Propidium Iodide Fluorescence Assay Research Articles

Protective Effect of Arzanol against H2O2-Induced Oxidative Stress Damage in Differentiated and Undifferentiated SH-SY5Y Cells

Oxidative stress can damage neuronal cells, greatly contributing to neurodegenerative diseases (NDs). In this study, the protective activity of arzanol, a natural prenylated α-pyrone-phloroglucinol heterodimer, was evaluated against the H2O2-induced oxidative damage in trans-retinoic acid-differentiated (neuron-like) human SH-SY5Y cells, widely used as a neuronal cell model of neurological disorders. The pre-incubation (for 2 and 24 h) with arzanol (5, 10, and 25 μM) significantly preserved differentiated SH-SY5Y cells from cytotoxicity (MTT assay) and morphological changes induced by 0.25 and 0.5 mM H2O2. Arzanol reduced the generation of reactive oxygen species (ROS) induced by 2 h oxidation with H2O2 0.5 mM, established by 2′,7′-dichlorodihydrofluorescein diacetate assay. The 2 h incubation of differentiated SH-SY5Y cells with H2O2 determined a significant increase in the number of apoptotic cells versus control cells, evaluated by propidium iodide fluorescence assay (red fluorescence) and NucView® 488 assay (green fluorescence). Arzanol pre-treatment (2 h) exerted a noteworthy significant protective effect against apoptosis. In addition, arzanol was tested, for comparison, in undifferentiated SH-SY5Y cells for cytotoxicity and its ability to protect against H2O2-induced oxidative stress. Furthermore, the PubChem database and freely accessible web tools SwissADME and pkCSM-pharmacokinetics were used to assess the physicochemical and pharmacokinetic properties of arzanol. Our results qualify arzanol as an antioxidant agent with potential neuroprotective effects against neuronal oxidative stress implicated in NDs.

Semi-Synthesis of Chloroxaloterpin A and B and Their Antifungal Activity against Botrytis cinerea.

Chloroxaloterpin A (1) and B (2) are two diterpenoids with potent inhibitory activities against spore germination of Botrytis cinerea, which were identified from Streptomyces sp. SN194 as minor products previously. In order to overcome the poor yields, 1 and 2 were synthesized using viguiepinol (3), the major metabolite of Streptomyces sp. SN194, as the precursor compound. Scanning electron microscope and transmission electron microscope observations revealed that after treating B. cinerea spores with 1 and 2, spores were obviously aberrant, the cytoplasm appeared withdrawn, and plasma membranes were blurred. Propidium iodide fluorescence assay indicated that 1 and 2 damaged plasma membranes of B. cinerea spores. In vivo assays showed that compounds 1 and 2 could effectively inhibit the sporulation of B. cinerea on tomato fruits, with sporulation inhibitory rates reaching 98.8% at 100 μg/mL. These findings provide new insights into chloroxaloterpin A and B and demonstrate their potential as lead candidates for novel fungicides.

Evaluation of Ligustrazine-Based Synthetic Compounds for their Antiproliferative Effects.

Ligustrazine and chalcones have been reported previously for various biological activities including anticancer effects. Based on the multitargeted biological activities approach of ligustrazine-based chalcones, in the current study 18 synthetic ligustrazine-containing α, β-unsaturated carbonyl-based 1, 3- Diphenyl-2-propen-1-one derivatives were evaluated for their inhibitory effects on the growth of five different types of cancer cells. All the compounds were evaluated for anticancer effects on various cancer cell lines by propidium iodide fluorescence assay and various other assays were performed for mechanistic studies. A majority of compounds exhibited strong inhibition of cancer cells, especially synthetic compounds 4a and 4b, bearing 1-Pyridin-3-yl-ethanone as a ketone moiety in the main structural backbone were found to be most powerful inhibitors of cancer cell growth. Nine most active compounds among the whole series were selected for further studies related to different cancer targets, including EGFR TK kinases, tubulin polymerization, KAF and BRAFV600E. Synthetic derivatives, including 4a-b and 5a-b showed a multitarget approach and strong inhibitory effects on EGFR, FAK and BRAF while three compounds, including 3e bearing methoxy substitution, 4a and 4b with 1-pyridin-3-yl-ethanone moiety showed the inhibition of tubulin polymerization.

MicroRNA-138-5p regulates pancreatic cancer cell growth through targeting FOXC1.

PurposeThe prognosis of pancreatic cancer ranks among the worst of all cancer types, which is primarily due to the fact that during the past decades little progress has been made in its diagnosis and treatment. Here, we set out to investigate the role of microRNA 138 (miR-138-5p) in the regulation of pancreatic cancer cell growth and to assess its role as putative therapeutic target.MethodsqRT-PCR was used to examine the expression of miR-138-5p in 8 pancreatic cancer cell lines and 18 primary human pancreatic cancer samples. A lentivirual vector containing miR-138-5p mimics (lv-miR-138-5p) was used to exogenously over-express miR-138-5p in the pancreatic cancer cells lines Capan-2 and PANC-1. The effect of this over-expression on cell proliferation was examined using an in vitro propidium iodide fluorescence assay. Capan-2 cells exogenously over-expressing miR-138-5p were transplanted into nude mice to examine its in vivo effect on tumor growth. A predicted target of miR-138-5p (FOXC1) was first validated using a luciferase assay and, subsequently, down-regulated by siRNA to assess its effect on pancreatic cancer cell growth.ResultsWe found that miR-138-5p was markedly down-regulated in both pancreatic cancer cell lines and primary human pancreatic cancer samples, compared to a human pancreas ductal epithelial (HPDE) cell line and normal pancreatic tissues, respectively (P < 0.05). In addition, we found that in the pancreatic cancer cells lines Capan-2 and PANC-1 lentiviral transfection of miR-138-5p mimicked up-regulation of the endogenous expression of miR-138-5p and, concomitantly, inhibited cancer cell proliferation (P < 0.05). The exogenous over-expression of miR-138-5p also led to a significant inhibition of tumor formation in vivo. Using a luciferase assay, we found that miR-138-5p directly targets FOXC1. In conformity with this notion, we found that FOXC1 was down-regulated upon miR-138-5p over-expression in pancreatic cancer cells. Finally, we found that silencing of FOXC1 by siRNA had an inhibitory effect on pancreatic cancer cell growth.ConclusionsOur data indicate that miR-138-5p may play an important role in regulating pancreatic cancer cell growth, possibly through targeting FOXC1. Over-expression of miR-138-5p may serve as a novel approach for the treatment of patients with pancreatic cancer.

A simple mechanistic way to increase the survival of Mammalian cells during processing for dry storage.

Recently, there has been considerable interest in developing processing methods that enable storage of cells in a dry state. Most of these studies describe cell viability following processing as a function of the final moisture content reached or the duration of drying. Recently, a cumulative osmotic stress model has been proposed, which takes both final moisture content and duration of drying into consideration in an effort to account for the effects of cumulative processing stresses. The present study demonstrates the applicability of this approach and elucidates a simple mechanistic technique to reduce cumulative osmotic stress during processing. Mouse macrophage cells (J774) were exposed to increasing concentrations of trehalose-containing 0.33× phosphate-buffered saline (PBS) by step-changing the extracellular solution in 2 increments of 0.7 Osm, using only trehalose as the additive solute. Three minutes was provided for equilibration prior to drying in a traditional low-humidity chamber. The data were compared with that of cells dried directly in isotonic 0.2 M trehalose in 0.33× PBS. Following dehydration, cells were rehydrated and viability was assessed 45 min postrehydration using a combination of trypan blue staining for membrane integrity of detached cells and calcein AM-propidium iodide fluorescence assay for live-dead staining of the attached cells. Cells that were preprocessed to higher trehalose concentrations in step changes prior to drying had higher viability scores at comparable final moisture levels when compared with cells dried in iso-osmotic solution, up to a limit of ∼8 Osm, at which point cells processed by both methods approached zero viability.

Up-regulation of Receptor Activator Nuclear Factor–Kappa B Ligand Expression by Root Canal Sealers in Human Osteoblastic Cells

Histologic investigations have demonstrated that root canal sealers can induce mild to severe inflammatory alternations. A recently identified tumor necrosis factor family molecule, receptor activator of nuclear factor-κB ligand (RANKL), plays a critical role in the development of osteoclasts that result in bone resorption. The aim of this study was to investigate the effects of root canal sealers AH26, Canals, and N2 on the expression of RANKL in human osteoblast cell line U2OS cells. Freshly mixed materials were filled in glass rings (4 mm height and 10 mm in diameter) and eluted in 10 mL of culture medium for 1 day. Subsequently, various dilutions (final dilution: 1/2, 1/4, and 1/8) of these extraction media were prepared for this study. Cytotoxicity was measured by the propidium iodide fluorescence assay. The expression of RANKL was measured by reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that AH26, Canals, and N2 were cytotoxic to U2OS cells in a concentration-dependent manner ( P < .05). The exposure of U2OS cells to root canal sealers resulted in the up-regulation of RANKL mRNA and protein expression ( P < .05). The expression of RANKL was up-regulated by root canal sealers in the following order: N2 > AH26 > Canals. Taken together, the activation of RANKL might play an important role in the pathogenesis of periapical bone destruction induced by root canal sealers.

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly( l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL −1, the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly( l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.

Correlation of the acid-sensitivity of polyethylene glycol daunorubicin conjugates with their in vitro antiproliferative activity

Polyethylene glycol conjugates with linkers of varying acid-sensitivity were prepared by reacting five maleimide derivatives of daunorubicin containing an amide bond ( 1) or acid-sensitive carboxylic hydrazone bonds ( 2– 5) with α-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000) or α,ω-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000). The polymer drug derivatives were designed to release daunorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. In subsequent cell culture experiments, the order of antitumor activity of the PEG daunorubicin conjugates correlated with their acid-sensitivity as determined by HPLC (cell lines: BXF T24 bladder carcinoma and LXFL 529L lung cancer cell line; assay: propidium iodide fluorescence assay). The acid-sensitivity of the link between PEG and daunorubicin is therefore an important parameter for in vitro efficacy.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Albumin conjugates of the anticancer drug chlorambucil: synthesis, characterization, and in vitro efficacy.

In our efforts to improve the selectivity and toxicity profile of antitumor agents, four maleimide derivatives of chlorambucil (1-4) were bound to thiolated human serum albumin which differ in the stability of the chemical link between drug and spacer. 1 is an aliphatic maleimide ester derivative of chlorambucil, whereas 2-4 are acetaldehyde, acetophenone, and benzaldehyde carboxylic hydrazone derivatives. HPLC stability studies at pH 5.0 with the related model compounds 5, 7, 8, and 9, in which chlorambucil was substituted by 4-phenylbutyric acid, demonstrated that the carboxylic hydrazone derivatives have acid-sensitive properties; the acid lability of 7 was particular prominent with a half-life of only a few hours. The alkylating activity of albumin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)-pyridine (NBP), demonstrating that on average three equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective albumin conjugates in the MCF7 mamma carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugate in which chlorambucil was bound to albumin through an ester bond was not as active as chlorambucil. In contrast, the conjugates in which chlorambucil was bound to albumin through carboxylic hydrazone bonds were as or more active than chlorambucil in both cell lines. In particular, the conjugate in which chlorambucil was bound to albumin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 4-fold (MCF7) to 13-fold (MOLT4) lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil.

Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays.

A propidium iodide fluorescence assay (PIA) was developed to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of standard compounds. Propidium iodide (PI) was used as a dye which penetrates only damaged cellular membranes. Intercalation complexes are formed by PI with double-stranded DNA which effect an amplification of the fluorescence. Incubation of the total cell population with PI and subsequent fluorescence detection allowed assessment of the number of non-vital cells (first measurement). After freezing the cells at -20 degrees C for 24 h PI had access to total DNA leading to total cell population counts (second measurement). The number of viable cells was calculated by the difference between these two measurements. In the proliferation and cytotoxicity assays 5 x 10(3) cells per well were plated in 96 multiwells and finally stained with 50 micrograms/ml PI in 25 microliters for 10 min. A correlation between the log of cell number and the log of fluorescence units could be demonstrated over a 2.5-3 log range (r = 0.97). The lower limit of cell detection was 150-500 cells/wells. In cytotoxicity assays eight clinically used cytostatics were tested which effected a clear dose-response relationship (r = 0.93-0.98) and high reproducibility (r = 0.92). In conclusion, this assay is a simple and rapid test system, the main advantages are the absence of any washing steps and the small number of tumor cells necessary for drug testing. The PIA can easily be used for cell number determinations in biological and pharmacological studies.