Thiazolyl Blue Research Articles

Acetal-Linked Paclitaxel Polymeric Prodrug Based on Functionalized mPEG-PCL Diblock Polymer for pH-Triggered Drug Delivery.

The differences in micro-environment between cancer cells and the normal ones offer the possibility to develop stimuli-responsive drug-delivery systems for overcoming the drawbacks in the clinical use of anticancer drugs, such as paclitaxel, doxorubicin, and etc. Hence, we developed a novel endosomal pH-sensitive paclitaxel (PTX) prodrug micelles based on functionalized poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL) diblock polymer with an acid-cleavable acetal (Ace) linkage (mPEG-PCL-Ace-PTX). The mPEG-PCL-Ace-PTX5 with a high drug content of 23.5 wt % was self-assembled in phosphate buffer (pH 7.4, 10 mM) into nanosized micelles with an average diameter of 68.5 nm. The in vitro release studies demonstrated that mPEG-PCL-Ace-PTX5 micelles was highly pH-sensitive, in which 16.8%, 32.8%, and 48.2% of parent free PTX was released from mPEG-PCL-Ace-PTX5 micelles in 48 h at pH 7.4, 6.0, and 5.0, respectively. Thiazolyl Blue Tetrazolium Bromide (MTT) assays suggested that the pH-sensitive PTX prodrug micelles displayed higher therapeutic efficacy against MCF-7 cells compared with free PTX. Therefore, the PTX prodrug micelles with acetal bond may offer a promising strategy for cancer therapy.

Treatment with sodium nitroprusside improves the endothelial function in aortic rings with endothelial dysfunction

PurposeVerify if sodium nitroprusside (SNP) is able to improve endothelial function and if this effect is independent of nitric oxide (NO) release of the compound. MethodsNormotensive (2K) and hypertensive (2K-1C) wistar rats were used. Intact endothelium aortas were placed in a myograph and incubated with SNP: 0.1nM; 1nM or 10nM during 30min. Cumulative concentration-effect curves for acetylcholine (Ach) were realized to measure the relaxing capacity. Intracellular NO were measured (by DAF-2DA probe) in HUVEC treated with SNP 0.1nM or DETA/NO 0.1μM. The detection of intracellular superoxide radical (O2•-) was obtained by using DHE probe. ResultsTreatment of 2K-1C aortic rings with SNP (0.1; 1.0 and 10nM) improved endothelium dependent relaxation induced by acetylcholine. This improvement induced by SNP was verified at the concentration of 0.1nM, which does not release NO, suggesting that this effect was not induced due to NO release by SNP compound. Besides, we show that the cell treatment with 0.1nM of SNP decreased the fluorescence intensity to DHE in cells stimulated with angiotensin II. These results indicate that SNP decreases the concentration of O2•- in HUVEC cells. ConclusionsThe SNP at a concentration that does not release NO inside the cells is able to attenuate endothelial dysfunction. Drugs and chemicalsAcetylcholine (Ach) (PubChem CID:6060); angiotensin II human (Ang II) (PubChem CID: 16211177); diethylenetriamine/nitric oxide (DETA-NO) (PubChem CID 4518); dihydroethidium (DHE) (PubChem CID: 128682); phenylephrine (Phe) (PubChem CID: 5284443); sodium nitroprusside (SNP) (PubChem CID: 11963579); Thiazolyl Blue Tetrazolium Bromide (MTT) (PubChem CID: 64965); 4,5-diaminofluorescein diacetate (DAF-2DA); 4-hidroxy-Tempo (Tempol) (PubChem CID: 137994), were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Isolation and Characterisation of a Proanthocyanidin With Antioxidative, Antibacterial and Anti-Cancer Properties from Fern Blechnum orientale.

Background:Blechnum orientale Linn. (Blechnaceae), a fern, is traditionally used in the treatment of various ailments, such as skin diseases, stomach pain, urinary bladder complaints, and also as a female contraceptive. Previously, we reported a strong radical scavenging activity, antibacterial activity and cytotoxicity against HT29 colon cancer cells by aqueous extract of B. orientale.Objective:In this study, we attempted to isolate and identify the active compound from the aqueous extract of B. orientale.Materials and Methods:Aqueous extract of B. orientale was subjected to repeated MCI gel chromatography, Sephadex-LH-20, Chromatorex C18 and semi-preparative high performance liquid chromatography and was characterized using nuclear magnetic resonance and electrospray ionization mass-spectrometry spectroscopic methods. Antioxidant activity was determined using 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay. Antibacterial assays were conducted using disc diffusion whereas the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined using the broth microdilution assay. Cytotoxicity was assessed using thiazolylblue tetrazoliumbromide.Results:A polymeric proanthocyanidin consisting of 2-12 epicatechin extension units and epigallocathecin terminal units linked at C4-C8 was elucidated. Bioactivity studies showed strong radical scavenging activity (IC50 = 5.6 ± 0.1 µg/mL), antibacterial activity (MIC = 31.3-62.5 µg/mL) against five gram-positive bacteria and selective cytotoxicity against HT29 colon cancer cells (IC50 = 7.0 ± 0.3 µg/mL).Conclusion:According to our results, the proanthocyanidin of B. orientale demonstrated its potential as a natural source of antioxidant with antibacterial and anti-cancer properties.SUMMARY A bioactive proanthocyanidin was isolated from the aqueous extract of medicinal fern Blechnum orientale Linn and the structure was elucidated using NMR and ESI-MS spectral studies.The proanthocyanidin compound possessed strong radical scavenging activity (IC50 5.6 ± 0.1 µg/mL)The proanthocyaniding compound showed bactericidal activity against five gram-positive bacteria inclusive of MRSA (minimum inhibitory concentration, MIC and minimum bactericidal concentration, MBC 31.3-62.5 µg/mL).The proanthocyanidin compound is strongly cytotoxic towards cancer cells HT29 (IC50 7.0 ± 0.3 µg/mL), HepG2 (IC50 16 µg/mL) and HCT116 (IC50 20 µg/mL) while weakly cytotoxic towards the non-malignant Chang cells (IC50 48 µg/mL). Abbreviation used: CC: Column chromatography, DP: degree of polymerization, DPPH: 2,2-diphenyl-1-picrylhydrazyl, ESI-MS: electronsprayionisation mass-spectrometry, MBC: Minimum bactericidal concentration, MIC: Minimum inhibitory concentration, MTT: Thiazolyl Blue Tetrazolium Bromide, MRSA: methicillin-resistant Staphylococcus aureus, NMR: nuclear magnetic resonance, TLC: thin layer chromatography, PD: prodelphinidin

Phytochemical composition, anti-inflammatory activity and cytotoxic effects of essential oils from three Pinus spp.

Context: Inflammation and cell differentiation lead to a number of severe diseases. In the recent years, various studies focused on the anti-inflammatory and anticancer activity of essential oils (EOs) of numerous plants, including different Pinus species. Objective: The phytochemical composition, anti-inflammatory and cytotoxic activity of EOs from needles and twigs of Pinus heldreichii Christ (Pinaceae) and P. peuce Griseb., and from needles, twigs and cones of P. mugo Turra were determined. Materials and methods: For separation and identification of the EOs, gas chromatography/flame ion detector (GC/FID) and GC/mass spectrometry were performed. The amount of secreted IL-6 in a lipopolysaccharide (LPS)-stimulated macrophage model was quantified (concentration of oils: 0.0001–0.2%, 3 h incubation). Cytotoxicity on the cancer cell lines HeLa, CaCo-2 and MCF-7 were determined using a MTT (Thiazolyl Blue Tetrazolium Bromide) assay (concentration of oils: 0.001–0.1%, 24 h incubation). Results: The most prominent members in the oils include: δ-3-carene, α-pinene and linalool-acetate (P. mugo); α-pinene, β-phellandrene and β-pinene (P. peuce); limonene, α-pinene and (E)-caryophyllene (P. heldreichii). EOs showed significant cytotoxic effects on cancer cell lines (IC50 0.007 to >0.1%), with a reduction in cell viability with up to 90% at a concentration of 0.1%, and anti-inflammatory activity (IC50 0.0008–0.02%) with a reduction of IL-6 secretion with up to 60% at a concentration of 0.01%. Discussion and conclusion: The EOs of needles and twigs from P. peuce and P. heldreichii as well as of needles, twigs and cones of P. mugo can be considered as promising agents for anticancer and anti-inflammatory drugs.

The effects of up-regulating the expression of microRNA-1224-5p on hepatocellular carcinoma tumor endothelial cells

Objective To investigate the effect of microRNA-1224-5p (miR-1224-5p) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). Methods Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000. TECs were divided into 3 groups: a control (CON) group consisted of TECs without transfection, a negative control (NC) group consisted of TECs transfected with negative control oligonucleotides and Green Fluorescent Protein (GFP), and a micro-up (MU) group consisted of TECs transfected with miR-1224-5p mimic and GFP. The expression of miR-1224-5p was quantified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay. Apoptosis and cell cycle were detected via flow cytometry. The migration and invasion of TECs were detected using Ttranswell assay. The ability of tube fromation of TECs was detected using tube formation assay. RESULTS Oligonucleotides were successfully transduced into TECs, and specifically up-regulated the expression of miR-1224-5p. Results The results of RT-qPCR showed that the expression of miR-1224-5p was significantly increased in the MU group (2-ΔΔCt=3.27±0.15) compared with the CON group (2-ΔΔCt=1.00) and the NC group (2-ΔΔCt=1.08±0.11) (P<0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MU group for the 4 time points compared with the CON group and the NC group (P<0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MU group (19.29±0.95)% compared with the CON group (8.73±0.64)% and the NC group (9.51±0.56)% (P<0.01). The results of cell cycle detection showed that percentage of G1 phase cells in the MU group (73.3±2.0)% was significantly higher than the CON group (50.7±1.3)% and the NC group (51.7±1.9)% and the percentage of S phase cells in the MU group (23.0±3.0)% was significantly decreased compared with the CON group (31.4±2.7)%, and the NC group (29.1±1.6)%. (P<0.05 or P<0.01). The ability of migration was significantly inhibited in the MU group (51.0±3.6) compared with the CON group (77.7±2.5), and the NC group (79.2±3.5) (P<0.01). And the ability of invasion of TECs was also significantly inhibited in the MU group (9.8±1.3) compared with the CON group (15.8±0.8) and the NC group (15.4±0.9) (P<0.01). The ability of tube formation of TECs in the MU group was inhibited absolutely, but TECs in the CON group and the NC group were not affected. Conclusion Up-regulation of miR-1224-5p could decrease proliferation, induce apoptosis, inhibit migration and invasion, and supress tube formation of TECs of human HCC. Key words: Hepatocellular carcinoma; Vascular endothelial cells of human hepatocellular carcinoma; MicroRNA-1224-5p

Assessment of the Antiproliferative Activity of a BET Bromodomain Inhibitor as Single Agent and in Combination in Non-Hodgkin Lymphoma Cell Lines.

To evaluate the antiproliferative activity of a novel BET Bromodomain inhibitor as single agent and in combination with the BTK inhibitor ibrutinib in non-Hodgkin lymphoma cell lines, we performed the MTT proliferation assay. This assay is based on the direct correlation between absorbance (measured colorimetrically at a wavelength of 570 nm) and cell proliferation. Thiazolyl Blue Tetrazolium Blue (MTT) is a yellowish solution that distinguishes between proliferating and dead cells since it is converted to water-insoluble MTT-formazan of dark blue color by mitochondrial dehydrogenases of living cells only.

Effect of microRNA-1 on hepatocellular carcinoma tumor endothelial cells.

To investigate the effect of microRNA-1 (miR-1) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). MiR-1 specific short hairpin RNA (shRNA) was synthesized and cloned into a recombinant lentiviral vector. TECs were then infected by the miRNA-1-shRNA recombinant lentivirus. TECs were divided into three groups: a control (CON) group consisting of normal TECs without lentiviral infection, a negative control (NC) group consisting of normal TECs infected with a negative control virus, and a micro-down (MD) group consisting of normal TECs infected with the miR-1-inhibition virus containing the target gene. Silencing of miR-1 expression was quantified via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay; the observations were continued for 5 d, and the optical density value at 490 nm was detected every day. Apoptosis was detected via flow cytometry using Annexin V-APC single staining. The migration and invasion of TECs were detected using transwell assays. Lentiviral miR-1 shRNA was successfully transduced into TECs, and specifically silenced the expression of miR-1. The results of qRT-PCR showed that the expression of miR-1 was significantly decreased in the MD group (2(-ΔΔCt) = 0.57 ± 0.14) compared with the CON group (2(-ΔΔCt) = 1) and the NC group (2(-ΔΔCt) = 1.05 ± 0.13) (P < 0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MD group in the 5 days compared with the CON and NC groups (P < 0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MD group (6.32% ± 0.33%) compared with the CON group (2.03% ± 0.30%) and the NC group (2.18% ± 0.15%) (P < 0.01). The ability of cell migration was significantly inhibited in the MD group (62.0 ± 5.48) compared with the CON group (99.8 ± 3.11) and the NC group (97.2 ± 3.70) (P < 0.01). The ability of invasion of TECs was also significantly inhibited in the MD group (29.8 ± 2.39) compared with the CON group (44.6 ± 3.36) and the NC group (44.4 ± 5.17) (P < 0.01). MiR-1 might be a potential tumor activator. Inhibiting its expression could decrease proliferation, induce apoptosis, and inhibit the migration and invasion of TECs of human HCC.

Effect of Bcl-2-siRNA on proliferation and apoptosis of pediatric acute B lymphoblastic leukemia (A-BLL) cells.

This study analyzed the effect of small interfering RNA specific for the Bcl-2 gene (siRNA Bcl-2) on the proliferation and chemotherapeutic sensitivity of pediatric A-BLL cells. Marrow samples were obtained from sixty newly-diagnosed A-BLL pediatric patients. The Bcl-2 mRNA expression in these samples was quantified by real time polymerase chain reaction. The Bcl-2 mRNA re-expression was analyzed by RNA interference using Bcl-2-siRNA. Cellular proliferation was detected using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay. The cell apoptosis was quantified by flow cytometry. The Bcl-2 mRNA expression was significantly higher in the drug-resistance group than in the chemotherapy sensitivity group prior to chemotherapy (P < 0.05). In addition, the Bcl-2 mRNA expression in the chemotherapy sensitivity group was significantly higher before chemotherapy than that after chemotherapy (P < 0.05). The Bcl-2 mRNA expression significantly decreased in the leukemic cells of the Bcl-2-siRNA transfection group. We observed statistically significant differences in the relative mRNA expression levels among the Bcl-2-siRNA transfection, blank control, liposome empty transfection, and unrelated sequence oligonucleotide groups (P < 0.05). The rate of apoptosis in pediatric A-BLL leukemic cells was observed to increase significantly after transfection with Bcl-2-siRNA compared to the control, liposome empty transfection, and unrelated sequence oligonucleotide groups (P < 0.05). Therefore, we concluded that Bcl-2-siRNA can successfully inhibit the multiplicative capacity of A-BLL leukemic cells and promote apoptosis.

Screening of Antitumor Bioactivity of Fungi Associated with Macro Algae and Sponge from Indrayanti Beach, Jogjakarta

This research was aimed to isolate marine derived-fungi which associated with macro algae and sponge from Indrayanti Beach, Jogjakarta and to screen the antitumor (T47D and HeLa) bioactivity of the fungi extracts. Three solid media of MEA (malt extract agar), GPY (glucose peptone yeast) and MFM (minimal fungi medium) were used as isolation medium. Each of the pure fungi isolates was then cultivated in 100 ml of liquid medium for 4 weeks at room temperature (27-28°C) in static conditions. The antitumor activity of the fungi extracts were tested against breast tumor cells (T47D) and cervical cancer cells (HeLa) using Thiazolyl Blue Tetrazolium Blue (MTT) assay method. A number of 21 isolates of fungi were isolated from 4 macro algae and 1 sponge samples. The identification of fungi isolate was conducted using combination of molecular approach (ITS1-5.8S-ITS4 DNA regions) and macro-micro morphological characteristics. Among those 21 marine fungi species isolated, MFGK-21 extract showed the best anti-servical tumor (HeLa) with an IC50 value of 240.1 µg/ml and MFGK-27 extract showed the best anti-breast tumor (T47D) with an IC50 value of 59.6 µg/ml. The MFGK-21 fungi isolate was identified as Penicillium steckii, while the MFGK-27 fungi isolate was identified as Aspergillus sydowii.

The relevance of testing the efficacy of anti-angiogenesis treatments on cells derived from primary tumors: a new method for the personalized treatment of renal cell carcinoma.

Despite the numerous available drugs, the most appropriate treatments for patients affected by common or rare renal cell carcinomas (RCC), like those associated with the Xp11.2 translocation/transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) gene fusion (TFE3 RCC), are not clearly defined. We aimed to make a parallel between the sensitivity to targeted therapies on living patients and on cells derived from the initial tumor. Three patients diagnosed with a metastatic RCC (one clear cell RCC [ccRCC], two TFE3 RCC) were treated with anti-angiogenesis drugs. The concentrations of the different drugs giving 50% inhibition of cell proliferation (IC50) were determined with the Thiazolyl Blue Tetrazolium Bromide (MTT) assay on cells from the primary tumors and a reference sensitive RCC cell line (786-O). We considered the cells to be sensitive if the IC50 was lower or equal to that in 786-O cells, and insensitive if the IC50 was higher to that in 786-O cells (IC 50 of 6±1 µM for sunitinib, 10±1 µM for everolimus and 6±1 µM for sorafenib). Based on this standard, the response in patients and in cells was equivalent. The efficacy of anti-angiogenesis therapies was also tested in cells obtained from five patients with non-metastatic ccRCC, and untreated as recommended by clinical practice in order to determine the best treatment in case of progression toward a metastatic grade. In vitro experiments may represent a method for evaluating the best first-line treatment for personalized management of ccRCC during the period following surgery.

A Comparative In Vitro Study of the Effects of Irsha and Chlorhexidine Mouthwashes and Acyclovir on HSV-1

Objective: Being able to cause disease in human, herpes simplex viruses (HSVs) clinically demonstrate themselves as intra-oral, extra-oral or recurrent lesions. The existing acyclovir has the selective anti-herpetic drug to control HSV infections. Due to emerged resistance to this drug and limitations of using it in especial situations, there is a need for alternative treatments such as available mouthwashes. This study aimed to compare two mouthwashes (Irsha and Chlorhexidine) with Acyclovir on HSV-1 in vitro.Methods: In this experimental research, we used MTT (Thiazolyl Blue Tetrazolium Bromide) colorimetric test to determine the cytotoxicity level of three solutions consecutively, antiseptic and nonalcoholic Irsha mouth wash (blue-colored), chlorhexidine 0.2% mouthwash, and acyclovir and absorbed wavelengths were recorded by Eliza Reader. After infecting the cells with different dilutions of HSV-1 in different concentrations of Irsha and chlorhexidine mouthwashes, we analyzed their antiherpetic effects on Vero cells. By using suitable statistical tests in version 15 of SPSS the results were then analyzed.Results: The results showed that in the concentrations of 0.38% for Irsha and 0.003% for chlorhexidine these mouthwashes kill 50% of Vero cells (CC50). After determining CC50, we detected the antiviral effects of Irsha and chlorhexidine mouthwashes and acyclovir solutions. We observed a significant difference between 0.5% concentration of Irsha mouthwash and other concentrations of it. The least logarithm of virus titration was observed in 0.002% concentration of/ chlorhexidine mouthwash. Both tested acyclovir concentrations (1250 µgr Mililiter and 2500µg/mL Mililiter µgr/ ) had a similar effect on decreasing virus titreConclusion: According to our results, anti-herpetic effect of Irsha is less than chlorhexidine and anti-herpetic effect of Acyclovir and Chlorhexidine is stronger than Irsha.

Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10-4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30-60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4-48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection.

Mixed ligand–silver(i) complexes with anti-inflammatory agents which can bind to lipoxygenase and calf-thymus DNA, modulating their function and inducing apoptosis

A new mixed ligand-silver(I) complex of formula [Ag(tpp)(2)(p-Hbza)] (1) (p-HbzaH = 4-hydroxybenzoic acid and tpp = triphenylphosphine) has been synthesized and characterized by elemental analysis, mp, vibrational spectroscopy (mid- and far-FT-IR), (1)H-NMR, UV-vis, ESI-MS spectroscopic techniques and X-ray crystallography. Complex 1 and the already known mixed ligand-silver(I) complexes of formulae [Ag(tpp)(2)(salH)] (2) (salH(2) = salicylic acid or 2-hydroxy-benzoic acid) and {[Ag(tpp)(3)(asp)](dmf)} (3) (aspH = o-acetylsalicylic acid) were used for the clarification of the cytostatic activity mechanism. Thus, 1-3 were tested for their in vitro cytotoxic activity against leiomyosarcoma (LMS) and human breast adenocarcinoma (MCF-7) cells with trypan blue and Thiazolyl Blue Tetrazolium Bromide (MTT) assays. For both cell lines, complexes 1-3 were found to be more active than cisplatin. Due to the morphology of the LMS cells after incubation with 1-3, the type of cell death was evaluated by flow cytometry assay and DNA fragmentation. The results show that LMS cells undergo programmed cell death (apoptosis). DNA binding tests indicate the ability of complexes 1-3 to modify the activity of the cells. The binding constants of 1-3 towards calf-thymus DNA (CT-DNA) ((27.7 ± 7.9) × 10(4) (1), (13.3 ± 6.5) × 10(4) (2) and (11 ± 2.8) × 10(4) (3) M(-1)) indicate strong interaction. Moreover, the influence of complexes 1-3 on the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically studied. Finally, docking studies on DNA binding interactions were performed.

Effect of lithium chloride and antineoplastic drugs on survival and cell cycle of androgen-dependent prostate cancer LNCap cells

Objective:Glycogen synthase kinase-3β (GSK-3β) has been reported to be required for androgen receptor (AR) activity. This study sought to determine the usefulness of lithium chloride (LiCl) as a highly selective inhibitor of GSK-3β to increase the sensitivity of LNCap cells to doxorubicin (Dox), etoposide (Eto), and vinblastine (Vin) drugs.Materials and Methods:Thiazolyl Blue Tetrazolium Blue (MTT) assay was used to determine the cytotoxic effect to LiCl alone or in combination with low dose and IC50 doses of drugs. Subsequently, cell cycle analysis was performed by using flow cytometry.Results:LiCl showed cytotoxic effect in a dose- and time-dependent manner (P<0.001). Both Dox (100 or 280 nM) and Vin IC50 (5 nM) doses caused G2/M-phase arrest (P<0.001) compared with control. However, low dose (10 μM) or IC50 (70 μM) Eto doses showed G2/M or S-phase arrests, respectively (P<0.001). Combination of low dose or IC50 dose of Eto with LiCl showed increased apoptosis as revealed by high percent of cells in SubG1 (P<0.05, P<0.01, respectively). Moreover, Eto (10 μM) led to decreased percent of cells in G2/M phase when combined with LiCl (P<0.05).Conclusion:This study showed that LiCl increases apoptosis of (LNCap) Lymph Node Carcinoma of the Prostate cells in the presence of Eto, which is S- and G2-phase-specific drug.

Exploration of novel in vitro assays to study drugs against Trichuris spp.

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50μg/ml) for 72h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.

Synthesis, structural characterization and biological studies of novel mixed ligand Ag(I) complexes with triphenylphosphine and aspirin or salicylic acid

Two new mixed ligand silver(I) complexes of formulae {[Ag(tpp)3(asp)](dmf)} (1) (aspH=o-acetylsalicylic acid and tpp=triphenylphosphine) and [Ag(tpp)2(o-Hbza)] (2) (o-HbzaH=o-hydroxy-benzoic acid) were synthesized and characterized by elemental analyses, spectroscopic techniques and X-ray crystallography at ambient conditions. Three phosphorus and one carboxylic oxygen atoms from a de-protonated aspirin ligand in complex 1 and two phosphorus and two carboxylic oxygen atoms from a chelating o-Hbza anion in complex 2 form a tetrahedral geometry around Ag(I) ions in both complexes.Complexes 1 and 2 and the silver(I) nitrate, tpp, aspNa and o-HbzaH were tested for their in vitro cytotoxic activity against leiomyosarcoma cells (LMS), human breast adenocarcinoma cells (MCF-7) and normal human fetal lung fibroblasts (MRC-5) cells with Thiazolyl Blue Tetrazolium Bromide (MTT) assay. For both cell lines 1 and 2 were found to be more active than cisplatin. Additionally, 1 and 2 exhibit lower activity on cell growth proliferation of MRC-5 cells. The type of LMS cell death caused by 1 and 2 were evaluated in vitro by use of flow cytometry assay. The results show that at concentrations of 1.5 and 1.9μΜ of complex 1, 44.1% and 69.4%, respectively of LMS cells undergo programmed cell death (apoptosis). When LMS cells were treated with 1.6 and 2.3μM of 2, LMS cells death was by 29.6% and 81.3%, respectively apoptotic. Finally, the influence of the complexes 1 and 2, upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied. The binding of 1 and 2 towards LOX was also investigated by Saturation Transfer Difference (STD) 1H NMR experiments.

Synthesis, structural characterization and biological studies of the triphenyltin(IV) complex with 2-thiobarbituric acid

The reaction between 2-thiobarbituric acid (H 2TBA), which was treated with an equimolar amount of potassium hydroxide, in a water with triphenytin chloride in methanol, results in the formation of the {[Ph 3Sn(O-HTBA)]} n ( 1) complex. Crystals of the hydrated 1 with formula {[Ph 3Sn(O-HTBA)]·0.7(H 2O)} n were growth from methanol/acetonitrile solution, of the white precipitation, filtered off, from the reaction. The crystal structure of complex 1 has been determined by X-ray diffraction at 120 K. Complex 1 is polymeric. The geometry around the tin(IV) ions is trigonal bi-pyramidal with coordination to three C atoms from phenyl groups and one O atom from a de-protonated HTBA ligand. Complex 1 and the already known [(n-Bu) 3Sn(O-HTBA)·H 2O] ( 2) were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (cervical), OAW-42 (ovarian), MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (renal) and additionally, the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line MTSV17 with a Trypan Blue assay. Moreover complex 1 was evaluated for its in vitro cell growth proliferation activity against leiomyosarcoma cells (LMS), MCF-7 and MRC-5 cells with a Thiazolyl Blue Tetrazolium Bromide (MTT) assay. The type of cell death caused by complexes 1 and 2 was also evaluated by use of flow cytometry assay. The results showed that these compounds mediate a strong cytotoxic response to normal and cancer cell lines tested through apoptosis and induce cell cycle arrest in S phase of the cell cycle, suggesting DNA intercalation (direct or indirect) with the complexes. Finally, the influence of these complexes 1 and 2 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied.

Extraction equilibria and spectrophotometric determination of vanadium(V) with 4-nitrocatechol and the ion-pair reagent thiazolyl blue tetrazolium

The formation and liquid-liquid extraction of a yellow ternary complex of vanadium(V) with 4-nitrocatechol (NC) and the ion-pair reagent Thiazolyl Blue Tetrazolium ⨑ub;3-(4,5-dimethyl-2-thiazol)-2,5diphenyltetrazolium bromide, MTT⫂ub; with 1: 2: 3 stoichiometry (V: NC: MTT) was studied. The optimum extraction conditions (pH, concentration of the reagents, extraction time), spectrophotometric parameters of the extract and key constants (extraction constant, association constant, distribution constant) were determined. Beer’s law was obeyed for concentrations ranging from 0.12 to 1.2 µg/mL of vanadium(V) with a molar absorptivity of ɛ = 3.13 × 104 L/mol cm at λmax = 400 nm. The effect of diverse ions was studied and extraction-spectrophotometric procedures for determination of vanadium in catalysts and steels were proposed.

Structural properties, cytotoxicity, and anti-inflammatory activity of silver(I) complexes with tris(p-tolyl)phosphine and 5-chloro-2-mercaptobenzothiazole.

The synthesis and characterization of the silver(I) chloride complex of formula {[AgCI(CMBZT)(TPTP)2] · (MeOH)} (1) (CMBZT = 5-chloro-2-mercaptobenzothiazole, TPTP = tris(p-tolyl)phosphine) is described. Also the structure of the hydrate derivative {[AgCI(TPTP)3] · (0.5 · H2O)} (2) of the corresponding known anhydrous silver complex (Zartilas et al., 2009), and the polymorph 3 of the known [AgI(TPTP)3] complex (Zartilas et al., 2009) were determined and compared with the known ones. In addition, the structure of the known one silver(I) cluster {[AgI(TPTP)]4} (4) (Meijboom et al., 2009) was re-determined at 120(2) K and possible Ag-Ag interactions were analyzed. The compounds 1–4 were characterized by X-ray crystallography at r.t (1) and 120 K (2–4). All these complexes and {[(Et3NH)+]2 · [Ag6(μ 3-Hmna)4(μ 3-mna)2]2− · (DMSO)2 · (H2O)} (5) (Hmna = 2-mercaptonicotinic acid) were evaluated for cytotoxic and anti-inflammatory activity. The in vitro testing of cytotoxic activity of 1–5 against leiomyosarcoma cancer cells (LMS), were evaluated with Trypan Blue and Thiazolyl Blue Tetrazolium Bromide or 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assays. The flow cytometry assay for complex 1 and showed that at 15 μM of 1, 62.38% of LMS cells undergo apoptosis, while 7% of LMS cells undergo cell necrosis. The antitumor activity of 3 is comparable with that of its reported polymorph (Zartilas et al., 2009). The anti-inflammatory, activity of complexes 1–3 and 5 was also studied. The activity towards cell viability was 2 > 3 > 5 > 1 > 4, while the order of the inhibitory activity in cell growth proliferation follows the order, 2 > 3 > 1 > 4 > 5. The anti-inflammatory activity on the other hand is 1 > 2 > 5 > ⋯ >3.

Experimental study on in vitro tumor cell killing by focused bi-frequency ultrasound activated hematoporphyrin derivatives

As ultrasound has become an important therapeutic technique on tumor, in order to find the cell-killing effect of focused bi-frequency ultrasound activated hematoporphyrin derivatives, experimental study was processed in vitro. Tumor cells K562 and SW-480 were killed by focused bi-frequency ultrasound activated hematoporphyrin (Hp) derivatives under optimized ultrasonic parameters and the cell-killing effect was tested by MTT (Thiazolyl blue: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) technique. The lower frequency (0.6 MHz) was better than the higher (1.1 MHz) in activating hematoporphyrin derivatives; the bi-frequency was better than mono-frequency, and the cell-killing efficiency was 2-3 times of sum of the two kinds of ultrasounds which constitute bi-frequency. Incubation for the 16 h was better than 4 h after the cells exposed to the ultrasound. The method of killing tumor cells by focused bi-frequency ultrasound activated hematoporphyrin derivatives is presented and evaluated in this paper.