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Memantine is used to prevent glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease. As glutamine is one of the major source of anabolism in fast growing cancer cells, we aimed to interfere with the cancer cell metabolism in A549 lung cancer cells by using memantine. The effects of memantine on cell cycle progression and cell death in A549 cells were assessed by MTT assay and PI staining. Cells were treated with 0.25 mM memantine for 48 hours and then cell metabolism (AMPKA1, AMPKA2, HIF1A, B-catenin, PKM), apoptosis (p53, p21, Bax, Bcl-XL, NOXA, PUMA) and autophagy related (LC3B-I, LC3B-II, SQSTM1) mRNA and protein expressions were investigated by RT-qPCR and western blotting. Memantine decreased cell viability significantly in a concentration-dependent manner by inducing G0/G1 cell cycle arrest. Our results suggest that memantine activates AMPK1/2 significantly (p=0.039 and p=0.0105) that led cells through apoptosis and autophagy by decreasing cancer cell metabolism regulators like HIF1A, B-catenin and PKM as the consequence of this energetic shift. Memantine represents a useful tool to target metabolism in cancer cells. Therefore, it might be used a new repurposed drug in cancer treatment., EXCLI Journal; 20:Doc223; ISSN 1611-2156

FoxM1, pek çok kanserde aşırı ifade edilen, hücre göçü, invazyonu, anjiogenez ve metastaz süreçlerinde önemli rolü olan onkogenik bir transkripsiyon faktörüdür. Bu çalışmada metastatik özellikte olan 4T1 hücreleri ve bu hücreler ile oluşturulmuş allograft Balb/C fare modelinde FoxM1 inhibisyonunun tek başına ve FoxM1'in çekirdeğe göçünde etkili olan MEK inhibisyonunun, hücre canlılığı ve metastaz parametreleri üzerine etkisini inceledik. Hücre canlılığı MTT testi kullanılarak, in vitro ve in vivo protein ifadeleri western blot ve immunohistokimya yöntemleriyle, uPA aktivitesi enzimatik yöntemlerle belirlenmiştir. MTT sonuçlarına göre Thiostrepton'un (FoxM1 inhibitörü) 24 saat ve 48 saat IC50 değeri 4 μM olarak belirlenirken, Selumetinib'in (MEK inhibitörü) IC50 değeri 24 saat için 8 μM, 48 saat için 6 μM olarak belirlenmiştir. Her iki inhibitörün de IC50 konsantrasyonları tek başına ve kombine verildiğinde FoxM1, ERK, p-ERK, Twist, E-cadherin, Vimentin ifadeleri üzerinde etkili olduğu belirlenmiştir. In vivo sonuçlarımıza göre Thiostrepton tek başına metastazla ilişkili proteinlerin çoğunda etkili olurken, Selumetinib ile kombine uygulamalarda sadece Twist, MMP-2 ve MMP-9 proteinlerin ifadesini azaltmıştır. İmmunhistokimyasal olarak ise Thiostrepton ve kombine grupta FoxM1 ifadesinin tümörde boyama şiddetini azalttığı görülürken, FoxM1'in daha çok lenfoid hücrelerdeki varlığı belirlenmiş ve sitoplazmik ve membranöz lokasyonda olduğu gözlenmiştir. Sonuçlarımıza göre FoxM1 inhibisyonunun Thiostrepton ile sağlanması üçlü negatif meme kanseri hücrelerinin metastaz potansiyelini azaltabilir ve MEK inhibisyonu veya diğer kemoterapötiklerle bu etkinin artırılabileceği düşünülebilir.Anahtar Kelimeler : FoxM1, Selumetinib, Thiostrepton, üçlü negatif memekanseri, metastaz FoxM1 is an oncogenic transciption factor that is overexpressed in many cancer types. It plays important roles in cell migration, invasion, angiogenesis and metastasis. In this study, we investigated the effects of FoxM1 inhibition alone and in combination with MEK inhibition which is a FoxM1's nuclear localization regulator on cell viability and metastasis parameters in 4T1 cells and its allograft Balb/C mouse model. Cell viability was determined by MTT assay, in vitro and in vivo protein expressions were analyzed by western blot and immunohistochemical methods, and uPA activity was determined by enzymatic methods. According to MTT results, the IC50 value of Thiostrepton (FoxM1 inhibitor) was determined as 4 μM for 24 h and 48 h, IC50 value of Selumetinib was determined to be 8 μM for 24 hours and 6 μM for 48 hours. Both inhibitors were found to be effective on the expressions of FoxM1, ERK, p-ERK, Twist, E-cadherin and Vimentin when IC50 concentrations of both inhibitors were given alone and in combination. According to our in vivo results, Thiostrepton was effective alone in most of the metastases related proteins. However, Thiostrepton and Selumetinib combination was only effective in Twist, MMP-2 and MMP-9 expressions. Immunohistochemically, FoxM1 expression intensity was decreased with Thiostrepton alone and in combination with Selumetinib in tumors where as FoxM1 was detected more in lymphoid cells and cytoplasmic and membranous loci. Our results suggest that FoxM1 inhibition with Thiostrepton may reduce the potentials for metastasis of triple-negative breast cancer and this effect can be increased by MEK inhibition or other chemotherapeutics.Keywords : FoxM1, Selumetinib, Thiostrepton, triple negative breastcancer, metastasis 112

Bu çalışmada kullanılan titanyum dioksit nanopartikülleri (TiO2 NP) ve çinko oksit nanopartiküllerinin (ZnO NP) taramalı elektron mikroskobunda küresel yapıda ve 10-300 nm arasında değişen partiküller içerdiği gözlenmiştir. Bu nanopartiküllerin, dinamik ışık saçılımı ile negatif elektrik yüklü ve ortalama hidrodinamik çaplarının TiO2 NP'nde 804,30±15,06 nm, ZnO NP'nde ise 495,80±51,15 nm ebatlarında olduğu tespit edilmiştir. Genotoksik etkilerin değerlendirilmesi için TiO2 NP'nin 20, 40, 60, 80 ve 100 µg/ml'lik, ZnO NP'nin 1, 5, 10, 20 ve 30 µg/ml'lik konsantrasyonları kullanılmıştır. TiO2 NP kromozom anormallikleri (KA) frekansını 24 saatlik uygulamada en düşük iki konsantrasyonda (20 ve 40 ?g/ml) önemli düzeyde artırmış, 48 saatlik uygulamada ise önemli bir artış oluşturmamıştır. ZnO NP, KA frekansını 24 saatlik uygulamada en yüksek iki konsantrasyonda (20 ve 30 ?g/ml), 48 saatlik uygulamada ise tüm konsantrasyonlarda önemli düzeyde artmıştır. TiO2 NP kardeş kromatid değişimi (KKD) frekansını her iki uygulama süresinde de negatif kontrole göre önemli düzeyde artırmıştır. Bu artışlar çözücü kontrol ile kıyaslandığında, 24 saatlik uygulamada 20, 60 ve 100 µg/ml'lik, 48 saatlik uygulamada ise 20, 60, 80 ve 100 µg/ml'lik konsantrasyonlarda anlamlıdır. ZnO NP, KKD frekansını her iki uygulama süresinde de doza bağlı olarak artırmıştır. Bu artış 24 saatlik muamelede en yüksek üç dozda, 48 saatlik uygulamada ise en yüksek iki dozda anlamlıdır. TiO2 NP, mikronükleus (MN) frekansında en düşük üç konsantrasyonda anlamlı olmayan bir artış oluşturmuştur. ZnO NP MN frekansını tüm konsantrasyonlarda artırmıştır, fakat bu artış en yüksek iki konsantrasyonda (20 and 30 ?g/ml) anlamlıdır. TiO2 NP 24 saatlik uygulamada mitotik indeksi kontrole göre düşürmüştür, ancak bu düşüş 60 ve 80 µg/ml'lik konsantrasyonlarda anlamlıdır. TiO2 NP 48 saatlik uygulamada mitotik indekste önemli bir değişikliğe sebep olmamıştır. ZnO NP, mitotik indeksi her iki uygulama süresinde de düşürmüştür. Bu düşüş 24 saatlik uygulamada 5, 10 ve 30 µg/ml'lik konsantrasyonlarda, 48 saatlik uygulamada 30 µg/ml'lik konsantrasyonda anlamlıdır. TiO2 NP, replikasyon indeksi üzerinde önemli bir etki göstermez iken, ZnO NP, her iki uygulama süresinde de en yüksek konsantrasyonda (30 µg/ml) anlamlı olmayan bir azalmaya sebep olmuştur. TiO2 NP ve ZnO NP, nükleer bölünme indeksini etkilememiştir. TiO2 NP kuyruk uzunluğunda en yüksek konsantrasyonda (100 µg/ml) kontrole göre anlamlı bir artışa sebep olmuştur, fakat kuyruk yoğunluğu ve kuyruk momenti üzerinde önemli bir etki göstermemiştir. ZnO NP uygulaması ile kuyruk yoğunluğu en yüksek konsantrasyonda önemli düzeyde azalmıştır. Kuyruk yoğunluğu ve kuyruk momenti benzer artış ve azalışlar gösterirken, kuyruk uzunluğu ZnO NP'nin 5 ve 20 µg/ml'lik konsantrasyonlarında önemli ölçüde artmıştır. Bu sonuçlar, TiO2 NP'lerinin nisbeten zayıf bir genotoksik etkiye sahip olduğunu, bunun aksine ZnO NP'nin yüksek konsantrasyonlarda hem güçlü bir genotoksik etkiye ve hem de sitotoksik etkiye sahip olduğunu göstermektedir. Titanium dioxide nanoparticles (TiO2 NPs) and zinc oxide nanoparticles (ZnO NPs), used in this study, were observed to be spherical in shape and contained particles ranging from 10-300 nm by scanning electron microscopy. These nanoparticles were found to be negatively charged and the mean hydrodynamic diameters were found to be 804.30±15.06 nm for TiO2 NPs and 495.80±51.15 nm for ZnO NPs by dynamic light scattering. 20, 40, 60, 80 ve 100 µg/ml concentrations of TiO2 NPs and 1, 5, 10, 20 ve 30 µg/ml concentrations of ZnO NPs were used to assess genotoxic effects. TiO2 NPs significantly increased the frequency of chromosomal aberrations (CAs) at two lowest concentrations (20 ve 40 ?g/ml) at 24 h treatment, however they did not show a significant increase at 48 h treatment. ZnO NPs significantly increased the frequency of CAs at two highest concentrations (20 and 30 ?g/ml) at 24 h and at all concentrations at 48 h treatment. TiO2 NPs significantly increased the frequency of sister chromatid exchanges (SCEs) at both treatment periods compared to negative control. These increases were significant at 20, 60 ve 100 µg/ml concentrations for 24 h treatment and, 20, 60, 80 ve 100 µg/ml concentrations for 48 h treatment compared to solvent control. ZnO NPs, increased the frequency of sister chromatid exchanges (SCEs) at both treatment periods in a dose dependent manner. These increases were significant at the three highest concentrations for 24 h and at the two highest concentrations for 48 h treatment. TiO2 NPs induced a non-significant increase in the the frequency of micronuclei at three lowest concentrations. ZnO NPs increased the frequency of MN at all concentrations, however these increases were significant at the two highest concentrations (20 and 30 ?g/ml). TiO2 NPs caused reduction in mitotic index at 24 h treatment compared to control. However these reductions were significant at 60 and 80 µg/ml concentrations. TiO2 NPs did not induced a significant effect on mitotic index at 48 h treatment. ZnO NPs reduced mitotic index at both treatment periods. These decreases were significant at 5, 10 and 30 µg/ml concentrations at 24 h and 30 µg/ml concentrations at 48 h treatments. While TiO2 NPs did not show any effect on replication index, ZnO NPs caused a non-significant decrease at the highest concentrations at both treatment periods. Neither TiO2 NPs nor ZnO NPs effect nuclear division index. TiO2 NPs caused a significant increase in tail lenght at the highest concentration (100 µg/ml) compared to control, however they did not show a significant effect on tail intensity and tail moment. Tail intensity reduced significantly at the highest concentrations with ZnO NPs treatment. Although tail intensity and tail moment displayed similar decreases and increases, tail lenght increased significantly at 5 and 20 µg/ml concentrations in ZnO NPs treatment. These results show that TiO2 NPs have a relatively weak genotoxic effect, by contrast, ZnO NPs have a strong genotoxic effect as well as cytotoxic effect at high concentrations. 116

Objectives Drug repurposing studies enable shorter routes to the clinic by skipping the steps like in vitro in vivo screening, chemical optimization and toxicological studies. In our study, we investigated the potent anti-cancer effect of Alzheimer's drug Memantine on 4T1 breast cancer cells. Methods Memantine's effect on proliferation of 4T1 cells was evaluated by using the MTT assay. Memantine inhibited 4T1 cell proliferation in a concentration- dependent manner at 24 and 48 hours. We investigated the drug's effect on the protein expressions of Bax, Bcl-2, Casp-3, Casp-9, E-Cad, Vimentin, B-Cat, GSK3B, p-ERK, ERK, p-GS, GS that are involved in apoptosis, metastasis and cell survival. Results Memantine altered the Bcl-2, Bax, Casp3, Casp-9 apoptotic protein expression levels. We found that memantine inhibited p-Erk expression and that result suggested a plausible mechanism of action for memantine's antineoplastic effect. Memantine also inhibited wound closure at 24 h, significantly (p = 0.0055). Conclusions Memantine inhibited 4T1 breast cancer cell proliferation at significantly lower doses than mostly studied re-purposed drug Metformin. Therefore, we believe that memantine might hold a great promise as a new repositioned drug in cancer treatment and it is our further interest to investigate its effects in vivo (Fig. 3, Ref. 22).

Bulbul, Y. Emre/0000-0003-1885-5026; Tort, Serdar/0000-0003-4945-5420; DEMIRTAS KORKMAZ, FUNDA/0000-0003-3978-9427 WOS: 000469332700010 PubMed: 31063838 Potential usage of biodegradable and biocompatible polymeric nanofibers is the most attention grabbing topic for the drug delivery system. In order to fabricate ultrafine fibers, electrospinning, one of the well-known techniques, has been extensively studied in the literature. In the present study, the objective is to achieve the optimum blend of hydrophobic and hydrophilic polymers to be used as a drug delivery vehicle and also to obtain the optimum amount of doxycycline (DOXH) to reach the optimum release. In this case, the biodegradable and biocompatible synthetic polymers, poly(e-caprolactone) (PCL) and poly(ethylene oxide) (PEO), were blended with different ratios for the production of DOXH-loaded electrospun PCL/PEO membranes using electrospinning technique, which is a novel attempt. The fabricated membranes were subsequently characterized to optimize the blending ratio of polymers by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD) and water contact angle analysis. After the characterization studies, different amounts of DOXH were loaded to the optimized blend of PCL and PEO to investigate the release of DOXH from the membrane used as a drug delivery vehicle. In vitro drug release studies were performed, and in vitro drug release kinetics were assessed to confirm the usage of these nanofiber materials as efficient drug delivery vehicles. The results indicated that 3.5% DOXH-loaded (75:25 w/w) PCL/PEO is the most acceptable membrane to provide prolonged release rather than immediate release of DOXH. Gazi University Research FundGazi University [BAP 06-2018/15]; Scientific and Technical Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TUBITAK-BIDEB 2211] This work was supported by the Gazi University Research Fund [BAP 06-2018/15]; and S. Melda Eskitoros-Togay was awarded scholarships by the Scientific and Technical Research Council of Turkey [TUBITAK-BIDEB 2211].

Alzheimer's disease (AD) is a major progressive neurodegenerative disorder of the aging population. High post-menopausal levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) are strongly associated with the onset of AD, and we have shown recently that FSH directly activates the hippocampal Fshr to drive AD-like pathology and memory loss in mice. To establish a role for FSH in memory loss, we used female 3xTg;Fshr +/+ , 3xTg;Fshr +/- and 3xTg;Fshr -/- mice that were either left unoperated or underwent sham surgery or ovariectomy at 8 weeks of age. Unoperated and sham-operated 3xTg;Fshr -/- mice were implanted with 17β-estradiol pellets to normalize estradiol levels. Morris Water Maze and Novel Object Recognition behavioral tests were performed to study deficits in spatial and recognition memory, respectively, and to examine the effects of Fshr depletion. 3xTg;Fshr +/+ mice displayed impaired spatial memory at 5 months of age; both the acquisition and retrieval of the memory were ameliorated in 3xTg;Fshr -/- mice and, to a lesser extent, in 3xTg;Fshr +/- mice- -thus documenting a clear gene-dose-dependent prevention of hippocampal-dependent spatial memory impairment. At 5 and 10 months, sham-operated 3xTg;Fshr -/- mice showed better memory performance during the acquasition and/or retrieval phases, suggesting that Fshr deletion prevented the progression of spatial memory deficits with age. However, this prevention was not seen when mice were ovariectomized, except in the 10-month-old 3xTg;Fshr -/- mice. In the Novel Object Recognition test performed at 10 months, all groups of mice, except ovariectomized 3xTg;Fshr -/- mice showed a loss of recognition memory. Consistent with the neurobehavioral data, there was a gene-dose-dependent reduction mainly in the amyloid β40 isoform in whole brain extracts. Finally, serum FSH levels < 8 ng/mL in 16-month-old APP/PS1 mice were associated with better retrieval of spatial memory. Collectively, the data provide compelling genetic evidence for a protective effect of inhibiting FSH signaling on the progression of spatial and recognition memory deficits in mice, and lay a firm foundation for the use of an FSH-blocking agent for the early prevention of cognitive decline in postmenopausal women., Competing Interests: COMPETING FINANCIAL INTERESTS M.Z. is inventor on issued and pending patients on the use of FSH as a target for osteoporosis, obesity and Alzheimer’s disease. The patents will be held by Icahn School of Medicine at Mount Sinai, and M.Z. would be recipient of royalties, per institutional policy. The other authors declare no competing financial interests.

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow--these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain., Competing Interests: Conflict of Interest: M.Z. consults for Gershon Lehmann, Guidepoint and Coleman groups. T.Y. is a senior editor of eLife. None of the other authors have any conflicts.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.