MTT Colorimetric Assay Research Articles

Cytotoxic Impact of N-Oleoylethanolamine on Bone Cancer Cells.

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. This study investigates the cytotoxic and apoptosis-inducing activities of NOE on human osteosarcoma Saos-2 cells. Cytotoxic effects were investigated by MTT colorimetric assay. For the detection of morphological and ultrastructural indicators of apoptosis, confocal and TEM techniques were used. 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. The results revealed that NOE exerts its cytotoxicity on Saos-2 cells through changing the ultrastructure and morphology of cells with clear apoptotic sparks.

Synthesis, antimicrobial, anticancer activities, PASS prediction, molecular docking, molecular dynamics and pharmacokinetic studies of designed methyl α-D-glucopyranoside esters

Carbohydrates are abundant naturally occurring biomolecules that play essential roles in a wide variety of biological processes. In this paper, we describe the synthesis of a series of methyl α-D-glucopyranoside (α-MGP) esters through regioselective acylation, by modification of the hydroxyl (-OH) group. Synthesized 6-O-acyl derivatives were further converted to 2,3,4-tri-O-acyl esters, in good yield. Structures of the newly designed α-MGP esters (termed compounds 1–8) were elucidated by spectroscopic, physicochemical, and elemental analyses. PASS (prediction of activity spectra for substances) and in vitro screening of compounds 1–8 against human and plant pathogenic microorganisms revealed substantial antibacterial and antifungal activities. Most of them showed >70% inhibition of fungal mycelial growth. In vitro effect of compound 7 against Ehrlich ascites carcinoma (EAC) cells, by MTT colorimetric assay, showed 23.97% of cell growth inhibition with an IC50 value of 1101.34 µg/ml. Molecular docking and nonbonding interaction studies were performed for three pathogen proteins Escherichia coli lectin FimH, Bacillus subtilis Obg GTP-binding protein, and Aspergillus flavus urate oxidase to evaluate binding affinities and interactions of compounds 1–8 with receptor proteins. A 100-ns molecular dynamics (MD) simulation study revealed the stable conformation and binding patterns/energy in a stimulating environment. In silico pharmacokinetics prediction and drug-likeness analysis of the compounds revealed improved kinetic parameters and satisfaction of all drug-likeness rules in regard to biological activity. Structure-activity relationship (SAR) studies, in combination with in vitro and in silico findings, indicated that among various acyl chains, lauroyl (C-12) and myristoyl (C-14), combined with α-MGP, were most effective against the tested bacterial and fungal pathogens. In conclusion, the designed α-MGP esters displayed enhanced pharmaceutical activities, and strong potential for development as antimicrobial agents.

Chemical and cytotoxicity evaluation of apprehended formulations of anabolic androgenic steroids

Apprehensions of formulations of anabolic-androgenic steroids (AASs) have been significantly increasing worldwide. It is known that the consumption of synthetic steroids can lead to several side effects including liver damage, heart failure and psychological disorders. Nonetheless, there are few studies evaluating the toxicity of anabolic agents’ formulations. In this sense, the aims of this work were to determine the chemical profile of AASs apprehended in Brazil by Gas Chromatography-Mass Spectrometry as well as to determine their cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed that most formulations had their constitution adulterated or falsified by the lack of active ingredients. By the colorimetric MTT assay, it could be observed that most anabolic agents had cytotoxicity in concentrations of 0.5 mg mL−1 in a general dose-dependent manner. Product #3 can be cited as displaying minimum cell viability (5.67 ± 0.25%) at 0.5 mg mL−1 increasing to 91.82 ± 4.78% at 0.125 mg mL−1. Therefore, in this study, apprehended formulations of AASs were mostly counterfeit materials with in vitro cytotoxicity, which shows the importance of analyzing these materials and indicating the adverse effects they pose to human health.

Promising Antiproliferative Compound From the Green Microalga Dunaliella tertiolecta Against Human Cancer Cells

Dunaliella tertiolecta is a green flagellated microalga with a high tolerance to salinity and high production of pigments such as zeaxanthin, a xanthophyll carotenoid present in higher plants known for its antioxidant potential. In the current study, the antiproliferative activity of raw extracts and fractions of D. tertiolecta (clone CCMP 1320) was evaluated against four different human cancer cell lines: melanoma, hepatocellular liver carcinoma, and two lung adenocarcinoma cell lines. In addition, a normal cell line (lung fibroblast) was used as toxicity control. The activity was evaluated by treatment with the extracts/fractions following the MTT colorimetric assay procedures. HPLC-UV-HRMS based dereplication helped to identify the bioactive metabolites. A glycoglycerolipid was identified in the active fraction, being involved in the bioactivity of this microalga. This compound, glycerol 1-(9Z,12Z,15Z-octadecatrienoate)-2-(4Z,7Z,10Z,13Z-hexadecatetraenoate)-3-O-β-D-galactopyranoside (1), was purified and its antiproliferative activity was confirmed. This work gives new insights on the antiproliferative activity of the green alga D. tertiolecta and its potential industrial applications.

Carpaine Promotes Proliferation and Repair of H9c2 Cardiomyocytes after Oxidative Insults.

Carpaine has long been identified as the major alkaloid in Carica papaya leaves that possess muscle relaxant properties. Limited study on the molecular signaling properties of carpaine urges us to conduct this study that aims to elucidate the mechanism underlying the cardioprotective effect of carpaine in embryonic cardiomyocytes of the H9c2 cell line. The 50% inhibitory concentration (IC50) of carpaine was first determined using a colorimetric MTT assay to establish the minimum inhibitory concentration for the subsequent test. Using a 1 µM carpaine treatment, a significant increase in the H9c2 proliferation rate was observed following 24 and 48 h of incubation. A Western blot analysis also revealed that carpaine promotes the upregulation of the cell cycle marker proteins cyclin D1 and PCNA. Carpaine-induced H9c2 cell proliferation is mediated by the activation of the FAK-ERK1/2 and FAK-AKT signaling pathways. In the setting of ischemia-reperfusion injury (IRI), carpaine provided a significant protective role to recover the wounded area affected by the hydrogen peroxide (H2O2) treatment. Furthermore, the oxidative-stress-induced reduction in mitochondrial membrane potential (MMP) and overproduction of reactive oxygen species (ROS) were attenuated by carpaine treatment. The current study revealed a novel therapeutic potential of carpaine in promoting in vitro cardiomyocyte proliferation and repair following injury.

MiR-557 downregulation lowered cell proliferation and malignancy in glioblastoma by targeting ADAM17


 
 
 
 Purpose: To examine the role of miR-557 in the pathogenesis and development of glioblastoma (GBM) and its potential regulatory mechanism.
 Methods: Dysregulation of miR-557 in GBM cells was determined using quantitative (real-time) PCR (qPCR). Cell proliferative ability, migratory activity and invasive ability were measured using the MTT colorimetric assay, wound healing assay and Transwell chambers assay. The direct target mRNA of miR-557 was predicted, and further validated using TargetScan and luciferase assay, respectively.
 Results: Compared with human astrocytes (HAs), the downregulation of miR-557 was observed in human GBM cell lines. In U-251MG and A172 cells that overexpressed miR-557 mimics, the level of proliferation was significantly reduced, the wound width was larger, and the number of invaded cells wan decreased than that of NC mimics. Predictive results from TargetScan and results of luciferase assay demonstrated that miR-557 directly targets the 3’-untranslated region (3’-UTR) of ADAM17. In A172 cells and U-251MG cells, the upregulation E-cadherin (E-cad) and the downregulation of N- cadherin (N-cad) and vimentin (Vim) were caused by miR-557 mimics as compared with NC mimics. Expression of ADAM17, NICD, HES1, and EGFR was downregulated by miR-557 mimics and upregulated by miR-557 inhibitors in A172 cells and U-251MG cells.
 Conclusion: Downregulation of miR-557 accelerates GBM cell growth and malignancy by directly targeting ADAM17 3’-UTR, providing a prognostic and potential therapeutic factor for new drug discovery in GBM.
 
 
 

Effects of Mitomycin-C and 5-Fluorouracil on Ocular Adnexal Sebaceous Carcinoma Cells

To investigate the effects of mitomycin-C (MMC) and 5-fluorouracil (5-FU) on the viability, proliferation, and migratory capacity of cultured ocular adnexal sebaceous carcinoma (SC) cells. Laboratory investigation. Human SC cell lines (Bascom Palmer 50 and 52 [BP50 and BP52]) and human limbal stem cells (LSCs) were treated with various concentrations of MMC and 5-FU. Cytotoxicity was assessed with the tetrazolium MTT colorimetric viability assay on normal corneal vs tumor cells. Growth curves and scratch assays were performed to characterize the effects of these chemotherapeutic agents on SC proliferation and migration, respectively. MMC decreased BP52 cell viability in a dose-dependent manner with a half-maximal effective dose (EC50) of 11.8 μM after 72 hours. SC viability decreased >50% at 80 mM 5-FU after 72 hours. MMC reduced LSC viability in a dose-dependent manner with an EC50 value of 3.24 μM, and 5-FU decreased LSC viability >50% at 160 μM. MMC decreased SC cell proliferation and migration in a dose-dependent manner. 5-FU displayed antiproliferative effects but did not affect cell migration at concentrations below 1000 μM. Our in vitro data corroborate clinical observations that MMC is efficacious for treating ocular adnexal SC, albeit at the expense of LSC viability. Our findings also demonstrate that topical 5-FU exhibits antiproliferative effects that supersede its cancer-killing and antimigratory effects on cultured SC cells.

Cytotoxicity and Antibacterial Activity of Mineral Trioxide Aggregate Cement with Radiopacity Introduced by ZrO2.

The article presents the results of in vitro studies on cytotoxicity and antibacterial activity of new MTA-type cements, developed on the basis of the sintered tricalcium silicate enriched with ZnO, along with an agent introducing the radiopacity in the form of ZrO2. The new materials have been developed to ensure that their physical and chemical properties are suited for endodontic applications. The cements were evaluated via characterisation of setting time, compressive strength, as well as translucency on X-ray images, and bioactivity in the simulated body fluid (SBF). The μCT was used to test the influence of the ZrO2 grains in the powder component on the microstructure of the produced cement. Then, the cytotoxic action of the cements was evaluated by applying a reference L-929 cell line. The conditions of the culture upon contact with the tested materials or with extracts from the cements were assessed using image analysis or an MTT colorimetric assay. Two strains of streptococci, Streptococcus mutans and Streptococcus sanguinis, were used to study the antibacterial activity of the tested cements with ZrO2 acting as the agent introducing the radiopacity. The new cements are characterised by appropriate properties as far as retrograde root canal filling is concerned.

Anti-proliferative Effect of Nine Resins from Medicinal Plants of Burkina Faso

Exudates are substances that ooze out of the pores of diseased and/or damaged tissue in plant that are well-used in flourishing international and local uses in Burkina Faso. Previously, the ethnopharmacological investigations in Center region showed that there are lot of medicinal uses like as old wounds, skin disorders diseases treatment. The current study aims to highlight the antitumoral potential of nine resins form central region. One hundred grams of each resin were placed at -80°C before extraction. The resins were grinded, and the powder was stirred twice with 1L and 500 mL of CHCl₃ respectively for 24h. The antiproliferative effect was determined by using MTT colorimetric assay with chloroformic extracts from resin of each plant and the concentrations which inhibited 50% of cell proliferation (IC₅₀) were determinated graphically. The results showed that the IC₅₀ of A549 proliferative inhibition was form 5.78 (<i>Lannea microcarpa</i>) to 32.48 µg/mL (<i>Daniellia oliveri</i>) and U373 inhibition IC₅₀ was from 12.90 (<i>L. macrocarpa</i>) to 41.12 µg/mL (<i>Boswellia dalzielli</i>). The Keratinocytes IC₅₀ was form 1.57 (<i>L. macrocarpa</i>) to 20.4 µg/mL (<i>Boswellia dalzielli</i>). Interestingly, the resin extract form <i>L. macrocarpa</i> resin was a best source of anticancer compounds for the A549 and U373 following by <i>Bombax costatum</i> resin. In the contrast <i>Commiphora africana</i> (Burseraceae) resin was the less antiproliferation effect. The exudates resin from plants that were a source of bioactive compounds could explain of the medicinal uses like as anti-inflammatory, wound healing, and skin ailments in Burkina Faso.

The effect of temozolomide on apoptosis-related gene expression changes in glioblastoma cells.

Glioblastoma (GB) is the most common and biologically the most aggressive primary brain tumor of the central nervous system (CNS) in adults. Standard treatment for newly diagnosed GB consists of surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Despite numbers of studies, a resistance to chemotherapy is the major obstacle to successful GB treatment. The aim of our study was to detect the sensitivity of glioblastoma T98G cells to TMZ treatment and subsequently to determine the expression changes of apoptosis-associated genes in glioblastoma cells. The human glioblastoma cell line (T98G) was treated with specified concentrations of TMZ during different time periods. Their viability was measured by colorimetric MTT assay and the activation of the apoptotic pathway was determined by measuring the caspase 3/7 activity. Commercial pre-designed microfluidic array was used to quantify expression of human apoptosis-associated genes. The untreated control of T98G cell line against human brain total RNA standards reported significant changes in several apoptotic genes expression levels. We identified also a deregulation in geneexpression levels between the TMZ treated and untreated T98G cells associated with apoptotic pathways. After 48 hours of exposure of T98G cells to TMZ, we observed a significant deregulation ofseven genes: BBC3, BCL2L1, RIPK1, CASP3, BIRC2, CARD6 and DAPK1. These results can contribute to the importance of apoptosis in glioblastoma cells metabolism and effect of TMZ treatment. Identification of apoptotic gene panel in T98G cell line could help to improve understanding of brain tumor cells metabolism. Recognizing of the pro-apoptotic and anti-apoptotic genes expression changes could contribute to clarify the sensitivity to TMZ therapy and molecular base in healthy and tumor cells (Tab. 1, Fig. 2, Ref. 48).

Effects of the Wnt/β-Catenin Signaling Pathway on Proliferation and Apoptosis of Gastric Cancer Cells.

Objective To explore the effect of the Wnt/β-catenin signaling pathway on the proliferation and apoptosis of gastric cancer cells. Methods An MTT colorimetric assay was used to detect the inhibitory effect of the Wnt/β-catenin signaling pathway inhibitor FH535 on the proliferation of MKN45 gastric cancer cells. The cell proliferation index (PI) and apoptosis index (AI) were measured by flow cytometry. The expression levels of β-catenin, c-myc, and cleaved caspase-3 in MKN45 gastric cancer cells were detected. Results After using the Wnt/β-catenin signaling pathway inhibitor FH535, MKN45 gastric cancer cells showed obvious shrinkage, death, and cell density decrease. MTT showed that the A value of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The survival rate of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The cell cycle of gastric cancer was arrested in G0/G1 phase. The expression levels of β-catenin and c-myc protein in MKN45 gastric cancer cells in FH535 group decreased significantly (P < 0.05), while the expression level of cleaved caspase-3 protein increased significantly (P < 0.05). Conclusion The Wnt/β-catenin signal molecule can maintain the proliferation of gastric cancer cells. Inhibition of the Wnt/β-catenin signaling pathway can inhibit the proliferation of gastric cancer cells and promote the apoptosis of MKN45 gastric cancer cells.

Phytochemical and biological study of phenolic fraction from Geoffroea decorticans bark

The stem bark of Geoffroea decorticans (Gill.ex Hook. et Arn.) Burk. (Fabaceae) was used medicinally to cure several skin affections; however, previous phytochemical and biological antecedents that supported its vulnerary property were not found yet. A purified methanolic fraction from G. decorticans bark (PFGB), was derivatized by silylation for GC/MS analysis, and complementary analyses by two-dimensional TLC and UV-Vis spectroscopy against detected standards compounds. Nine phenolic compounds were characterized as gallic acid, quercetin, kaempferol, apigenin, protocatechuic acid, taxifolin, 4-Hydroxybenzoic acid; among these two methoxy-flavonoids. PFGB showed significant antibacterial activity (MICs= 125 μg/mL) against Staphylococcus aureus (25923) and Enterococcus faecalis (29212) ATCC strains. Cytotoxic effects on human lymphocytes activated or not by LPS were not observed up to 100 μg/mL, by MTT colorimetric assay. The resultsobtained through the ethnopharmacological approach of this work contribute to the scientific validation of the vulnerary medicinal use of G. decorticans.

Phytochemistry and Antiviral Properties of Two Lotus Species ‎Growing in Egypt

Background: Lotus arabicus L and Lotus glaber Mill. belong to the family Fabaceae, and they grow in the wild in ‎Egypt and have different therapeutic uses in folk medicine. Objectives: This study aimed to evaluate the phytochemical profile and ‎antimicrobial properties of the methanolic extracts of two Lotus spp. growing in Egypt, L. ‎arabicus and L. glaber. Material and methods: Gas chromatography-mass spectrometry was used to identify the compounds of the extracts of two Lotus species. An MTT colorimetric assay and the disc diffusion method were performed to investigate the antiviral and antimicrobial activities of two lotus species, respectively. Results: The n-hexane and methanol extracts of L. ‎arabicuscontained high percentages of alkane hydrocarbons, such as 5-methyloctadecane, ‎while L. glaber contained dodecane. The major compounds in the methanol extract of L. ‎arabicus were hexadecanoic acid methyl ester and dodecanoic acid,2,3-bis(acetyloxy)propyl ‎ester. The major compounds in the methanol extract of L. glaber were palmitic acid and ‎lucenin 2. The indole alkaloid ditaine was found only in L. arabicus. This alkaloid was ‎identified for the first time in the genus Lotus. The antimicrobial properties of the extracts of ‎the two Lotus species showed that the n-hexane extract of ‎both Lotus species may have potential antifungal activity against Candida parapsilosis and ‎Aspergillus flavus. Moreover, the methanolic extracts of both Lotus species have potential ‎antiviral activity against the coxsackie B virus, but only the L. arabicus extract showed ‎activity against the hepatitis A virus.‎ Conclusion: Lotus arabicus might have potential antifungal or antiviral activity greater than L. glaber.

Selective Targeting of the Novel CK-10 Nanoparticles to the MDA-MB-231 Breast Cancer Cells

The main objective of this project was to formulate novel decorated amphiphilic PLGA nanoparticles aiming for the selective delivery of the novel peptide (CK-10) to the cancerous/tumor tissue. Novel modified microfluidic techniques were used to formulate the nanoparticles. This technique was modified by using of Nano Assemblr associated with salting out of the organic solvent using K2HPO4. This modification is associated with higher peptide loading efficiencies, smaller size and higher uniformity. Size, zeta potential & qualitative determination of the adsorbed targeting ligands were measured by dynamic light scattering and laser anemometry techniques using the zeta sizer. Quantitative estimation of the adsorbed targeting ligands was done by colorimetry and spectrophotometric techniques. Qualitative and quantitative uptakes of the various PLGA nanoparticles were examined by the fluorescence microscope and the flow cytometer while the cytotoxic effect of the nanoparticles was measured by the colorimetric MTT assay. PLGA/poloxamer.FA, PLGA/poloxamer.HA, and PLGA/poloxamer.Tf have breast cancer MDA. MB321 cellular uptakes 83.8, 75.43 & 69.37 % which are higher than those of the PLGA/B cyclodextrin.FA, PLGA/B cyclodextrin.HA and PLGA/B cyclodextrin.Tf 80.87, 74.47 & 64.67 %. Therefore, PLGA/poloxamer.FA and PLGA/poloxamer.HA show higher cytotoxicity than PLGA/ poloxamer.Tf with lower breast cancer MDA-MB-231 cell viabilities 30.74, 39.15 & 49.23 %, respectively. The design of novel decorated amphiphilic CK-10 loaded PLGA nanoparticles designed by the novel modified microfluidic technique succeeds in forming innovative anticancer formulations candidates for therapeutic use in aggressive breast cancers.

Pluronic F127/carfilzomib-based nanomicelles as promising nanocarriers: synthesis, characterization, biological, and in silico evaluations

In this work, we designed a drug delivery nanosystem based on oil-in-water Pluronic F127 nanomicelles to encapsulate and control the release of the antineoplasic drug carfilzomib (CFZ), and hypothesized that CFZ entrapment might result in a better effective and more localized cytotoxic effect on cancerous cells and to reduce its undesirable side effects for in vitro and in vivo applications. Pluronic F127/CFZ nanomicelles showed a high drug encapsulation efficiency (74.8%) and were allowed to achieve a sustained release pattern. Computational analyses were conducted to increase understanding of the nature and strength of mutual interactions between Pluronic F127 and CFZ. Simulation data indicated that the position of heteroatoms on CFZ influences the strength of attractive interactions in the Pluronic F127/CFZ nanomicelles. Cytotoxic activity of free and encapsulated CFZ was assayed in several cancerous cell lines, SHY-SY5Y human neuroblastoma, MCF7 breast, and HeLa cervical cancer cells employing the MTT colorimetric assay. We found that Pluronic F127/CFZ nanomicelles exhibited greater anti-proliferative activity than free CFZ (lower IC50s) and induced evident morphological alterations in cancer cells. Caspase-3 activity was assessed to determine the main mechanism of drug-induced cell death. Compared to free CFZ, our formulation only increased caspase-3 activity in HeLa cells. To examine the short-term toxicity of F127/CFZ nanomicelles, intravenous injections of free CFZ and F127/CFZ nanomicelles at three doses of 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg for five consecutive days were administered to an animal model. The potential anti-bactericidal activity of this new formulation was also assayed against four standard bacteria, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Unfortunately, no significant bacteria-killing effects were detected despite other drugs of the same family showing promising results. Still, our new formulation showed desirable anti-cancer effects and might be a good therapeutic modality for cancer treatment.

Two new benzophenones from Dobinea delavayi

• Two new benzophenones 1 and 2 were isolated from Dobinea delavayi . • Complete spectral data of athyriol ( 4 ) were reported for the first time. • α -Hydroxycaffeic acid ( 6 ) was isolated as a natural product for the first time. • α -Hydroxycaffeic acid ( 6 ) exhibited significant in vitro antimalarial activity. Two new benzophenones, 2,6,3′,4′-tetrahydroxy-4-methoxybenzophenone ( 1 ) and 2,6,4′-trihydroxy-4,3′-dimethoxybenzophenone ( 2 ), together with four known phenolic compounds ( 3 - 6 ) were isolated from the roots of Dobinea delavayi (Anacardiaceae). Structures of the isolated compounds were determined by the comprehensive analyses of the physicochemical properties and spectral data (NMR and MS). In vitro antimalarial activity and cytotoxicity of isolated compounds were evaluated by β -hematin formation inhibition assay and MTT colorimetric assay, respectively. Compound 6 exhibited significant in vitro antimalarial activity with IC 50 value of 42.1 μg/mL. Compound 4 exhibited weak cytotoxicity against HepG2 and MCF-7 cells with IC 50 values of 169.2 and 192.0 μg/mL.

Evaluation of antileishmanial potential of the antidepressant escitalopram in Leishmania infantum

Neglected tropical diseases (NTDs) such as visceral leishmaniasis (VL) present a limited and toxic therapeutic arsenal, and drug repositioning represents a safe and cost-effective approach. In this work, we investigated the antileishmanial potential and the mechanism of lethal action of the antidepressant escitalopram. The efficacy of escitalopram was determined ex-vivo using the intracellular Leishmania (L.) infantum amastigote model and the mammalian cytotoxicity was determined by the colorimetric MTT assay. The cellular and molecular alterations induced by the drug were investigated using spectrofluorimetry, a luminescence assay and flow cytometry. Our data revealed that escitalopram was active and selective against L. infantum parasites, with an IC50 value of 25 µM and a 50% cytotoxic concentration (CC50) of 184 µM. By using the fluorescent probes SYTOX® Green and DiSBAC2(3), the drug showed no alterations in the plasma membrane permeability nor in the electric potential of the membrane (∆ψp); however, after a short-time incubation, the drug caused a dose-dependent up-regulation of the calcium levels, leading to the depolarization of the mitochondrial membrane potential (∆ψm) and a reduction of the ATP levels. No up-regulation of reactive oxygen (ROS) was observed. In the cell cycle analysis, escitalopram induced a dose-dependent increase of the parasites at the sub G0/G1 stage, representing fragmented DNA. Escitalopram presented a selective antileishmanial activity, with disruption of single mitochondrion and interference in the cell cycle. Approved drugs such as escitalopram may represent a promising approach for NTDs and can be considered in future animal efficacy studies.

Potential in vitro therapeutic effects of targeting SP/NK1R system in cervical cancer.

Cervical cancer, an aggressive gynecological cancer, seriously threatens women's health worldwide. It is recently reported that neuropeptide substance P (SP) regulates many tumor-associated processes through neurokinin-1 receptor (NK1R). Therefore, we used cervical cancer cell line (HeLa) to investigate the functional relevance of the SP/NK1R system in cervical cancer pathogenesis. Cellular proliferation and cytotoxicity were analyzed by colorimetric MTT assay. Quantitative real-time PCR (qRT-PCR) was used to measure mRNA expression levels of desired genes. Cell cycle distribution and apoptosis were evaluated by flow cytometry. A wound-healing assay was employed to assess migration ability. We found that the truncated isoform of NK1R(NK1R-Tr) is the dominantly expressed form of the receptor in Hela cells. We also indicated that that SP increased HeLa cell proliferation while treatment with NK1R antagonist, aprepitant, inhibited HeLa cell viability in a dose and time-dependent manner. SP also alters the levels of cell cycle regulators (up-regulation of cyclin B1 along with downregulation of p21) and apoptosis-related genes (up-regulation of Bcl-2 along with downregulation of Bax) while aprepitant reversed these effects. Aprepitant also induced arrest within the G2 phase of the cell cycle and subsequent apoptosis. Furthermore, SP promoted the migrative phenotype of HeLa cells and increased MMP-2 and MMP-9 expression while aprepitant exposure significantly reversed these effects. Collectively, our results indicate the importance of the SP / NK1R system in promoting both proliferative and migrative phenotypes of cervical cancer cells and suggest that aprepitant may be developed as a novel treatment for combating cervical cancer.

Pyrrole Plasma Polymer-Coated Electrospun Scaffolds for Neural Tissue Engineering.

Promising strategies for neural tissue engineering are based on the use of three-dimensional substrates for cell anchorage and tissue development. In this work, fibrillar scaffolds composed of electrospun randomly- and aligned-oriented fibers coated with plasma synthesized pyrrole polymer, doped and undoped with iodine, were fabricated and characterized. Infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction analysis revealed the functional groups and molecular integration of each scaffold, as well as the effect of plasma polymer synthesis on crystallinity. Scanning microscopy imaging demonstrated the porous fibrillar micrometric structure of the scaffolds, which afforded adhesion, infiltration, and survival for the neural cells. Orientation analysis of electron microscope images confirmed the elongation of neurite-like cell structures elicited by undoped plasma pyrrole polymer-coated aligned scaffolds, without any biochemical stimuli. The MTT colorimetric assay validated the biocompatibility of the fabricated composite materials, and further evidenced plasma pyrrole polymer-coated aligned scaffolds as permissive substrates for the support of neural cells. These results suggest plasma synthesized pyrrole polymer-coated aligned scaffolds are promising materials for tissue engineering applications.

Antimicrobial and cytotoxic capacity of pyroligneous extracts films of Eucalyptus grandis and chitosan for oral applications

Chitosan films containing distilled pyroligneous extracts of Eucalyptus grandis (DPEC), characterized and developed by Brazilian Agricultural Research Corporation—Embrapa Temperate Agriculture (EMBRAPA-CPACT), were evaluated for antimicrobial activity against Candida albicans, Streptococcus mutans, and Lactobacillus acidophilus by direct contact test. Further, their capacity for the prevention of teeth enamel demineralization and cytotoxicity in vitro were also determined. The natural polymers were tested at different concentrations (1500–7500 µg mL−1) and the formulation of an experimental fluoride varnish with antimicrobial activity was evaluated by direct contact test, whereas cytotoxicity was analyzed through the colorimetric MTT assay. Preliminary data showed no statistically significant differences in cytotoxicity to NIH/3T3 cell line when DPEC is compared to the control group. On the other hand, the antimicrobial capacity and demineralization effects were found between the test groups at the different concentrations tested. Chitosan films containing distilled pyroligneous extracts of E. grandis may be an effective control strategy to prevent biofilm formation related to dental caries when applied as a protective varnish. They may inhibit the colonization of oral microorganisms and possibly control dental caries through a decrease in pH and impairment of enamel demineralization.