Acridine Orange/ethidium Bromide Research Articles

Biomimetic mineralization on natural and synthetic polymers to prepare hybrid scaffolds for bone tissue engineering.

To mimic the mineral component and the microstructure of natural bone, nanohydroxyapatite (nHAP)/polymer (natural and synthetic materials) composite scaffolds with micropore structures and good mechanical performance were prepared via the assistance of sonication and amidation. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) showed the biomimetic homogeneous formation of nHAP in the polymer matrix. Fourier transform infrared spectroscopy (FTIR) indicated that the polymer matrix regulated the crystallization of nHAP. The integration of synthetic/natural polymers benefited nHAP crystallization in the polymer matrix and thus improved the mechanical performance of the chitosan (CS)/collagen (Col)/ poly (lactic acid) (PLA)/nHAP scaffolds. The in vitro bioactivity ability was investigated by incubating in a simulated body fluid (SBF). The methyl thiazolyl tetrazolium (MTT) test, the acridine orange/ethidium bromide (AO/EB) and Giemsa staining showed that the scaffolds had good biocompatibility and could maintain the cell growth. Therefore, the CS/Col/PLA/nHAP scaffold is promising for bone tissue engineering applications.

Apoptosis of mesenchymal stem cells is regulated by Rspo1 via the Wnt/β-catenin signaling pathway

ObjectiveThe aim of this study was to investigate the effect and possible mechanism of action of roof plate-specific spondin1 (Rspo1) in the apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). MethodsOsteogenic and adipogenic differentiation of BMSCs was identified by Alizarin Red and Oil Red O staining, respectively. BMSC surface markers (cluster of differentiation 29 [CD29], CD90, and CD45) were detected using flow cytometry. BMSCs were transfected with an adenoviral vector encoding Rspo1 (BMSCs-Rspo1 group). The expression levels of Rspo1 gene and Rspo1 protein in the BMSCs-Rspo1 group and the two control groups (untransfected BMSCs group and BMSCs-green fluorescent protein [GFP] group) were analyzed and compared by quantitative polymerase chain reaction and Western blot. The occurrence of apoptosis in the three groups was detected by flow cytometry and acridine orange-ethidium bromide (AO-EB) double dyeing. The activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the expression levels of the key proteins of the pathway (β-catenin, c-Jun N-terminal kinase [JNK], and phospho-JNK). ResultsOsteogenic and adipogenic differentiation was confirmed in cultured BMSCs by the positive expression of CD29 and CD90 and the negative expression of CD45. Significantly increased expression levels of Rspo1 protein in the BMSCs-Rspo1 group compared to those in the BMSCs (0.60 ± 0.05 vs. 0.13 ± 0.02; t=95.007, P=0.001) and BMSCs-GFP groups (0.60 ± 0.05 vs. 0.10 ± 0.02; t=104.842, P=0.001) were observed. The apoptotic rate was significantly lower in the BMSCs-Rspo1 group compared with those in the BMSCs group ([24.06 ± 2.37]% vs. [40.87 ± 2.82]%; t = 49.872, P = 0.002) and the BMSCs-GFP group ([24.06 ± 2.37]% vs. [42.34 ± 0.26]%; t = 62.358, P = 0.001). In addition, compared to the BMSCs group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.14 ± 0.14; t = −9.217, P = 0.000) and JNK (1.87 ± 0.17 vs. 0.61 ± 0.07; t = −22.289, P = 0.000) were increased in the BMSCs-Rspo1 group. Compared to the BMSCs-GFP group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.44 ± 0.14; t = −5.692, P = 0.000) and JNK (1.87 ± 0.17 vs. 0.53 ± 0.06; t = −10.589, P = 0.000) were also upregulated in the BMSCs-Rspo1 group. Moreover, the protein expression levels of phospho-JNK were increased in the BMSCs-Rspo1 group compared to those in the BMSCs group (1.89 ± 0.10 vs. 0.63 ± 0.09; t = −8.975, P = 0.001) and the BMSCs-GFP group (1.89 ± 0.10 vs. 0.69 ± 0.08; t = −9.483, P = 0.001). ConclusionThe Wnt/β-catenin pathway could play a vital role in the Rspo1-mediated inhibition of apoptosis in BMSCs.

An investigation of antibiofilm and cytotoxic property of MgO nanoparticles

Abstract MgO nanoparticles (MgONPs) have been widely used as antibacterial agents with the advantages of them being nontoxic and their unique biological properties. In this study, we synthesized MgONPs by co-precipitation method and characterized them by XRD, SEM and EDS analysis. The antibiofilm activity of MgONPs as observed quantitatively by crystal violet assay and their action on biofilm architecture were assessed in both Gram-positive and Gram negative uropathogenic bacteria. In addition, the cytotoxic effect of synthesized MgONPs was evaluated against human breast cancer, MCF-7 cells. The morphological changes of apoptosis were observed by Acridine Orange/Ethidium Bromide (AO/EB) staining using a fluorescent microscope. MgONPs inhibited more than 50% of the biofilms in most of the tested uropathogenic bacteria; notably 80% inhibition in the case of K. pneumoniae. MgONPs successfully inhibited the viability of MCF-7 cells at a concentration of 50 μg.mL−l. Given their antibiofilm properties; MgONPs could be used as a potential nanomaterial for in vivo applications such as coating for a medical implant.

Cyclopamine Suppresses Human Esophageal Carcinoma Cell Growth by Inhibiting Glioma-Associated Oncogene Protein-1, a Marker of Human Esophageal Carcinoma Progression.

BackgroundEsophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth.Material/MethodsGli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression.ResultsGli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 μM, (13.37±1.42)% at 5.0 μM, (22.3±2.92)% at 10.0 μM, and (33.57±1.75)% at 20.0 μM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent.ConclusionsGli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.

Endogenous H2S-Assisted Cancer-Cell-Specific Activation of Theranostics with Emission Readout.

Realizing the importance of activation of the anticancer drug, its distribution, and for cancer management, a new theranostic probe has been developed. Endogenous H2S stimulated the theranostic molecular prodrug (TP-HS) which is activated in cancer cells; it monitors the actual time of formation of therapeutic agent SN-38 in cellular milieu through fluorescence imaging. Upon exposure to H2S in a similar physiological condition, the azide functionality converted to amine (-NH2) in TP-HS which allows self-immolative scission of the labile benzyl-carbonate moiety for release of rhodol and SN-38 in a concerted manner. Thus, an intense emission band centered at 548 nm has appeared for quantifying the active therapeutic component. The fluorescence image revealed that the TP-HS preferentially releases rhodol and SN-38 in colon cancer (HCT116 cells) and lung cancer cells (A549 cells) compared to normal human fibroblast cells (WI-38). Further, the dose-dependent antiproliferative activity of TP-HS against various cells supports that TP-HS releases SN-38 based on endogenous H2S in cancer cells followed by its apoptotic progression monitored by (a) live-dead, i.e., acridine orange-ethidium bromide double staining assay, (b) APOPercentage apoptotic assay, and (c) Annexin V-FITC staining by flow cytometry. The theranostic prodrug TP-HS showed anticancer efficacy via the desirable apoptotic pathway. It is the first demonstration of a strategic theranostic molecular prodrug system that could be delivered chemotherapeutically with validating the real-time activation of chemotherapy in the cancer cells without the support of a cancer-directing ligand.

Synthesis of SiO2 nanostructures from Pennisetum glaucum and their effect on osteogenic differentiation for bone tissue engineering applications.

Silica nanostructures were fabricated from Pennisetum glaucum (pearl millet) seed husk by acid-pretreatment and calcination. The fabricated silica nanostructure (SN) functional groups, crystalline nature, surface morphology, and particle size were analyzed by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy, respectively. Additionally, the cytocompatibility of SNs was analyzed on human mesenchymal stem cells (hMSCs) in an MTT assay, propidium iodine (PI) staining, and acridine orange/ethidium bromide (AO/EB) staining. We observed peaks at 1090 and 800 cm-1, which were assigned to symmetric, asymmetric, and bending vibrations of O-Si-O. The SNs showed an amorphous nature with a spherical shape and were 20-60 nm in diameter. The MTT assay results indicated that SNs exhibited cytocompatibility in hMSCs. The PI staining and AO/EB staining results suggested that SNs do not affect nuclear morphology at up to 400 μg/mL. Furthermore, SNs effect on osteogenic differentiation in hMSCs was studied. These results indicate that SNs induced osteogenic differentiation in hMSCs by upregulation of ALP, BSP, ON and RUNX2 genes. Our process could valorize the Pennisetum glaucum agricultural residues to high value products for bone tissue engineering applications.

Induction of Cell Death in Human A549 Cells Using 3-(Quinoxaline-3-yl) Prop-2-ynyl Methanosulphonate and 3-(Quinoxaline-3-yl) Prop-2-yn-1-ol.

Despite major advancements in the development of various chemotherapeutic agents, treatment for lung cancer remains costly, ineffective, toxic to normal non-cancerous cells, and still hampered by a high level of remissions. A novel cohort of quinoxaline derivatives designed to possess a wide spectrum of biological activities was synthesized with promising targeted and selective anticancer drug activity. Hence, this study was aimed at determining in vitro anticancer activity effects of a newly synthesized class of 3-(quinoxaline-3-yl) prop-2-ynyl quinoxaline derivatives on A549 lung cancer cells. An assessment of the quinoxaline derivatives ferric reducing power, free radical scavenging activity, cytotoxic activity, and ability to induce reactive oxygen species (ROS) production was performed using the Ferric Reducing Antioxidant Power (FRAP), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assays, respectively. The ability of the quinoxaline derivatives to induce apoptosis in A549 cells was assessed using the Acridine Orange/Ethidium Bromide (AO/EB) and Annexin V-FITC/Dead Cell Assay. Of the four quinoxaline derivatives tested, 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate (LA-39B) and 3-(quinoxaline-3-yl) prop-2-yn-1-ol (LA-55) displayed a dose-dependent reducing power, free-radical scavenging activity, inhibition of cell viability, and stimulation of ROS production which was accompanied by induction of apoptosis in A549 lung cancer cells. None of the quinoxaline derivatives induced cell death or ROS production in non-cancerous Raw 267.4 macrophage cells. Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer, and MCF-7 breast cancer cells albeit inhibition was more pronounced in A549 cells. The results of the study suggest that 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate and 3-(quinoxaline-3-yl) prop-2-yn-1-ol induce apoptotic cell death in A549 lung cancer cells.

Synthesis, Characterization, and In Vitro Evaluation of Super Paramagnetic Nanoparticles Grafted with PAMPS for Controlled Delivery of Cationic Drugs

AbstractThe pH‐sensitive poly(2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) (PAMPS) grafted Fe3O4 nanoparticles (NPs), as the polyanionic nanocarriers, were synthesized and characterized by Fourier transform‐infrared (FTIR) spectroscopy, energy‐dispersive X‐ray spectroscopy (EDS), dynamic light scattering (DLS), thermo‐gravimetric analysis (TGA) and vibrating sample magnetometry (VSM). The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were used to explore the shape, morphology and size of the NPs. Doxorubicin (Dox), an anticancer agent as the model drug, was successfully loaded on the synthesized NPs. The Dox‐loaded NPs exhibited different release profiles at various pH values. For example, the cumulative Dox release at pH 5.5 was about 2.6 times higher than that at pH 7.4. The cytotoxicity of the synthesized NPs with or without Dox were investigated in the vicinity of the human adipose derived mesenchymal stem cells (hADSCs) and HeLa cells using the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay and the AO/EB (acridine orange/ethidium bromide) staining method.

Marine sponge alkaloid aaptamine enhances the anti-bacterial and anti-cancer activity against ESBL producing Gram negative bacteria and HepG 2 human liver carcinoma cells

Abstract Marine sponge alkaloid aaptamine is a promising anti-bacterial and anti-cancer drug against ESBL producing Gram negative bacteria and HepG 2 human liver carcinoma cell line. The antibacterial activity of aaptamine against selected ESBL producing Gram negative bacteria was inhibited at the concentration of 55 µg/mL. At the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) of 50 µg/mL, the aaptamine was significantly effective against all the ESBL positive uropathogens. Importantly, the inhibition was much high compared to other marine compounds through intracellular damage and morphological modification by confocal laser scanning electron microscope (CLSM) and scanning electron microscope (SEM). Further, the cytotoxic efficacy of aaptamine against HepG 2 cells was showed at 75 µg/mL very effectively and this concentration was indicated as IC50 dose. The morphological and intracellular nuclear damage of aaptamine treated HepG 2 cell was detected by florescence microscope using acridine orange/ethidium bromide (AO/EB) combination and Hoechst 33342 staining assays. The nuclear fragmentation of aaptamine treated HepG 2 cells were confirmed by expression of Caspase-9, Caspase-8, Caspase-3 cleavage, which provide the suggestion for the induction of intrinsic and extrinsic apoptosis pathways. In addition, the western blot analysis of appatamine treated HepG 2 cells also confirmed that the expression genes were damaged and the aaptamine showed dose and time dependent relationship. Hence, our findings suggest, the marine natural compounds aaptamine might be a potential alternative agents for ESBL producing uropathogens and human liver cancer therapy.

Taurine Prevented Hypoxia Induced Chicken Cardiomyocyte Apoptosis Through the Inhibition of Mitochondrial Pathway Activated by Calpain-1.

Objective To determine whether taurine has protective effects on chicken myocardial apoptosis induced by hypoxic condition through inhibiting calpain-1 derived mitochondrial apoptotic pathway. Methods Chicken primary embryonic myocardial cells were isolated and cultured at 37°C under a 5% CO2 atmosphere. Firstly the optimum concentration of taurine or PD150606 was chosen by detecting the cell viability. Chicken cardiomyocytes were cultured in 95% N2-5% CO2 atmosphere for 12h to produce hypoxic conditions. Before hypoxic treatment, 10mM taurine and 10uM PD150606 (a specific calpains inhibitor) were added separately or together. The cell apoptosis was detected by acridine orange/ethidium bromide (AO/EB) double staining. Western blotting was used to determine the protein expressions of calpain-1, cytochrome c, Bcl-2, procaspase-9 and procaspase-3 in the cardiomyocytes. Results Taurine administration effectively attenuated the myocardial apoptosis under hypoxic condition, reduced the calpain-1 protein level. In addition, pre-treated taurine could up-regulate the protein expressions of Bcl-2 and procaspase-3 in hypoxic myocardial cells, down-regulate protein expression levels of cytochrome c and procaspase-9. Moreover, taurine exhibited same inhibition effect as PD150606 on the cell apoptosis and proteins express under hypoxic condition. Conclusions Taurine could attenuate the chicken cardiomyocyte apoptosis impaired by hypoxia through inhibiting calpian-1-derived mitochondrial apoptotic pathway in vitro.

Proteomic Study of a Parkinson's Disease Model of Undifferentiated SH-SY5Y Cells Induced by a Proteasome Inhibitor.

Parkinson's disease (PD) is one of the most common nervous system degenerative diseases. However, the etiology of this disease remains elusive. Here, a proteasome inhibitor (PSI)-induced undifferentiated SH-SY5Y PD model was established to analyze protein alterations through proteomic study. Methods: Cultured undifferentiated SH-SY5Y cells were divided into a control group and a group treated with 2.5 µM PSI (PSI-treated group). An methyl thiazolyl tetrazolium (MTT) assay was applied to detect cell viability. Acridine orange/ethidium bromide (AO/EB), α-synuclein immunofluorescence and hematoxylin and eosin (H&E) staining were applied to evaluate apoptosis and cytoplasmic inclusions, respectively. The protein spots that were significantly changed were separated, analyzed by 2D gel electrophoresis and DIGE De Cyder software, and subsequently identified by MALDI-TOF mass spectrometry and database searching. Results: The results of the MTT assay showed that there was a time and dose dependent change in cell viability following incubation with PSI. After 24 h incubation, PSI resulted in early apoptosis, and cytoplasmic inclusions were found in the PSI-treated group through H&E staining and α-synuclein immunofluorescence. Thus, undifferentiated SH-SY5Y cells could be used as PD model following PSI-induced inhibition of proteasomal function. In total, 18 proteins were differentially expressed between the groups, 7 of which were up-regulated and 11 of which were down-regulated. Among them, 5 protein spots were identified as being involved in the ubiquitin proteasome pathway-induced PD process. Conclusions: Mitochondrial heat shock protein 75 (MTHSP75), phosphoglycerate dehydrogenase (PHGDH), laminin binding protein (LBP), tyrosine 3/tryptophan 5-monooxygenase activation protein (14-3-3ε) and YWHAZ protein (14-3-3ζ) are involved in mitochondrial dysfunction, serine synthesis, amyloid clearance, apoptosis process and neuroprotection. These findings may provide new clues to deepen our understanding of PD pathogenesis.

Bacillomycin D-C16 triggers apoptosis of gastric cancer cells through the PI3K/Akt and FoxO3a signaling pathways.

Bacillomycin D can inhibit the growth of Aspergillus ochraceus in food samples. In addition, it can induce apoptosis in and inhibit the proliferation of cancer cells, although the details of this mechanism are unknown. In this study, we separated bacillomycin D-C14, D-C15, D-C16 monomers from the Bacillus subtilis strain fmbJ. The bacillomycin D monomers containing longer fatty acid chains better induced apoptosis in Bgc-823, Sgc-7901, and Hgc-27 gastric cancer cells. The Bgc-823 cell line was the most sensitive. Acridine orange-ethidium bromide staining indicated that bacillomycin D-C16-induced Bgc-823 cell death by triggering apoptosis, characterized by membrane blebbing, cellular shrinkage, and DNA fragmentation. Flow cytometric analysis showed a bacillomycin D-C16 dose-dependent trigger of Bgc-823 apoptosis. Bacillomycin D-C16-induced the mitochondrial pathway, as indicated by a reduced Bcl-2/Bax expression ratio, enhanced cytochrome C release, and higher levels of cleaved caspase-3. Furthermore, bacillomycin D-C16 effectively repressed phosphorylation of the serine-threonine protein kinase Akt at Ser-473 and increased the levels of the FoxO3a protein. The combination of the PI3K/Akt-inhibitor BEZ235 with bacillomycin D-C16 enhanced the apoptosis of Bgc-823 cells. Together, these findings indicated that bacillomycin D-C16 induces apoptosis through the PI3K/Akt and FoxO3a signaling pathways.

Ternary Copper (II) complex based chemical probes for DNA targeting: Cytotoxic activity under visible light

Among the bio‐metals, copper derivatives of O, N, S donor salicylaldehyde thiosemicarbazones have been obtained large interest due to their potential biological applications. Multisubstituted thiosemicarbazone ligand HL derived, new ternary Cu (II) complexes of [Cu(L)(bpy)] (1) and [Cu(L)(phen)] (2) (where, bpy is 2,2′‐bipyridine and phen is 1,10‐phenanthroline) have been synthesized and characterized using different physico‐chemical techniques. Complexes 1 and 2 are structurally characterized by single crystal X‐ray diffraction analysis, which reveals the trigonal bipyramidal distorted square based pyramid geometry of both the complexes 1 and 2 with ONS‐donor thiosemicarbazone bonded at the upper plane. The ground state electronic structures of complexes 1 and 2 have been investigated by using DFT/B3LYP theoretical evaluation with 6‐31G (d,p) and LANL2DZ basis set. The affinity towards DNA and human serum albumin has been evaluated using computational docking analysis and complex 2 exposes significant binding ability towards DNA and human serum albumin, because of its immense hydrophobicity. Consequently, complex 2 have higher antimicrobial in addition to the cytotoxic activity than complex 1 and free ligand HL under visible light. Along with, their apoptosis pathway of cytotoxicity has been evaluated by fluorescent microscopic analysis using acridine orange/ethidium bromide (AO/EB) stains. From these preliminary investigations, we believe that complex 2 can play a role as a more robust pharmacological agent.

Mitochondrial division inhibitor-1 potentiates cisplatin-induced apoptosis via the mitochondrial death pathway in cholangiocarcinoma cells

AimsMdivi-1, a selective Drp-1 inhibitor, impedes mitochondrial dynamics and suppresses cancer proliferation and progression. Cholangiocarcinoma (CCA) is a very aggressive malignancy which is refractory to chemotherapy. The study investigated the mechanism of the chemosensitizing effect of mdivi-1 in cholangiocarcinoma. Main methodsCCA cells and HEK293 T cells were employed in the study. Cell viability and induction of apoptotic cell death were determined by the MTT and acridine orange-ethidium bromide methods. Cellular glutathione content and reactive oxygen species (ROS) formation were assessed using thiol green and 2′,7′-dichlorofluorescin diacetate fluorescent probes, respectively. Mitochondrial transmembrane potential and autophagy were detected by JC-1 dye and autophagy assay. Cell cycle progression was analyzed by flow cytometry. Cell migration was measured using the wound healing assay. Proteins involved in cell proliferation and cell cycle were analyzed by western immunoblotting. Key findingsMdivi-1 enhanced cisplatin-induced cytotoxicity in CCA cells but not in HEK293 T cells. Mdivi-1 enhanced cisplatin induced glutathione redox stress, ROS formation, and loss of mitochondrial transmembrane potential. Moreover, mdivi-1 also inhibited autophagic flux and suppressed CCA cell migration. SignificanceMdivi-1 sensitized CCA cells to cytotoxicity of cisplatin in association with increases of oxidative stress and autophagosomes, and induced cell death via the mitochondrial pathway. Disruption of mitochondrial dynamics may be a novel strategy to improve the efficacy of chemotherapy to treat CCA.

Syzygium cumini extract induced reactive oxygen species-mediated apoptosis in human oral squamous carcinoma cells.

Syzygium cumini (L.) Skeels (jambolan) is commonly used in Indian traditional medicine to treat a variety of diseases such as obesity, diabetes etc. The cytotoxic potential of S. cumini (SC) against oral cancer cell line remains elusive. Therefore, in this study, we evaluated the cytotoxic effect of S. cumini in human oral squamous cell carcinoma (OSCC) cell line (SCC-25 cells). Oral squamous cell carcinoma cells are treated with different concentrations (10, 20, and 40 μg/mL) of S. cumuni for 24 hours and cytotoxicity was analyzed by MTT assay. The intracellular reactive oxygen species (ROS) was measured using the indicator dye, 2',7'-dichlorofluorescin diacetate staining. Apoptosis-related morphological changes were evaluated by dual acridine orange/ethidium bromide (AO/EB) fluorescent staining and phosphatidylserine externalization was measured by annexin V assays. The protein and gene expression of cadherin-1 was evaluated by western blotting and PCR analysis. Syzygium cumini treatments caused cytotoxicity of OSCC cell line and induced intracellular ROS accumulation. This treatment also caused apoptosis-related morphological changes and externalization of phosphatidylserine in OSCC cells. Further, S. cumini treatments increased protein and gene expression of cadherin-1. Syzygium cumini extract inhibits the proliferation of OSCC cells and induces apoptosis through ROS accumulation and therefore, it could be used for the prevention of OSCC.

Greener approach for synthesis of novel steroidal prodrugs using ionic liquid, their DFT study and apoptosis activity in prostate cancer cell line

In the present work Ionic liquid (IL), N-methyl-2-pyrolidonehydrogensulphate, (NMP+HSO4−) has been used as the green catalyst for the synthesis of novel biologically active diosgenin prodrugs 2 and 3. The prodrugs were synthesized by coupling of non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and mefenamic acid with diosgenin in the presence of ionic liquid (NMP+HSO4−). A high conversion of diosgenin (90%) and prodrugs yield (86.57 ± 1.78%) were achieved using (NMP+HSO4−) as catalyst under the following conditions: 40 °C, NSAID/diosgenin 1:1 (mol/mol), catalyst load (47.56 mg, 0.241 mmol). Re-usability test showed that (NMP+HSO4−) catalyst has excellent utility for repeated use, the ease of its separation from reaction mixture by simple filtration, is thus beneficial from economical point of view. Diosgenin along with its prodrugs 2 and 3 were investigated for cytotoxicity and apoptosis through MTT assay and ethidium bromide/acridine orange (EB/AO) staining, respectively, of prostate cancer cell line PC-3. The results were very promising as these prodrugs have shown significant anticancer activity against prostate cancer cells. Density functional theory (DFT) studies were also carried out for prodrugs 2 and 3 using B3LYP/6-31G (d,p) basis set. Intramolecular interactions were analysed by AIM (Atoms in Molecule) approach. Molecular electrostatic potential surface (MEPS) was used to indicate nucleophilic and electrophilic sites. Electronic properties such as HOMO-LUMO energies were measured with the help of time dependent DFT method. Global reactivity descriptors of prodrugs 2 and 3 were also calculated.

Metformin sensitizes cholangiocarcinoma cell to cisplatin-induced cytotoxicity through oxidative stress mediated mitochondrial pathway

AimsMetformin (Met), an essential antidiabetic agent, shows antitumor activity in some cancers. A previous study showed that Met enhanced cytotoxic activity of cisplatin (Cis) in cholangiocarcinoma (CCA) in association with the activation of AMP-activated protein kinase and suppression of Akt-mTOR. However, these effects do not entirely explain the observed chemosensitizing effect. The present study investigated the interaction of Met and Cis over the enhanced antitumor effect. Main methodsKKU-100 and KKU-M156 cells were used in the study. Cytotoxicity was assessed by acridine orange-ethidium bromide staining. Reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm) were measured by dihydroethidium and JC-1 fluorescent methods. Cellular glutathione (GSH) and redox ratio were analyzed by enzymatic coupling assay. Proteins associated with antioxidant system and cell death were evaluated by western immunoblot. Key findingsCytotoxicity of Cis was enhanced by Met in association with ROS formation and GSH redox stress. The antioxidants, N-acetylcysteine and TEMPOL, and MPTP inhibitor, cyclosporine, attenuated cytotoxicity in association with suppression of ROS formation and the losses of Δψm. Met in combination with Cis suppressed expression of Nrf2 and altered the expression of Bcl2 family proteins. SignificanceThe chemosensitizing effect of Met in combination with Cis is causally associated with increased oxidative stress-mediated mitochondrial cell death pathway. Met may improve the efficacy of Cis in the treatment of cancer.

Metformin enhances inhibitive effects of carboplatin on HeLa cell proliferation and increases sensitivity to carboplatin by activating mitochondrial associated apoptosis signaling pathway.

Cervical cancer has become the fourth most common cancer in developing countries. This study aimed to investigate anti-tumor effects of Metformin combining with carboplatin in cervical cell line, HeLa cell. Human cervical cancer cell line, HeLa cell, was treated with Metformin (5 mmol/l or 10 mmol/l) or/and carboplatin (25 mg/l or 50 mg/l) at different final concentrations, and divided into 8 groups. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell viability. Acridine orange/ethidium bromide (AO/EB) staining was used to examine nuclear fragments and cell apoptosis. Annexin V/propidium iodide (PI) staining was employed to detect apoptosis of HeLa cells. Mitochondrial membrane potential of the HeLa cells was evaluated by staining with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) reagent. MTT results showed that Metformin combining carboplatin significantly reduced HeLa cell viability compared to that of no-drug treatment group (p<0.05). Metformin combining carboplatin significantly increased the amounts of nuclear fragments compared to that of no-drug treatment group (p<0.05). The flow cytometry assay results indicated that Metformin combining carboplatin significantly enhanced the apoptotic rates compared to that of no-drug treatment group (p<0.05). The JC-1 staining findings illustrated that Metformin combining carboplatin significantly decreased the mitochondrial membrane potential compared to that of no-drug treatment group (p<0.05). Metformin enhanced the inhibitive effects of carboplatin on HeLa cell proliferation. Metformin increased the sensitivity of HeLa cell to the treatment of Carboplatin by activating mitochondrial-associated apoptosis signaling pathway.

Investigation of Camphor Effects on Fusarium graminearum and F. culmorum at Different Molecular Levels.

Fusarium graminearum and F. culmorum are phytopathogens, which cause destructive diseases in cereals. Epidemics of these phytopathogens are caused by mycotoxin contamination and the reduction of crop quality. In this study, the alteration due to in vitro camphor treatment on F. culmorum 9F and F. graminearum H11 isolates was investigated in terms of epigenetic, cellular, and transcription levels. Camphor with different concentrations (0.2, 0.4, 0.8, 1, 2, and 4 µg/µL) was applied to potato dextrose agar (PDA) growth media. The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC50) were calculated as 2 and 1 µg/µL, respectively. hog1, mst20, CAT, POD, mgv1, stuA, and tri5 genes, which are related to various cellular processes and pathogenesis, were examined by qPCR assay. qPCR analysis showed that camphor treatment leads to the downregulation of tri5 expression but the upregulation of the remaining genes. Apoptosis and oxidative stress were confirmed via acridine orange/ethidium bromide (AO/EB) and dichlorofluorescin diacetate (DCF-DA) staining, respectively. Moreover, coupled restriction enzyme digestion-random amplification (CRED-RA) assay, used for DNA methylation analysis, was carried out to evaluate epigenetic alterations. The decrease in genomic template stability (GTS) values, which resulted due to the alterations in random amplified polymorphic DNA (RAPD) profiles caused by camphor treatment, were detected as 97.60% in F. culmorum 9F and 66.27% in F. graminearum H-11. The outer and inner methylated cytosine profiles are determined by CRED-RA assay as type I–IV epigenetic alterations. The outcomes indicated that camphor could lead to alterations at several molecular levels of F. graminearum and F. culmorum.

In silico studies, synthesis and anticancer activity of novel diphenyl ether-based pyridine derivatives.

A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 ± 0.45µM) and 3p (IC50 10.57 ± 0.54µM) were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.