometry, apoptosis ratio has been determined as 29.65% in the experimental group of bax positive cells, as 13.98% in the experimental group of bax negative cells. Changes in the membrane potential has been established as 8.62% in the experimental group of bax positive cells, as 97.98% in the experimental group of bax negative cells. When the obtained data from Enspire device was reviewed; bax positive cells I?B phosphorylations were found as as 5.22 for experimental groups; MEK1 phosphorylations were found as and as 1.15 for experimental groups; STAT 3 phosphorylations were found as and as 2.52 for experimental groups. In HCT-116 bax negative cells, I?B phosphorylation were and 2.71 in experimental groups; MEK1 phosphorylation were 1.18 in experimental groups; STAT 3 phosphorylation were 1.54 in experimental groups. Our data show that Bax protein plays role in the apoptotic effect of resveratrol by altering mitochondrial membrane potential and mitochondrial membrane permeability, signal transduction and the absence of Bax increase the sensitivity of HCT-116 colon carcinoma cells to apoptosis.

Effect of D-penicillamine on rat lung elastin cross-linking during the perinatal period.

This study was designed to clarify the effects of D-penicillamine (DPA), a drug used for treatment of various pathological events, on lung elastin formation and maturation of the newborn in the perinatal period. The investigation was conducted on 20 newborn rats bred from 40 female and six male rats. DPA doses 400 mg kg(-1) day(-1) and physiological saline were given intraperitoneally (i.p) to experimental and control groups. To assess newborn maturation, their body and lung weights were determined. Serum Cu levels were measured by atomic absorption spectroscopy and ceruloplasmin (Cp) activities were measured spectrophotometrically. Newborn lung tissue elastin, desmosine (DES) and isodesmosine (IDES) levels were measured by HPLC. The results showed that DPA treatment caused loss of skin elasticity and reduction in body and lung weight in newborns of the experimental group. The serum Cu levels and Cp activity were found to be significantly lower in both maternal and newborn of the experimental groups compared with the control group. The lung DES, IDES and elastin values of newborns in the experimental group were decreased compared with the control group. In conclusion, our results indicate that 400 mg kg(-1) day(-1) DPA, a dose that is used in the treatment of Wilson's disease, rheumatoid arthritis and cystinuria, caused the retardation of newborn maturation, a decrease in DES-IDES cross-links and levels of lung elastin of offspring in the perinatal period. Another conclusion to be drawn from this study is that even low levels of Cu depletion due to DPA administration induces a change in cross-linking in lung elastin during the perinatal period.

Effect of melatonin cotreatment against kainic acid on coenzyme Q10, lipid peroxidation and Trx mRNA in rat hippocampus.

Oxidative stress is a likely molecular mechanism in the neurotoxicity of kainic acid (KA), an excitotoxic substance. The aim of this report was to assess the effect of melatonin co-treatment against KA by measuring the levels of Coenzyme Q10 (CoQ 10), lipid peroxidation (LPO), and Thioredoxin (Trx) mRNA in the rat hippocampus. The male rats were divided into three groups as saline, KA treatment (15 mg/kg), and KA plus melatonin (20 mg/kg). The levels of LPO and CoQ10 were determined by high pressure liquid chromatography (HPLC) consisting of fluorescence and electro-chemical detectors, respectively. The expression of the Trx gene was quantified using reverse transcription followed by real-time polymerase chain reaction (RT-PCR). The results show that the level of LPO increased although the level of CoQ10 decreased both in homogenates and mitochondria in KA-treated rats However, melatonin co-treatment attenuated the level of LPO and partially restored the level of CoQ10. Melatonin co-treatment against KA did not affect the regulation of Trx. Finally, in the context of the decreased LPO and the increased CoQ10, the results suggest that melatonin may be protective against central nervous system pathologies involving excitotoxicity or where oxidative damage may contribute to mitochondrial dysfunction.