μM Dose Research Articles

Anti-Cancer Potential of Afzelin towards AGS Gastric Cancer Cells.

Afzelin demonstrates anti-inflammatory and anti-cancer properties. Our purpose was to assess its influence on apoptosis, Bax, caspases, MUC1, cancer-related carbohydrate antigens, enzymes participating in their formation, and galectin-3 in AGS gastric cancer cells. A total of 60 and 120 μM afzelin was used in all experiments. Flow cytometry was applied to determine apoptotic response. Western blotting and RT PCR were used to detect the expression of mentioned factors. Flavonoid at higher concentration revealed slight apoptotic respond. Bax, caspase-3, -8, -9 increased upon afzelin action. Stimulatory effect of the flavonoid on MUC1 cytoplasmic tail and extracellular domain in cell lysates and on MUC1 gene was revealed. MUC1 release into the culture medium was inhibited by the flavonoid. The 60 μM afzelin dose stimulated GalNAcTL5 protein expression and inhibited C1GalT1. ST6GalNAcT mRNA was inhibited by both flavonoid doses. ST3GalT was inhibited by 120 μM afzelin on protein and mRNA level. Lewisa/b protein was reduced by both afzelin concentrations. FUT3 and FUT4 mRNA was inhibited by 120 μM dose of afzelin. Galectin-3 protein increased in cell lysates and decreased in culture supernatant by 60 and 120 μM flavonoid. Galectin-3 gene expression was stimulated by two used concentrations of afzelin in comparison to control. We conclude that afzelin can be considered as the potential anti-cancer agent, supporting conventional cancer treatment.

Influence of curcumin and carbazole on ovine ovarian preantral follicle and granulosa cell functions

The present study was undertaken to study the effect of plant bioactive compounds curcumin and carbazole on sheep ovarian functions. In the present study, both the bioactive compounds were tested at different levels (Control, T1-1 μM, T2-5 μM, T3-10 μM, T4- 25 μM, T5- 50μM, T6-100 μM) on preantral follicle (PF) growth rate, survival rate (6 days culture), granulosa cell (GC) number increment (2 days culture) and estradiol production (5 days GC culture spent media). Curcumin had shown a significantly higher PF survival rate (%), i.e. 74.3±1.5, 76.3±1.4 at 10 and 25 μM levels respectively. Similarly, higher PF growth rates (μm per day), i.e. 16.1±0.9 was observed at 50 μM levels. Similarly, curcumin was effective @ 50 μM level to increase the granulosa cell number as well as estradiol production with a mean granulosa cell number (×105) and estradiol production (pg) values of 1.55±0.04 and 85.3±3.3 respectively. Likewise, carbazole was effective at the level of 25 μM to increase the PF growth rate (μm per day), survival rate (%)with mean values of 74.3±1.3, 12.1±0.9. Similarly, carbazole was effective at 50 μM dose levels in the granulosa cell number increment (×105) with a mean value of 1.57±0.02. No significant change in estradiol production was observed in carbazole treated group.

Effect of Hydrated Carbon 60 Fullerene on Frozen Ram Semen Quality.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C60HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 μM C60HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at -140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 μM C60HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C60HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 μM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 μM doses) and catalase (between 1 and 40 μM doses) activities. In conclusion, results of this study show that the C60HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze-thawing.

Flavanols and Flavanes from Crinum asiaticum and Their Effects on LPS Signaling Pathway Through the Inhibition of NF-κB Activation.

Three new flavanols, (2R,3S)-7-methoxy-flavan-3-ol (1: ), (2R,3S)-7-hydroxy-flavan-3-ol (2: ), and (2R,3S)-2'-hydroxy-7-methoxy-flavan-3-ol (3: ), together with two known flavans (4: and 5: ), were isolated from the chloroform extract of Crinum asiaticum. Their structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-MS, and CD data. The isolated compounds 1: and 3: -5: showed inhibitory activity toward LPS-induced nitric oxide (NO) production. Further investigation of the NF-κB pathway mechanisms indicated that 1: and 3: -5: inhibited the LPS-induced IL-6 production and p65 subunit phosphorylation of NF-κB in RAW264.7 cells, with an effective dose of 10 µM.

Oxymatrine hydrazone (OMTH) synthesis and its protective effect for rheumatoid arthritis through downregulation of MEK/NF-κB pathway.

Rheumatoid arthritis (RA) is one of the inflammatory diseases detected in more than 1% of the world population. In the present study, oxymatrine hydrazone (OMTH) was synthesized and investigated for treatment of RA in vitro in TNF-α induced fibroblast-like synoviocyte cell model. Cell viability and apoptosis were detected using MTT and flow cytometry assays, respectively. ELISA was used for determination of inflammatory cytokines and western blotting for evaluation of protein expression. Pretreatment of HFLS-RA cells with 0.5, 1.0, 1.5, 2.0, and 2.5μM doses of OMTH suppressed TNF-α induced promotion of proliferative potential in dose-based manner. The OMTH pretreatment of TNF-α exposed HFLS-RA cells significantly increased apoptotic cell proportion. In TNF-α exposed HFLS-RA cells OMTH pretreatment elevated Bax and suppressed Bcl-2 expression. Treatment of HFLS-RA cells with OMTH prevented TNF-α mediated elevation of IL-1β, IL-6 and IL-8. Moreover, OMTH treatment of HFLS-RA cells effectively suppressed TNF-α mediated elevated levels of MMP-1 and MMP-13. Pretreatment of HFLS-RA cells with OMTH reversed TNF-α mediated promotion of iNOS and COX-2 levels. The MEK/1/2 and p65 phosphorylation in TNF-α exposed HFLS-RA cells was reduced by OMTH pre-treatment in dose-based manner. Thus, OMTH successfully inhibited TNF-α-mediated increased viability of RA synovial cells and activated apoptosis. Pretreatment of TNF-α exposed synovial cells with OMTH targeted phosphorylation of MEK/NF-κB. Therefore, OMTH may act as potential therapeutic agent for RA treatment.

Powerful toxic activity of citrinin, a fungal phytotoxin, and its mode of action in onion cells.

The dose-related effects of citrinin (CTN) on various physiological, cytogenetic, biochemical, and anatomical parameters using Allium cepa L. bulbs as a test material were researched in the present study. The physiological parameters examinated were fresh weight, root length, root number, and germination percentage; the cytogenetic parameters were micronucleus (MN) frequency, chromosome aberration (CA), and mitotic index (MI); the biochemical parameters were catalase (CAT), superoxide dismutase (SOD) activities, malondialdehyde (MDA) level, and free proline contents. And the anatomical changes in root tip cells were investigated by cross-sections. For this aim, onion bulbs were splitted four groups as three applications and one control. The bulbs in the control group were treated with distilled water; the bulbs in the application groups were treated with 1 μM, 5 μM, and 10 μM doses of CTN for 7 days. CTN application caused a decrease in the physiological parameters compared to the control group. This treatment created an increase in the frequency of MN and CA, and a reduce in the MI. In addition, it induced a dose-dependent increase in CAT and SOD activities and MDA and proline contents compared to the control group. Moreover, after CTN application, anatomical changes such as flattened nucleus, cell wall thickening, and cell deformation were identified and it was found that these changes reached their maximum at 10 mg/L dose CTN. Concequently, CTN caused inhibitory effects and the Allium test material was found to be a useful bioindicator for monitoring these effects.

Effects of different doses of melatonin on rat adipose derived mesenchymal stem cells

Stem cell treatment is based on Melatonin which is crucial for lots of pathological and physiological pathways. Our aim is determining the most appropriate dose of melatonin affecting the rat adipose tissue mesenchymal stem cells. Stem cells were isolated from male rat adipose tissue. Differentiation and characterization experiments were performed. Cell viability analyses in stem cells were used the XTT [2,3-Bis-(2-methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide] assay. After 24h incubation, different concentrations (0.5, 1, 5, 10, 50µM) of extract were treated to the stem cells for 24h, 48 and 72h considering time and dose dependent manner. Total antioxidant status (TAS) and the total oxidant status (TOS) in control cells and melatonin treated cells (5, 10µM) were determined Rel Assay commercial kits. In 24h, melatonin increased cell viability in all groups. When we evaluate the effect of melatonin in 48h, the most proliferation increase was seen at 5, 10µM doses. When the total oxidant activity melatonin was found to be significantly lower in 5 and 10µM dose groups of melatonin. Melatonin increases the survivor of stem cells and the most effective dose is 5 and 10µM. The reduction of the oxidative stress index as a result of treating melatonin to mesenchymal stem cells showed that melatonin is a powerful antioxidant for stem cells.

Evaluation of the Effect of Orlistatorlistat on Expression of OCT4, Nanog, SOX2, and KLF4 Genes in Colorectal Cancer SW40 Cell Line

Background and Objective:Orlistat drug is one of the most criticalanti-obesity drugs that widely used around the world. The aim of this study was evaluation the effect of orlistat on the expression of OCT4, Nanog, SOX2, and KLF4 genes in the colorectal cancer SW40 cell line. Materials and Methods:SW40 cell line was cultured in DMEM medium contained orlistat for 24h, and cell viability was assessed by MTT assay. The fold changes of expression of OCT4, NANOG, KLF4, and SOX2 at mRNA level against β-actin were determined by real-timePCR. Two-sample t-test and one-way ANOVA were used to compare the mean of expression of different genes in different groups and different concentrations; a significant level of 0.05 was considered in all tests. Results:Our results showed a significant difference in cell viability, when different doses of Orlistat were used for 24 hour. concentrations of 25 and 100 μM reduce significantly the expression of OCT4 (P<0.05) and SOX2 (P<0.05) in the treated group in comparison to control (P<0.05). Also, the mRNA expression of KLF4 and Nanog was reduced significantly after treatment of SW40 cell lines was performed with 100 μM doses of Orlistat (P<0.05). Conclusion:It appears that after further studies in animal and human phases, orlistat can be used for the treatment of Colorectal Cancer.

Anemoside B4 inhibits enterovirus 71 propagation in mice through upregulating 14-3-3 expression and type I interferon responses.

Enterovirus 71 (EV71) is the major pathogens of human hand, foot, and mouth disease (HFMD). EV71 efficiently escapes innate immunity responses of the host to cause infection. At present, no effective antiviral drugs for EV71 are available. Anemoside B4 (B4) is a natural saponin isolated from the roots of Pulsatilla chinensis (Bunge) Regel. P. chinensis extracts that shows a wide variety of biological activities. In this study, we investigated the antiviral activities of B4 against EV71 both in cell culture and in suckling mice. We showed that B4 (12.5-200 μM) dose dependently increased the viability of EV71-infected RD cells with an IC50 value of 24.95 ± 0.05 μM against EV71. The antiviral activity of B4 was associated with enhanced interferon (IFN)-β response, since knockdown of IFN-β abolished its antiviral activity. We also confirmed that the enhanced IFN response was mediated via activation of retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) pathway, and it was executed by upregulation of 14-3-3 protein, which disrupted the interaction between yes-associated protein (YAP) and interferon regulatory factor 3 (IRF3). By using amino acids in cell culture (SILAC)-based proteomics profiling, we identified the Hippo pathway as the top-ranking functional cluster in B4-treated EV71-infected cells. In vivo experiments were conducted in suckling mice (2-day-old) infected with EV71 and subsequently B4 (200 mg · kg-1 · d-1, i.p.) was administeredfor 16 days. We showed that B4 administration effectively suppressed EV71 replication and improved muscle inflammation and limb activity. Meanwhile, B4 administration regulated the expressions of HFMD biomarkers IL-10 and IFN-γ, attenuating complications of EV71 infection. Collectively, our results suggest that B4 could enhance the antiviral effect of IFN-β by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

Arabidopsis Hypocotyl Adventitious Root Formation Is Suppressed by ABA Signaling.

Roots are composed of different root types and, in the dicotyledonous Arabidopsis, typically consist of a primary root that branches into lateral roots. Adventitious roots emerge from non-root tissue and are formed upon wounding or other types of abiotic stress. Here, we investigated adventitious root (AR) formation in Arabidopsis hypocotyls under conditions of altered abscisic acid (ABA) signaling. Exogenously applied ABA suppressed AR formation at 0.25 µM or higher doses. AR formation was less sensitive to the synthetic ABA analog pyrabactin (PB). However, PB was a more potent inhibitor at concentrations above 1 µM, suggesting that it was more selective in triggering a root inhibition response. Analysis of a series of phosphonamide and phosphonate pyrabactin analogs suggested that adventitious root formation and lateral root branching are differentially regulated by ABA signaling. ABA biosynthesis and signaling mutants affirmed a general inhibitory role of ABA and point to PYL1 and PYL2 as candidate ABA receptors that regulate AR inhibition.

Hydroxychloroquine induces oxidative DNA damage and mutation in mammalian cells

Since the early stages of the pandemic, hydroxychloroquine (HCQ), a widely used drug with good safety profile in clinic, has come to the forefront of research on drug repurposing for COVID-19 treatment/prevention. Despite the decades-long use of HCQ in the treatment of diseases, such as malaria and autoimmune disorders, the exact mechanisms of action of this drug are only beginning to be understood. To date, no data are available on the genotoxic potential of HCQ in vitro or in vivo. The present study is the first investigation of the DNA damaging- and mutagenic effects of HCQ in mammalian cells in vitro, at concentrations that are comparable to clinically achievable doses in patient populations. We demonstrate significant induction of a representative oxidative DNA damage (8-oxodG) in primary mouse embryonic fibroblasts (MEFs) treated with HCQ at 5 and 25 μM concentrations (P = 0.020 and P = 0.029, respectively), as determined by enzyme-linked immunosorbent assay. Furthermore, we show significant mutagenicity of HCQ, manifest as 2.2- and 1.8-fold increases in relative cII mutant frequency in primary and spontaneously immortalized Big Blue® MEFs, respectively, treated with 25 μM dose of this drug (P = 0.005 and P = 0.012, respectively). The observed genotoxic effects of HCQ in vitro, achievable at clinically relevant doses, are novel and important, and may have significant implications for safety monitoring in patient populations. Given the substantial number of the world’s population receiving HCQ for the treatment of various chronic diseases or in the context of clinical trials for COVID-19, our findings warrant further investigations into the biological consequences of therapeutic/preventive use of this drug.

Abstract 1003: NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells

Abstract Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.

Abstract 2736: Decreasing colorectal cancer extracellular pH dampens immune cell killing and is partially ameliorated by treatment with NHE1 inhibitor Cariporide

Abstract Tumor cells upregulate a plethora of proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment. Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how alterations in pH modulate the interactions between immune cells and tumors cells could help elucidate opportunities for new therapeutic approaches. To determine how pH modification impacts immune cell-mediated killing of tumor cells, co-culture experiments were conducted with GFP+ SW480 colorectal cancer cells and either NK-92 natural killer cells or TALL-104 T cells at various pHs in a 48-well plate. Manipulation of pH occurred using the compounds lactic acid and sodium bicarbonate. We observed that both NK-92 cells and TALL-104 cells exhibited decreasing amounts of cell killing in response to decreasing pH. Next, to determine if therapeutic manipulation of pH via alteration of pH pump activity could overcome the negative changes in immune cell activity associated with pH, we added Cariporide, a selective NHE1 inhibitor, to the co-culture experiments. Treatment with Cariporide bolstered T cell killing in a dose-dependent manner, however the doses used did not impact natural killer cell-mediated killing. Given the importance of cell signaling on immune function, and because we saw potentially significant changes in immune killing with Cariporide, we then evaluated how changes in pH using Cariporide affected these signaling molecules. Colorectal cancer cells (HCT116, HT29, KM12C) were treated with Cariporide at 5 µm and 10 µm doses and cancer cell culture supernatants were collected and frozen down at -80 °C. Cytokine, chemokine, and growth factor levels were analyzed using a Luminex 200 multiplexing instrument. Overall cytokine levels showed a decreasing trend in response to increasing doses of Cariporide. There were notable decreases in the levels of IL-8/CXCL8, VEGF, CCL3/MIP-1 alpha, and M-CSF with increasing doses of Cariporide. All of these factors, as well as extracellular acidosis, are known to promote epithelial mesenchymal transition (EMT), thus, promoting metastatic dissemination. A therapeutic that targets factors that lead to EMT could help prevent metastasis. Moreover, we saw that pH impacts the function of immune cells and this is relevant in the context of a highly acidic TME. Therapeutically modifying the pH of the TME using Cariporide treatment may be a way to improve immune response and prevent EMT. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response. Citation Format: Kelsey E. Huntington, Aaron W. Maxwell, Anna Louie, Shengliang Zhang, Lanlan Zhou, Wafik S. El-Deiry. Decreasing colorectal cancer extracellular pH dampens immune cell killing and is partially ameliorated by treatment with NHE1 inhibitor Cariporide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2736.

Physiological and biochemical effects of 2.4-D herbicide in wheat (Triticum aestivum L.) varieties

In this work were investigated the physiological and biochemical responses depended on toxic effect caused by different concentrations of herbicides called as 2.4-Dichlorophenoxyacetic acid (2.4-D) herbicide in the seedlings that belong to three wheat varieties. The seeds which belong to wheat (Triticum aestivum L. Bayraktar cv., İkizce cv. and Tosunbey cv.) were used as plant material. 15-day old seedlings for each wheat variety were divided into 4 groups consisting of the same number of seedlings and four variety doses of herbicide (0, 100 µM, 300 µM ve 1000 µM) were applied for them. In these applications for seedlings was preferred hydroponic surrounding to root. Although three varieties increased the growth of root and shoot elongation from the growth parameters of the seedlings, it leaded to a decrease in growth parameters in general in this herbicide. Although 2.4-D in the leaves caused an increase in 100 μM dose for only the Bayraktar in amount of chlorophyll a+b. In ones except these, there has always been a decreased. The amount of carotenoid resulted in the reduction of three varieties. Amount of MDA increased in all three varieties. Glutathione (GSH) / oxidized glutathione (GSSG) ratios in leaves increased in three varieties. The superoxide dismutase (SOD) activity in the leaves increased at the Bayraktar and decreased in the İkizce and the Tosunbey. Catalase (CAT) activity showed to a decrease in three varieties. As a result, it was determined that the 2.4-D, which was toxic for wheat plants even in very low concentrations.

Hydrogen sulfide alleviates apoptosis and autophagy induced by beryllium sulfate in 16HBE cells.

Beryllium and its compounds are systemic toxicants that are widely applied in many industries. Hydrogen sulfide has been found to protect cells. The present study aimed to determine the protective mechanisms involved in hydrogen sulfide treatment of 16HBE cells following beryllium sulfate-induced injury. 16HBE cells were treated with beryllium sulfate doses ranging between 0 and 300 μM BeSO4 . Additionally, 16HBE cells were subjected to pretreatment with either a 300 μM dose of sodium hydrosulfide (a hydrogen sulfide donor) or 10 mM DL-propargylglycine (a cystathionine-γ-lyase inhibitor) for 6 hr before then being treated with 150 μM beryllium sulfate for 48 hr. This study illustrates that beryllium sulfate induces a reduction in cell viability, increases lactate dehydrogenase (LDH) release, and increases cellular apoptosis and autophagy in 16HBE cells. Interestingly, pretreating 16HBE cells with sodium hydrosulfide significantly reduced the beryllium sulfate-induced apoptosis and autophagy. Moreover, it increased the mitochondrial membrane potential and alleviated the G2/M-phase cell cycle arrest. However, pretreatment with 10 mM DL-propargylglycine promoted the opposite effects. PI3K/Akt/mTOR and Nrf2/ARE signaling pathways are also activated following pretreatment with sodium hydrosulfide. These results indicate the protection provided by hydrogen sulfide in 16HBE cells against beryllium sulfate-induced injury is associated with the inhibition of apoptosis and autophagy through the activation of the PI3K/Akt/mTOR and Nrf2/ARE signaling pathways. Therefore, hydrogen sulfide has the potential to be a promising candidate in the treatment against beryllium disease.

Exogenous melatonin reduces water deficit-induced oxidative stress and improves growth performance of Althaea rosea grown on coal mine spoils.

Coal mining activities are responsible for significant land degradation and often long-term irreversible effects on ecosystem functioning. To better understand how coal mined sites could be re-vegetated and ecosystem functioning restored, we address the role of the signalling hormone melatonin, which controls plant growth and development under adverse environmental conditions. We assessed the effects of exogenous melatonin on the plant species Althaea rosea by measuring morphological growth attributes, photosynthetic efficiency, reactive oxygen species (ROS)-induced oxidative damage and antioxidant defence developed by the seedlings when grown on coal-mined spoils under various water regimes. Water deficit and negative effects of coal mine spoils significantly decreased morphological growth attributes (i.e. plant height, root length and dry biomass), gas-exchange traits (i.e. net photosynthesis rate, inter intercellular concentration of CO2, transpiration rate, stomatal conductance and water use efficiency) and photosynthetic pigments (chlorophyll and carotenoid contents) by increasing the ROS-induce oxidative damage and decreasing antioxidant enzyme activities of A. rosea seedlings. However, melatonin applications increased photosynthetic performance and antioxidant enzyme activities and decreased hydrogen peroxide and malondialdehyde contents and ultimately improved growth performance of A. rosea in coal-mined spoils. Overall, our findings show how the application of optimum water (63.0 %field capacity equivalent to 1.67 mm day-1) and melatonin (153.0 μM dose) significantly improves the re-vegetation of coal-mined spoils with A. rosea. Our study provides new insight into melatonin-mediated water stress tolerance in A. rosea grown on coal-mined spoils, and this strategy could be implemented in re-vegetation programmes of coal mine-degraded areas under arid and semiarid conditions of the north-western part of China and perhaps across other arid areas worldwide.

Antifungal imidazole: Potential new antineoplastic agent-cytotoxic effects on non-small cell lung cancer cell lines (A549).

e21037 Background: There are many drugs that can be applied to the treatment of lung cancer. These therapeutics include classical chemotherapeutics, targeted drugs against driver mutations, and immunotherapeutics. However, still, new agents are required to better results and patients outcomes. Recently, imidazole and its compounds, a type of antifungal drugs, were found to have antitumor efficacy in several cancer types. Its effects on non-small-cell lung cancer cells are yet known. This study aimed to detect anti-cancer properties of imidazole on non-small-cell lung cancer cells and suitability for clinical usage as an anti-cancer agent. Methods: We used A549 cell lines that are non-small-cell lung cancer cells in this study. A549 cells were treated with imidazole (molecular grade) at 1, 5, 10, 20, 40, 80 mM doses for 24, 48 and 72 hours. Cytotoxicity and IC50 values (the half-maximal inhibitory concentration) were calculated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis. Colony formation assay was performed to detect the effect of imidazole on cancer cell colony formation ability. The cellular morphological alterations were observed on bright-field microscopy using Giemsa staining. Cellular migration status of A549 cells was defined with in vitro scratch assay up to 48th hour. Results: Cytotoxicity assay results showed that low-level imidazole induced cell proliferation. However, high-level imidazole treatment decreased the cell viability of A549 cells in a dose and time-dependent manner. The IC50 value was calculated as 60 mM, 28 mM, and 15,9 µM doses respectively at 24, 48, 72 hours in A549 cells. Also, we determined that the number of colonies (number of colonies: 42.7 ± 3.06) formed in A549 cell lines treated with imidazole at IC50 dose was statistically less than the colony number of the control group (number of colonies: 70.7 ± 5.13) (p &lt; 0.01). Interestingly, we observed that colony number increased at a low dose (at 5 mM) imidazole treated group, statistically significant (p &lt; 0.05). Cellular morphology was not affected at low doses; however, at the IC50 dose, A549 cells changed their cellular morphology, lost cell-cell contact, decreased cytoplasmic volume, and differentiated from parental morphology. In addition to these effects, we observed that imidazole treated cells decreased their migration capabilities compared with control group cells (p &lt; 0.05). Conclusions: Our results have shown that antifungal imidazole treatment inhibits cancer cell biological responses such as proliferation, colony formation ability, and motility in non-small lung cancer cell lines in a dose and time-dependent manner. These results suggest that imidazole would be the right candidate for the synergy with other therapeutic options such as immunotherapy. This introductory study allows us further studies exploring the synergy and its mechanism.

Molecular Targets of Phyto-bioactive Compounds in Female Reproductive System of Mammals: A Review

Phytochemicals present in the plants are divided into primary (Alcohol Amino acids, nucleotides. etc) and secondary metabolites (Alkaloids, Saponins etc.). Carotenoids (reduces reactive oxygen species formation, decreases apoptotic cells, restoration of actin capping expression proteins etc.), Phenolics (inhibits extracellular signal-regulated kinase signalling pathway), Isoflavones (inhibits tyrosine kinase pathway) and alkaloids (downregulation of vascular endothelial growth factor, tumor necrosis factor-alpha and hypoxia-inducible factor 1-alpha messengers) are the major phytochemicals, having the potential effects towards ovarian function. Likewise, bioactive compounds are the chemicals that can interact with certain components of live tissue to exert their various effects (antioxidant, antineoplastic, receptor inhibition, gene expression etc.) respective to female fertility. Similarly, bioactive compounds: Kaempferol [phosphatidylinositol -3- kinase (PI3K)/protein kinase B (Akt) pathway], Quercetin (controlling the release of 17β-estradiol etc.), Myricetin (PI3K/Akt and MAPK signalling pathway), Galngin (inhibition of angiogenesis via decreasing the VEGF and p-Akt) and Resveratrol (regulation of Foxo3a and SIRT1 genes etc.) shows its effects by targeting different molecules and/or pathways at the ovarian microenvironment. However, Genistein (binding to estrogen receptors: ESRα and ESRβ etc.) and Diadzein (disrupting the endocrines etc.) emphatically interfere with the ovarian functions. Besides this, molecular effects exerted by these phyto-bioactive compounds on the in vivo and/or in vitro ovarian culture systems entirely depend on their dosage: Kaempferol @10 μM increased the primordial follicle activation, Quercetin @4 µM improved the quality of oocytes whereas @8 µM reduced the quality), Resveratrol @ 2 µM increased the blastocyst formation, Myricetin @ 100 mg/kg/day feeding in rats induced estrogenic activity, Genistein, feeding in female mice @ 500 and 1000ppm increased the gestation time and Diazdein causes the inhibition of 3-hydroxysteroid dehydrogenase at 40 µM doses. The assessment was done via the systemic collection of literature from sources such as newspapers, conference papers, journals, theory and dissertation articles, electronic databases, manuals, encyclopedia and annual reviews, as well as e-books and reporting. As a result, the preceding discussion focuses on the key phyto-bioactive compounds and their molecular targets in female fertility. This will aid in the successful and secure application of plant bioactive compounds in the field of female reproductive health.

Exploring NCX4040, an Aspirin Derivative, as a Potential Treatment for Benign Prostatic Hyperplasia

Benign Prostatic hyperplasia (BPH) is a leading cause of lower urinary tract symptoms which affects men above 50 years of age. Chronic inflammation and abnormal proliferation of stromal and epithelial cells are implicated in BPH disease onset. The symptoms of BPH include back pain and difficulty in emptying bladder. Finasteride, sildenafil and tamsulosin are some of the drugs used to ease difficulty urinating and relax the muscles of the gland. Transurethral resection of the prostate or laser surgery can be performed to treat severe symptoms. However, these therapies have deleterious effects such as low blood pressure, ejaculatory dysfunction, and lump formation. Hence, there is an unmet need for potential drugs against BPH. Nonsteroidal anti-inflammatory drugs (NSAIDs) have proven to be effective in cancers but their applicability in BPH condition is yet to be fully explored. Aspirin, one of the NDSAIDs, has anti-tumor and anti-inflammatory properties at higher doses. NCX4040, a nitric oxide releasing derivative of aspirin, could prove to be effective against BPH, since it can inhibit abnormal cell proliferation and serve as a vasodilator. We hypothesize that NCX4040 would be an effective drug to treat BPH. BPH-1 epithelial and WPMY-1 stromal cells were used as in vitro models of BPH. MTT assay was performed to check the inhibitory effect of NCX4040 and blocking agents like catalase and N-acetyl-L-cysteine (NAC) were explored on cells after treatment. Clonogenic assay was done to explore the colony formation ability of cells. Spheroid assay was performed to analyze the anti-proliferative effect of NCX4040. Annexin V/PI and cell cycle analysis was performed to check for apoptosis and cell cycle arrest in the cells. Western blot was done to assess the signaling molecules altered by NCX4040 in BPH-1 cells. Confocal immunofluorescence was employed to analyze the dynamics of actin filaments after treatment in cells. Our studies revealed that NCX4040 inhibited the cell viability of BPH-1 and WPMY-1 in a dose dependent manner with IC50 predicted at 5µM and 2.5µM respectively. Of note, catalase and NAC blocked the effect of NCX4040 on prostate cells. Colony formation assay result implied a gradual decrease in the number of colonies of cells treated with NCX4040 with 2.5µM and 5µM doses. Spheroid assay in BPH-1 cells showed inhibitory effects after treatment. Cell cycle analysis by flowcytometry inferred that cell cycle arrest at G2/M phase and annexin V analysis indicated that activation of apoptosis in cells following treatment. Phalloidin staining showed decrease in the actin filament intensity in cells. At the molecular level, NCX4040 downregulated the expression of key markers such as RhoA, p65, COX-2, PCNA, Cyclin D3, and PDE-5 in BPH-1 cells. Taken together, NCX4040 could be used as a potential agent to manage BPH with minimal side effects, which needs further evaluation in animal models.

Roles of Bak and Sirt3 in Paraquat-Induced Cochlear Hair Cell Damage.

Paraquat, a superoxide generator, can damage the cochlea causing an ototoxic hearing loss. The purpose of the study was to determine if deletion of Bak, a pro-apoptotic gene, would reduce paraquat ototoxicity or if deletion of Sirt3, which delays age-related hearing loss under caloric restriction, would increase paraquat ototoxicity. We tested these two hypotheses by treating postnatal day 3 cochlear cultures from Bak±, Bak-/-, Sirt3±, Sirt3-/-, and WT mice with paraquat and compared the results to a standard rat model of paraquat ototoxicity. Paraquat damaged nerve fibers and dose-dependently destroyed rat outer hair cells (OHCs) and inner hair cells (IHCs). Rat hair cell loss began in the base of the cochlea with a 10μM dose and as the dose increased from 50 to 500μM, the hair cell loss increased near the base of the cochlea and spread toward the apex of the cochlea. Rat OHC losses were consistently greater than IHC losses. Unexpectedly, in all mouse genotypes, paraquat-induced hair cell lesions were maximal near the apex of the cochlea and minimal near the base. This unusual damage gradient is opposite to that seen in paraquat-treated rats and in mice and rats treated with other ototoxic drugs. However, paraquat always induced greater OHC loss than IHC loss in all mouse strains. Contrary to our hypothesis, Bak deficient mice were more vulnerable to paraquat ototoxicity than WT mice (Bak-/- > Bak± > WT), suggesting that Bak plays a protective role against hair cell stress. Also, contrary to expectation, Sirt3-deficient mice did not differ significantly from WT mice, possibly due to the fact that Sirt3 was not experimentally upregulated in Sirt3-expressing mice prior to paraquat treatment. Our results show for the first time a gradient of ototoxic damage in mice that is greater in the apex than the base of the cochlea.