A safety investigation into topical effects of naproxen sodium on nasal epithelial cells and potential toxicity in local application.

OBJECTIVES: We examined how topically-applied naproxen sodium affects human nasal epitheliocytes in culture. METHODS: Samples of healthy human primary nasal epithelium (NE) harvested during septoplasty from volunteers without rhinosinusitis were incubated in cell culture. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays may be utilised when assessing cellular damage (toxicity), as evidenced by DNA fragmentation, nuclear condensation, alteration in the outer plasma membrane and cytoskeletal alteration. This was the method used in the study. Cultured epitheliocytes were incubated with naproxen sodium for 24 h at 37 °C. The MTT assay was then performed and the cells' morphology was examined by confocal microscopy. Additionally, cellular proliferation was assessed by the artificial scratch method followed by light microscopy. RESULTS: The results indicated that naproxen sodium does not cause any cytotoxic effects upon nasal epithelial cells when applied topically. There was no evidence indicating cytotoxicity on the nasal epitheliocytes in culture for the 24 h period over which the drug was applied. In particular, there was no alteration in cellular morphology, damage to the intracellular organelles structure or the cytoskeleton secondary to naproxen sodium. Furthermore, cellular proliferation occurred normally in these conditions, as on scratch test. CONCLUSION: Topical naproxen sodium may be used on nasal epithelial cells without inducing toxicity. This agent is therefore suitable, given its known anti-inflammatory effects, for use in patients suffering from diseases involving nasal and paranasal sinusal inflammation, including rhinosinusitis (both chronic and acute) and nasal polyposis which should be investigated. In the future, topical medication forms for nasal usage should be developed.

Escin attenuates oxidative damage, apoptosis and lipid peroxidation in a model of cyclophosphamide-induced liver damage.

To investigate the effects of escin (ES) on acute damage induced by alkylating agent, experimental rats were injected with cyclophosphamide (CPM) to cause liver damage. The animals were divided into four groups: Control Group, CPM (200 mg/kg), ES (10 mg/kg), CPM, and ES Groups. Immunohistopathological, hepatic histopathological, and biochemical changes were analyzed. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), malondyaldehyde (MDA), glutathion (GSH), total oxidant status (TOS) and total antioxidant status (TAS) in serum were all determined. Serum and immunohistopathology analysis revealed that MDA, ALT, AST, LDH, TOC and OSI, caspase-3 and Bax levels had increased while GSH, TAC, Bcl- 2 and OSI levels decreased in CPM Group when compared to Control Group. These findings appear to account for the severe damage detected. In the CPM + ES treated group, positive improvements were found in biochemical parameters as well as in cell-death and tissue-related damage parameters.The results show that ES considerably protects the rat liver against CPM-induced hepatotoxicity thanks to because of its anti-oxidant and anti-apoptotic properties.

Cytotoxic Impact of N-Oleoylethanolamine on Bone Cancer Cells.

BACKGROUND: Cancer is a complex disease that derives from the uncontrolled proliferation of cells. Bone cancer is a type of prevalent cancer that occurs both in young and adults. Bone cancer is most common in the long bones of the pelvis, arms and legs. Statistically, more than 200 cases of osteosarcoma have been reported annually in our country. Classical treatment with chemotherapeutics remains ineffective in the cure of this cancer type. Recent studies have shown that ceramide induces apoptosis at its increased levels in the cells. Thus, many studies have been conducted to cause the accumulation of ceramide molecules in the cell by different ways to induce apoptosis. NOE (Noleoylethanolamine) is a specific inhibitor of ceramidase enzymes that hydrolyze intracellular ceramides and prevent apoptosis. OBJECTIVE: This study investigates the cytotoxic and apoptosis-inducing activities of NOE on human osteosarcoma Saos-2 cells. METHODS: Cytotoxic effects were investigated by MTT colorimetric assay. For the detection of morphological and ultrastructural indicators of apoptosis, confocal and TEM techniques were used. RESULTS: Our finding indicated that NOE is effective in the inhibition of the growth of Saos-2 cells. Confocal and TEM findings showed morphological and ultrastructural changes as chromatin condensation, fragmentation of nuclei and mitochondria as well as damaged cytoskeleton and cell shrinkage. CONCLUSION: The results revealed that NOE exerts its cytotoxicity on Saos-2 cells through changing the ultrastructure and morphology of cells with clear apoptotic sparks.

Correction to: Characteristics of apoptosis induction in human breast cancer cells treated with a ceramidase inhibitor.

[This corrects the article DOI: 10.1007/s10616-020-00436-1.].

Molecular docking analyses of Escin as regards cyclophosphamide-induced cardiotoxicity: In vivo and in Silico studies.

This study aims to investigate whether Escin (ES) can protect against Cyclophosphamide (CPM)-induced cardiac damage. The experimental rats were categorized as Control, CPM (200 mg/kg), ES (10 mg/kg), and CPM + ES Groups, each having 6 members. Their heart tissues were stained with Hematoxylin and Eosin and the structural changes were investigated under the light microscope. The biochemical markers of ischemia modified albumin (IMA), creatine kinase (CK-MB), antioxidant activity indicators Catalase (CAT), and superoxide dismutase (SOD) activities were measured using blood samples. Besides, the effects of CPM, ES, and CPM + ES upon CAT and SOD activities were shown via molecular docking studies. In the Single-Dose CPM group, CK-MB and IMA levels significantly increased while SOD and CAT levels significantly decreased. However, the heart tissues were damaged. CK-MB and IMA levels significantly decreased in CP+ ES Group. On the other hand, SOD, and CAT levels significantly increased and reduced the damage remarkably. Our findings showed that ES treatment successfully reduced the toxic effects upon the rats. The conclusion is that ES treatment can help protect the heart tissue against CPM-induced toxicity. Both in-vivo results and molecular modeling studies showed that the negative effects of CPM upon SOD activity were bigger than that of CAT.

Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters.

Ceranib-2 is a recently discovered, poorly water-soluble potent ceramidase inhibitor, with the ability to suppress cancer cell proliferation and delay tumor growth. However, its poor water solubility and weak cellular bioavailability hinder its use as a therapeutic agent for cancer. PEGylated rosin esters are an excellent platform as a natural polymer for drug delivery applications, especially for controlling drug release due to their degradability, biocompatibility, capability to improve solubility, and pharmacokinetics of potent drugs. In this study, stable aqueous amphiphilic submicron-sized PEG400-rosin ester-ceranib-2 (PREC-2) particles, ranging between 100 and 350 nm in a 1:1 mixture, were successfully synthesized by solvent evaporation mediated by sonication.Conclusion: Stable aqueous PEGylated rosin ester nanocarriers might present a significant solution to improve solubility, pharmacokinetic, and bioavailability of ceranib-2, and hold promises for use as an anticancer adjacent drug after further investigations.

Characteristics of apoptosis induction in human breast cancer cells treated with a ceramidase inhibitor.

Cancer is a complex disease with high mortality rates. Breast cancer is one of the most fatal diseases both for men and woman. Despite the positive developments on cancer treatment, a successful treatment agent/method has not been developed, yet. Recently, cancer research has been involved in sphingolipid metabolism. The key molecule here is ceramide. Ceramides mediate growth suppress, apoptosis and aging regulation. Ceramidases metabolize ceramide and decrease its level in cells and cause escape the death. Inhibition of ceramidases as new targets for cancer treatment is shown in the literature. Herein, we found that d-erythro-MAPP and its nanoparticle formulation, reduce the viability of MCF-7 cells in a dose-dependent manner with IC50 value of 4.4 µM, and 15.6 µM, respectively. Confocal and transmission electron microscopy results revealed apoptotic morphological and ultrastructural changes for both agents. Apoptosis and cell cycle arrest were supported by annexin-V, mitochondrial membrane potential changings and cell cycle analysis, respectively.

The effect of R547, a cyclin-dependent kinase inhibitor, on hepatocellular carcinoma cell death.

Hepatocellular carcinoma (HCC) is the third main cause of cancer-related death. Cyclin-dependent kinases (CDKs) and their cyclin partners regulate the cell cycle. Since inhibition of CDKs gives some guiding ideas for cancer studies, we aimed to determine the possible effects of R547, a cyclin kinase 1-2-4 inhibitor, on proliferation and apoptotic mechanisms of Hep G2 cells (human) and H-4-II-E cells derived from rat HCC. We determined in vitro survival rates with MTT assay, apoptosis with flow cytometry, morphological changes with confocal microscopy, and ultrastructural changes by transmission electron microscopy. Cisplatin was used as a positive control. After 24 h of culture with 0.1, 1, 10, 50, and 100 µM doses of R547, the corresponding percentages of live Hep G2 cells were 101%, 94%, 93%, 89%, and 79% (P < 0.001), respectively. However, with the same R547 doses the live Hep G2 cell percentages were 92%, 101%, 53.6% (P <0 .01), 47.4% (P < 0.001), and 41% (P < 0.001), respectively, after 48 h. After 24 h of incubation with the same doses of R547, the survival percentages of live rat cells were 90%, 80% (P < 0.01), 63% (P < 0.001), 47% (P < 0.001), and 43% (P < 0.001), respectively. The percentages of surviving H-4-II-E cells were 96%, 85% (P < 0.01), 46% (P < 0.001), 44% (P < 0.001), and 45% (P < 0.01), respectively, after 48 h. Since R547 did not significantly affect Hep G2 cell survival in 24 h, experiments of apoptosis were carried out with H-4-II-E cells. The early apoptotic rates of 38% and 45% (P < 0.05 for both) after applications of 10 and 25 µM R547 (control: 4.1%), respectively, indicated that R547 has an apoptotic effect on H-4-II-E cells in 24 h. The apoptosis morphology at 24 h of treatment was clearly observed with microscopic examinations. According to our results, it is obvious that R547 has antiproliferative action when compared to cisplatin.

Fe3O4 Nanopowders: Genomic and Apoptotic Evaluations on A549 Lung Adenocarcinoma Cell Line.

The magnetite nanoparticles are progressively used in a wide range of biological applications. In the present study, we purposed to show apoptosis-inducing ability of Fe3O4 nanopowders on A549 cells. In addition, the toxic effects of Fe3O4 nanopowders were researched on L929 cells. The cytotoxicity of Fe3O4 nanopowders were evaluated on A549 and L929 cells by MTT assay and inhibited cell proliferation by time and dose-dependent manner on A549 cells but was not toxic on L929 cells. According to these findings, IC30 value of Fe3O4 nanopowders was determined as 5 µM. The early and late apoptotic cells were detected by Annexin V-FITC/PI assay using IC30 concentration of Fe3O4 nanopowders. Furthermore, The IC30 value of Fe3O4 nanopowders was not effective in the activation of caspase-3 but was effective on loss of mitochondrial membrane potential. The apoptotic index of A549 cells was investigated and found out to increase by IC30 value of Fe3O4 nanopowders using TUNEL, BrdU, Bcl-2 immunocytochemical assays. The upregulated and downregulated genes were profiled and the presence of some apoptotic genes was determined with administration of IC30 value of Fe3O4 nanopowders by microarray assay. This work suggests that Fe3O4 nanopowders could be a good candidate for therapy of lung adenocarcinoma cells.

Effect of acrylamide on BEAS-2B normal human lung cells: Cytotoxic, oxidative, apoptotic and morphometric analysis.

Due to the broad toxic relevance of acrylamide, many measures have been taken since the 1900s. These measures increased day by day when acrylamide was discovered in foods in 2002, and its toxic spectrum was found to be wider than expected. Therefore, in some countries, the products with higher acrylamide content were restricted. On the other hand, the effects of acrylamide on the respiratory system cells have yet to be well understood. In this study, we aimed at investigating the effect of acrylamide on lung epithelial BEAS-2B cells. Initially, the cytotoxic effect of acrylamide on BEAS-2B was determined by MTT assay. Then, cellular oxidative stress was measured. Flow cytometry analysis was conducted for Annexin-V and caspase 3/7. Furthermore, Bax, Bcl-2 and Nrf-2 proteins were evaluated by immunocytochemistry. Finally, acrylamide-induced cellular morphological changes were observed under confocal and TEM microscopes. According to MTT results, the IC50 concentration of acrylamide was 2.00 mM. After acrylamide treatment, oxidative stress increased dose-dependently. Annexin V-labelled apoptotic cells and caspase 3/7 activity were higher than untreated cells in acrylamide-treated cells. Immunocytochemical examination revealed a marked decrease in Bcl-2, an increase in Bax and Nrf-2 protein staining upon acrylamide treatment. Furthermore, in confocal and TEM microscopy, apoptotic hallmarks were pronounced. In the present study, acrylamide was suggested to display anti-proliferative activity, decrease viability, induce apoptosis and oxidative stress and cause morphological changes in BEAS-2B cells.

Acrylamide exerts its cytotoxicity in NIH/3T3 fibroblast cells by apoptosis.

OBJECTIVE: Acrylamide is a chemical utilized in various industries, and many studies have demonstrated its toxicity. The NIH/3T3 mouse embryonic cell line is the standard cell line of fibroblasts, which have a pivotal role with their versatile functions in the body. However, only two studies have attempted to investigate the effect of acrylamide on these crucial cells. To fill this knowledge gap, we aimed to determine the effects of acrylamide on NIH/3T3 cells. METHOD: First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and calculated the IC50 dose of acrylamide. Then, we treated cells with the IC50 dose of acrylamide for 24 h and determined whether the dominant death mode of NIH/3T3 cells was apoptosis or necrosis by annexin V and caspase 3/7 assays. Finally, we performed confocal microscopy and transmission electron microscope (TEM) analysis for observing the morphological alterations. RESULTS: MTT assay results showed that acrylamide treatment reduced the viability of NIH/3T3 cells dose-dependently and that the IC50 of acrylamide was 6.73 mM. Based on annexin V and caspase 3/7 assays, the dominant death mode of NIH/3T3 cells was determined to be apoptosis. Also, caspase 3/7 activities of the acrylamide-treated NIH/3T3 cells were three times greater than those of the untreated NIH/3T3 cells. Furthermore, we observed membrane blebbing, nuclear chromatin clumping, and cytoplasmic vacuolization in TEM analysis and apparent apoptotic bodies, nuclear fragmentations, and condensations in confocal microscopy. CONCLUSIONS: In conclusion, our results suggested that the IC50 of acrylamide against NIH/3T3 cells for 24 h was 6.73 mM and that acrylamide exerted its cytotoxic and anti-proliferative effects on these cells mainly via apoptosis.

The apoptotic and genomic studies on A549 cell line induced by silver nitrate.

Lung cancer is the leading cause of male cancer deaths worldwide. Metal-based anticancer drugs have evolved significantly during the past decades. Recently, silver ions have been investigated for their anticancer effects. We aimed to study the time-course cytotoxic effects of silver nitrate on A549 adenocarcinomic human alveolar basal epithelial cells to provide insights into the molecular-level understanding of growth suppression mechanism involved in apoptosis. The influences of silver nitrate were studied via MTT assay, flow cytometry, immunocytochemical, confocal and transmission electron microscopy, and microarray assays. Silver nitrate showed inhibitory effects against A549 cells in a dose- and time-dependent manner for 24, 48, and 72 h and induced apoptosis. The early and late apoptotic cells and depolarized mitochondrial membrane potential were determined by the half-maximal inhibitory concentration (IC50) value of silver nitrate treated for 72 h. But cysteinyl aspartate proteinase-3 was not activated for 72 h. Furthermore, IC50 value of silver nitrate also induced apoptosis according to immunocytochemical assays for 72 h. The downregulated CCNY, HNRNPL, ASF1B, PIAS4, HNRNPH1, EIF2C2, TAF15, FOXC1, LEP, and PCB2 genes administered with silver nitrate IC50 were identified as apoptosis-leading genes. Silver nitrate may be a suitable therapeutic agent against lung cancer.

Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

Haloferax alexandrinus Strain TM JCM 10717T = IFO 16590T is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

Examining impacts of ceranib-2 on the proliferation, morphology and ultrastructure of human breast cancer cells.

Acid ceramidases are enzymes with a vital role in metabolizing ceramide to sphingosine-1-phosphate that is an antiproliferative metabolite in the ceramide pathway. Inhibition of exogenous ceramides with ceramidase inhibitors lead to augmented ceramide levels in cells and in turn lead to cell cycle arrest and apoptosis. Our study aimed at targeting ceramide metabolic pathway to induce apoptosis in human breast cancer cell line (MCF7) and we examined the antiproliferative and apoptotic activities of ceranib-2, an inhibitor of human ceramidase, on this cell line as well ultrastructural and mophological changes. Methods used for our examinations in this study were the colorimetric MTT assay, Annexin V/Propidium iodide and JC-1 staining, transmission electron microscopy and confocal microscopy. Ceranib-2 effectively inhibited the viability of MCF7 cells in 24 h in a dose dependent manner leading to apoptosis via the mitochondrial pathway by reducing the potential of mitochondrial membrane. Additionally, significant changes on cell morphology and ultrastructure were observed on MCF7 cells exposed to ceranib-2 indicating apoptotic cell death. Collectively, our data demonstrate that ceranib-2 exerts a great potential to be an antineoplastic compound and that the mechanism of its action rely on its apoptosis inducing ability.

Cytotoxic, anti-proliferative and apoptotic effects of silver nitrate against H-ras transformed 5RP7.

Metal based drugs have successfully been used in both the detection and treatment of different disease states. The antibacterial features of metal ion silver are well documented. Most recently, metal ion silver has been tested and applied in anticancer activity. The present study observed the cytotoxic, anti-proliferative and apoptotic effects of metal complex silver nitrate in H-ras transformed 5RP7 cell lines for 24 h. In addition, the toxic effects of silver nitrate was investigated on NIH/3T3 primary mouse embryonic fibroblast cells for 24 h. Cytotoxic effects were determined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Apoptosis and necrosis were evaluated by flow cytometric analysis (Annexin-V FITC/PI). Caspase-3 activation was researched by flow cytometric analysis. Apoptotic morphology was observed by DAPI staining. Structure and ultra-structure changes of cells were assessed using transmission electron microscopy. The results indicate silver nitrate has high cytotoxicity and a strong capacity to induce apoptosis in H-ras 5RP7 cells. Furthermore silver nitrate was not toxic against NIH/3T3 primary mouse embryonic fibroblast cells at low doses for 24 h.

The Potential Protective Effects of 2-aminoethyl Diphenylborinate against Inner Ear Acoustic Trauma: Experimental Study Using Transmission and Scanning Electron Microscopy.

OBJECTIVE: In this prospective experimental study, we investigated the preventive effects of 2-aminoethyl diphenylborinate (2-APB) in rats exposed to acoustic trauma (AT). Light microscopic, transmission electron microscopic (TEM), and scanning electron microscopic (SEM) examinations were performed. MATERIALS AND METHODS: Eighteen healthy Wistar albino rats were divided into the following three groups: groups 1 (control), 2 (AT), and 3 (AT+APB). The rats in groups 2 and 3 were exposed to AT; in group 3 rats, 2-APB at 2 mg/kg was also administered, initially transperitoneally, after 10 min. RESULTS: During the light microscopic, TEM, and SEM examinations, the structures of the cochlear hair cells, stereocilia, and Deiter's cells were normal in the control group. In the AT group, the organ of Corti and proximate structures were damaged according to the light microscopic examination. During the TEM examination, intense cellular damage and stereocilia loss were detected, while during the SEM examination, extensive damage and stereocilia loss were observed. Decreased damage with preserved cochlear structure was detected during the light microscopic examination in the AT+APB group than in the AT group. During the TEM and SEM examinations, although stereocilia loss occurred in the AT+APB group, near-normal cell, cilia, and tectorial membrane structures were also observed in the AT+APB group compared with the AT group. CONCLUSION: 2-APB may have protective effects against AT damage of the cochlea. The main mechanism underlying this effect is the inhibition of the vasoconstriction of the cochlear spiral modiolar artery, thereby improving cochlear blood flow. We conclude that 2-APB may also be effective if used immediately following AT.

Induction of apoptosis in prostate cancer cells by the novel ceramidase inhibitor ceranib-2.

Ceramidases are key enzymes that decrease ceramide levels in cells. A reduction in ceramide concentration impairs ceramide signalling, and results in apoptosis resistance in cancer cells. This study investigates the potential for ceranib-2, a novel ceramidase inhibitor, to affect the survival and/or promote apoptosis of prostate cancer cells (LNCaP and DU145) in vitro. Cell viability was determined using MTT, and apoptosis assessed via flow cytometry. We examined structural changes with both confocal and transmission electron microscopes. Ceranib-2 concentrations of 0.1, 1, 5, 10, 25 and 50 µM were applied to LNCaP and DU145 cell lines. The corresponding reduction in LNCaP cell viability (against the control) was 84%, 80%, 64%, 56%, 40% and 15% after 24 h, and 81%, 74%, 60%, 55%, 27% and 11% after 48 h. For DU145 cells, viability was reduced to 84%, 82%, 63%, 50%, 41% and 18% after 24 h, and 64%, 42%, 30%, 20%, 8% and 5% after 48 h. Following treatment with 25 and 50 µM ceranib-2, the respective observed rates of early apoptosis in LNCaP cells were 23% and 36% after 24 h and 27% and 58% after 48 h. The morphological and ultrastructural signs of apoptosis detected were fragmented nuclei, chromatin condensations and cytoskeleton laceration. The inhibitory effects of ceranib-2 on prostate cancer cell survival are dose and time dependent. For LNCaP cells, ceranib-2 toxicity was predominately apoptotic in nature, while for DU145 cells, cell death may be related to non-apoptotic mechanisms.

Isolation and identification of selenite reducing archaea from Tuz (salt) Lake In Turkey.

In this study, Tuz lake brine samples were investigated for isolation and identification of selenite resistant halophilic prokaryotes. Among the 20 strains of extremely halophilic Bacteria and Archaea, a Gram negative rod designated as strain 106, showed high capacity in the resistance to selenite (25 mM) under aerobic conditions. Phenotypic characterizations and phylogenetic analyses based on 16S rDNA sequence comparison indicated that strain 106 was Halorubrum xinjiangense. The ability of strain 106 to deposite selenium-containing particles were investigated by Transmission Electron Microscopy (TEM). Electron micrographs shows intact cells after selenite reduction and large amounts of selenium-containing particles are present in the culture medium indicating that strain 106 is able to efficiently transport elemental selenium out of the cell.