Tetrazolium Dye Research Articles

Sterols possess immunomodulatory property: Activity guided isolation and in vitro screening of phytoconstituents of Pongamia glabra and Ficus glomerata

ObjectiveTo isolate the phytoconstituents from the methanolic extracts of the stem bark of Pongamia glabra and Ficus glomerata, characterize spectroscopically and screen for in vitro immunomodulatory activity on human neurophils. MethodsA flavonoid (PGF) and an alkaloidal compound (PGA) from the extract of P. glabra and a steroidal compound (FGS) and tannin fraction (FGT) from the extract of F. glomerata were isolated using column chromatography technique and were subjected for the spectroscopic (FT-IR, 1HNMR and LC-MS) and TLC studies to identify the compounds. The isolated compounds were screened for in vitro immunomodulatory activity on human neutrophils using nitroblue tetrazolium (NBT) dye test, phagocytosis of Candida albicans and neutrophil locomotion and chemotaxis assay at the concentration range of 100, 50, 25, 12.50 and 5.00 µg/mL. ResultsFrom the spectroscopic and TLC studies data, the isolated compounds were identified as glabrin (PGA), karanjin (PGF), β-sitosterol (FGS), and tannin fraction (FGT). The isolated compounds PGA, PGF, FGS, and FGT exhibited significant (P < 0.05) in vitro immunomodulatory activity in all the parameters studied. ConclusionThe steroidal compound, i.e. FGS was found to be more immunopotent than all constituents alkaloid, flavonoid and tannins. Hence, these constituents could be attributed to the immunomodulatory property of the plants.

The Effect of Cobalt (II) Chloride in the Viability Percentage and the Induced Hypoxia Inducible Factor -1α of Human Adipose Mesenchymal Stem Cells (HAMSCs): An In Vitro Study

Introduction: The preconditioning of HAMSCs using Cobalt (II) Chloride (CoCl2) as a hypoxia mimicking agent (HMA) may provide the adaptive state of HAMSCs. Objective: To examine the effect of CoCl2 in the viability percentage and the induced Hypoxia Inducible Factor-1α (HIF-1α) expression of HAMSCs in vitro. Methods: HAMSCs were thawed, cultured, and then sub-cultured from the laboratory cell stock. The Immunocytochemistry (ICC) methods with Fluorescein isothiocyanate (FITC) antibody labelling kit was used to validate the HAMSCs employing its Cluster of Differentiation (CD) expression such as CD73(+), CD105(+) and CD45(-). HAMSCs were sub-cultured and then co-cultivated with or without CoCl2 in the different concentration (200mol, 150mol, 100mol, 75mol, 50mol, 25mol) for 24 hours. The cytotoxicity test was conducted by utilizing tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The formazan crystal was then observed, and the cell viability percentage was calculated. In examining CoCl2 induced hypoxia in HAMSCs, the expression of HIF-1α applied ICC-FITC in vitro. Analysis of Variance (ANOVA) was performed to analyze the obtained data statistically (p0.05). CoCl2 successfully induces the hypoxia confirmed by HIF-1α positive expression of HAMSCs after 24 hours incubation in vitro. Conclusion: CoCl2 does not affect the viability percentage of HAMSCs and it can be used as a biocompatible HMA in HAMSCs in vitro.

DNA photocleavage and melanoma cells cytotoxicity induced by a meso-tetra-ruthenated porphyrin under visible light irradiation

Porphyrins are used as photosensitizing agents in photodynamic therapy (PDT) for several pathologies. Here we demonstrate the DNA photocleavage and cytotoxicity properties of a free-base meso-tetra-ruthenated porphyrin (H2RuTPyP) in purified DNA samples and in a melanoma cell line, respectively. Cytotoxicity of H2RuTPyP was investigated by the tetrazolium dye (MTT) colorimetric assay and its genotoxic potential by direct plasmid DNA photocleavage after incubation with specific DNA repair enzymes. H2RuTPyP porphyrin efficiently induced DNA damage at the lower concentration of 5.0 μM, whereas it induced complete DNA degradation at 15 μM. The addition of different scavengers for reactive oxygen species (ROS) during the visible light exposures did not decrease the DNA damage formation, suggesting a hydrolytic mechanism for the induction of DNA breaks. Also, H2RuTPyP exhibited a much higher cytotoxicity in melanoma cells in comparison to a keratinocyte cell line. The detection of intracellular reactive oxygen species (ROS) produced by H2RuTPyP through the DCF-DA assay also suggests that ROS have a minor role in the induction of cytotoxicity. Therefore, H2RuTPyP seems to be a very effective photosensitizer, representing a promising alternative for the development of new skin cancer treatments using PDT process.

2-Vinyl-4H-1,3-Dithiin, a Bioavailable Compound from Garlic, Inhibits Vascular Smooth Muscle Cells Proliferation and Migration by Reducing Oxidative Stress.

Organosulfur compounds (OSCs) of garlic have various health benefits including anti-hypertensive effect. It has been described that volatile OSCs uptake occurs into a moderate extent. Among the bioavailable OSCs present in garlic, 2-vinyl-4H-1,3-dithiin (2VD) is a main component in garlic macerated oil and stir-fried garlic but has been poorly investigated compared with others OSCs, such allyl mercaptan (AM) and S-allyl cysteine (SAC). The aim of this study was to evaluate the effects of 2VD on vascular smooth muscle cells (VSMCs) isolated from spontaneous hypertensive rats (SHR) and compare them with those produced by AM and SAC. Cell viability and proliferation were measured using tetrazolium dye MTT assay and flow cytometry. Cell migration was evaluated by scrape-wound migration assay. OSCs anti-oxidative capacity was determined by measuring reactive oxygen species (ROS) generation, and total antioxidant status (TAS). Non-toxic plasmatic concentrations (10μgL-1) of 2VD and AM inhibited VSMCs proliferation stimulated with 5% fetal bovine serum and impaired cell migration. In further flow cytometry analysis 2VD treatment resulted in a partial cell cycle arrest at G2 phase. The OSCs tested were able to prevent ROS increase after angiotensin II stimulation and surprisingly 2VD induced higher total antioxidant status compared with AM and SAC. Our results showed that 2VD produces equivalent or superior beneficial effects on VSMCs to those reported for other bioavailable compounds of garlic. This preliminary evidence suggests that 2VD intake could also exert important protective effects against arterial remodeling in hypertension.

Phytochemical Screening and Effect of Cymodocea serrulata on HepG2-Human Hepatocellular Carcinoma Cell Line

Cymodocea serrulata, a seagrass commonly known as karumbupassi has been used as a food and also as a medicine by coastal region people and by fishermen while traveling in the sea. It is used as a tranquilizer for babies as it has a soothing quality, it helps during pregnancy and against cough and malaria. C.serrulata is seen abundant in South Indian coastal region. Although there is a report on the antibacterial, antioxidant, and anti-inflammatory property of C.serrulata, there are no evident details on phyto-compounds present in it. Invitro antiproliferative test was performed by MTT (methylthiozoltetrazolium) assay method against HePG2 celline of liver cancer cells. MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by liver cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570nm.

Anti-amoebic Activity of Leaf Extracts and Aporphine Alkaloids Obtained from Annona purpurea.

Annona purpurea has been traditionally used by indigenous and socioeconomically disadvantaged people to treat infectious and parasitic diseases, including amoebiasis. The goal of this study was to assess the effect of a crude methanolic extract, an alkaloid extract, and aporphine alkaloids from leaves of A.purpurea on the viability of Entamoeba histolytica trophozoite cultures and to identify the mechanism of action. Different concentrations of the extracts and alkaloids purpureine (1: ), 3-hydroxyglaucine (2: ), norpurpureine (3: ) glaziovine (4: ), and oxopurpureine (5: ) were added to the cultures, and dead parasites were counted after 24 h using a tetrazolium dye reduction assay and analyzed by flow cytometry. The crude extract did not affect the viability of amoebae, but the alkaloid extract and the derived alkaloid glaziovine (4: ) had important anti-amoebic activity with an IC50 of 33.5 µM compared to that shown by metronidazole (6.8 µM). The treatments induced significant morphological changes in the trophozoites, and most parasites killed by the alkaloid extract were positive for Annexin V, suggesting that apoptosis was the main mechanism of action. In contrast, glaziovine (4: ) induced less apoptosis with more amoebic lysis. This study supports the idea that aporphine alkaloids from A.purpurea, mainly (+)-(R)-glaziovine (4: ), could contribute to the development of new formulations for the treatment of amoebiasis. In addition, X-ray diffraction structural analysis and complete 1H and 13C NMR assignments of (+)-(R)-glaziovine (4: ) were performed and reported for the first time.

Abstract P5-05-14: Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression

Abstract Introduction Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a new paradigm in the treatment of hormone receptor (HR) positive and HER-2 negative metastatic breast cancer (MBC). However, despite their evident clinical benefit, the cellular and molecular mechanisms underlying treatment efficacy and drug failure are still largely unknown. CDK4/6 inhibitors block the cyclin D1-CDK4/6 interaction, leading to inhibition of cell cycle progression through the G1/S phase and inhibiting cell proliferation. SAMHD1 is a dNTPase that maintains the intracellular dNTP pool at levels adequate for DNA replication and repair. SAMHD1 function is regulated by phosphorylation, a process controlled by CDKs. Moreover, SAMHD1 has also been shown to modify the efficacy of different nucleotide analogues. Here, we aim to evaluate the capacity of selective CDK4/6 inhibitors to modify the efficacy of different antimetabolites currently used for cancer treatment. Material and methods: Cytotoxic activity of 5-fluorouracil, its prodrug capecitabine and the anti-folate pemetrexed was evaluated alone or in combination with the CDK4/6 inhibitor palbociclib in breast cancer cell lines (T47D and MDA-MB-468) and primary cells expressing or not SAMHD1 through HIV-2 Vpx mediated degradation. Cytotoxicity was measured either by colorimetric tetrazolium dye MTT method or flow cytometry. Drug combinations were evaluated by calculating the combination index (CI) through the ixobologram equation, considering a synergic drug combination a CI &amp;lt;1. Intracellular dNTP content was determined using a polymerase- based method. Expression of relevant genes was measured by qPCR mRNA detection. SAMHD1 expression and phosphorylation were measured by Western blot. Results Analysis of cell cycle profile and protein expression of drug-treated cells confirmed that CDK4/6 control SAMHD1 phosphorylation and decrease intracellular dNTPs. CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib blocked SAMHD1 phosphorylation, whereas SAMHD1 protein expression was not affected. In addition, we observed a concomitant dephosphorylation and decreased expression of Rb, the natural substrate of CDK6, and a known marker of CDK4/6 inhibitor activity. In combination, palbociclib enhanced the efficacy of pemetrexed or 5-fluorouracil up to 6-fold, indicating strong synergy (CI=0,0049-0,404). Interestingly, the synergistic effect was lost in cells not expressing SAMHD1. Palbociclib did not change antimetabolite efficacy in the breast cancer MDA-MB-468 cell line. However, Rb and pRb were not detected in MDA-MB-468 cells by mRNA qPCR or Western blot. Conclusion CDK4/6 inhibitors induce the activation of SAMHD1. Taken together, our results suggest that CDK4/6 inhibitor-mediated enhancement of antimetabolite activity is dependent on the regulation of SAMHD1 function. CDK4/6 inhibitors restrict cells in G1 but also prevent SAMHD1 phosphorylation that sensitizes tumoral cells to antimetabolite drugs. Thus, pharmacological modulation of SAMHD1 activity has the potential to improve the efficacy of cancer therapies and paves the way to the identification of malignancies that may be treated with new drug combinations. Combinations with selective CDK4/6 inhibitors plus pemetrexed or capecitabine could be effective in the treatment of cancer after first line drug-failure. SAMHD1 could be a useful biomarker to predict treatment response. Citation Format: Eudald Felip, Roger Badia, Mireia Margelí, Marc Castellví, Vanesa Quiroga, Iris Teruel, Beatriz Cirauqui, Margarita Romeo, Ifeanyi Jude Ezeonwumelu, Eva Riveira-Muñoz, José Esté, Ester Ballana. Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-14.

MiR-22-3p Regulates Amyloid β Deposit in Mice Model of Alzheimer's Disease by Targeting Mitogen-activated Protein Kinase 14.

Morris water maze test was performed to test the spatial memory. Quantitative polymerase chain reaction (qPCR) was used to assess the expression level of miR-22-3p. The enzymelinked immunosorbent assay (ELISA) was used to assess the levels of Aβ40 and Aβ42. Immunoblotting analysis was performed to detect the protein expression levels of amyloid precursor protein (APP), mitogen-activated protein kinase 14 (MAPK14) and beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1). Luciferase assay was used to identify the interaction between miR- 22-3p and MAPK14. The tetrazolium dye (MTT) colorimetric assay was used to test the influence of miR-22-3p overexpression on cell viability. Flow cytometry analysis was performed to evaluate the effect of miR-22-3p overexpression on cell apoptosis. Morris water maze test showed that mice model of AD had impaired spatial memory, which was able to be ameliorated by miR-22-3p overexpression. Immunoblotting analysis revealed that the protein expression levels of APP, MAPK14 and BACE1 were enhanced in AD model, which could be prevented by miR-22-3p overexpression. ELISA showed that Aβ40 and Aβ42 levels were dramatically increased in AD model, which were inhibited by miR-22-3p overexpression. Luciferase assay and immunoblotting analysis indicated that miR-22-3p targeted and regulated MAPK14 expression. MiR-22-3p overexpression reduced Aβ deposit and alleviated AD symptoms by targeting and regulating MAPK14 expression, which ameliorated AD symptoms.

Ginsenoside Re Mitigates 6-Hydroxydopamine-Induced Oxidative Stress through Upregulation of GPX4.

Ginsenosides are active components found abundantly in ginseng which has been used as a medicinal herb to modify disease status for thousands of years. However, the pharmacological activity of ginsenoside Re in the neuronal system remains to be elucidated. Neuroprotective activity of ginsenoside Re was investigated in SH-SY5Y cells exposed to 6-hydroxydopamine (6-OHDA) to induce cellular injury. Ginsenoside Re significantly inhibited 6-OHDA-triggered cellular damage as judged by analysis of tetrazolium dye reduction and lactose dehydrogenase release. In addition, ginsenoside Re induced the expression of the antioxidant protein glutathione peroxidase 4 (GPX4) but not catalase, glutathione peroxidase 1, glutathione reductase, or superoxide dismutase-1. Furthermore, upregulation of GPX4 by ginsenoside Re was mediated by phosphoinositide 3-kinase and extracellular signal-regulated kinase but not by p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Ginsenoside Re also suppressed 6-OHDA-triggered cellular accumulation of reactive oxygen species and peroxidation of membrane lipids. The GPX4 inhibitor (1S,3R)-RSL3 reversed ginsenoside Re-mediated inhibition of cellular damage in SH-SY5Y cells exposed to 6-OHDA, indicating that the neuronal activity of ginsenoside Re is due to upregulation of GPX4. These findings suggest that ginsenoside Re-dependent upregulation of GPX4 reduces oxidative stress and thereby alleviates 6-OHDA-induced neuronal damage.

Co-Administration of Curcumin and Bromocriptine Nano-liposomes for Induction of Apoptosis in Lung Cancer Cells

Background:In recent years, nanotechnology with modern advances in the macromolecular design of nano-carriers has proved to be helpful in the development of drugs delivery systems. This research represents a novel co-administration of nano-vehicles, known as liposomes. Liposomes efficiently encapsulate curcumin and BR in a polymer structure, which results in enhanced aqueous solubility of the mentioned hydrophobic agents and higher bioavailability of the drugs. Methods:Preparation of curcumin and BR liposomes were carried out by the thin film method, and the amounts of purified drug and its release were analyzed. After dose determination, the human lung cancer cells (QU-DB) were exposed to BR and curcumin liposomes for 12, 24, and 48 h. Then the viability and apoptosis assays were carried out by using MTT and flow cytometry technique, respectively. Results:In this research, in vitro anti-cancer effects of former nano-formulations on lung cancer cells was confirmed, and no cytotoxicity effects of these nano-preparations were observed in the normal cells (HFLF-PI5). Conclusion:Our findings suggest the nano-liposomal drugs as effective anti-cancer agents; however, additional clinical examinations are required.

Mining Xylose Isomerase Producing Microbes

An aerobic, rod shaped, mesophilic, gram positive xylitol producing bacterial isolates were made from different enriched substrates. Xylose isomerase is an enzyme that catalyzes the interconversion of D-xylose and D-xylulose. The microbes producing xylulose from xylose via xylose isomerase enzyme were screened for the xylose isomerase activity based on 2, 3, 5-Triphenyl tetrazolium dye retention capacity. Among the fourteen isolates screened, only six isolates showed the positive results by retaining the dye of which XLY3 was the best. Phylogenetic and sequence analysis of 16SrRNA gene showed 98 % homology to Bacillus endophyticus

Metabolic Adaptation of Airway Smooth Muscle Cells to an SPHK2 Substrate Precedes Cytostasis.

Thickening of the airway smooth muscle is central to bronchial hyperreactivity. We have shown that the sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) can reverse preestablished airway hyperreactivity in a chronic asthma model. Because sphingosine analogs can be metabolized by SPHK2 (sphingosine kinase 2), we investigated whether this enzyme was required for AAL-R to perturb mechanisms sustaining airway smooth muscle cell proliferation. We found that AAL-R pretreatment reduced the capacity of live airway smooth muscle cells to use oxygen for oxidative phosphorylation and increased lactate dehydrogenase activity. We also determined that SPHK2 was upregulated in airway smooth muscle cells bearing the proliferation marker Ki67 relative to their Ki67-negative counterpart. Comparing different stromal cell subsets of the lung, we found that high SPHK2 concentrations were associated with the ability of AAL-R to inhibit metabolic activity assessed by conversion of the tetrazolium dye MTT. Knockdown or pharmacological inhibition of SPHK2 reversed the effect of AAL-R on MTT conversion, indicating the essential role for this kinase in the metabolic perturbations induced by sphingosine analogs. Our results support the hypothesis that increased SPHK2 levels in proliferating airway smooth muscle cells could be exploited to counteract airway smooth muscle thickening with synthetic substrates.

Naphthalene substituted benzo[c]coumarins: Synthesis, characterization and evaluation of antibacterial activity and cytotoxicity

AbstractNovel congeners of naphthalene substituted benzo[c]coumarins (2a-f) were synthesized by reaction of various 3-coumarinoyl methyl pyridinium bromide salts (1a-d) with a selected set of acetyl naphthalene in the presence of sodium acetate in refluxing glacial acetic acid. Structures of the synthesized compounds were confirmed by elemental analysis and by various spectroscopic techniques such as 1H-NMR, 13C-NMR, DEPT, and MS spectral data. Synthesised compounds were screened for antibacterial activity and cytotoxicity against different human cancer cell lines including cervix cancer (HeLa), breast cancer (MCF-7) and lung cancer (A549) using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Although with varying degrees, a significant growth inhibitory and cytotoxic effects were observed on all three cancer cell lines. Compounds 2b and 2e showed significant growth inhibitory and cytotoxicity against aforementioned cancer cell lines.

Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro.

BackgroundSnake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells.MethodsThe crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry.ResultsMTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Antibacterial Activity of Bioflavonoid from Fruit Pulps of Acacia nilotica Willd

The emergence of multi-drug resistance in bacteria has led to call for research and development of new leads as antibiotics from medicinal plants. Acacia nilotica (Linn) is a plant of multipurpose medicinal uses, three bioactive flavonoids (methyl gallate, gallic acid and catechin) were isolated from its fruit pulps through a bioassay guided fractionation technique and characterized based on High Performance Liquid Chromatography, Liquid Chromatography-Mass Spectra and Nuclear Magnetic Resonance spectra. Antibacterial activity of these compounds was determined by microplate tetrazolium dye assay of broth microdilution technique against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and clinical isolates of Salmonella typhi, Klebsiella pneumonia, Candida albicans and Bacillus subtilis. Catechin, methyl gallate and gallic acid at 19.5, 39 and 39 µg/ml respectively caused a significant bio-reduction in cells of test organisms. Time kill kinetic study of the extract shows that there was percentage of growth reduction in test organisms at 2, 4, 6, 8 and 12 hrs of contact. The extent and rate of killing of the organism by the extract at 2 x MIC followed the same trend as rate of killing was time dependent. Antibacterial effects of these compounds are within the breakpoint of control drug chloramphenicol and could serve as leads in new drug development.

Inhibition of Phosphatidylinositol 3-kinase (PI3K) Signaling Synergistically Potentiates Antitumor Efficacy of Paclitaxel and Overcomes Paclitaxel-Mediated Resistance in Cervical Cancer.

Acquired paclitaxel (PTX) resistance limits its effectiveness and results in advanced cancer progression. This review investigated whether the inhibition of phosphatidylinositol 3-kinase (PI3K) signaling overcomes paclitaxel resistance in cervical cancer. It was established paclitaxel-resistant cell lines (PTX-R ME180/PTX-R HeLa) and determined the combination index for paclitaxel and PI3K inhibitors (BYL-719/ LY294002) by tetrazolium dye assay. Flow cytometry was used to detect the cell cycle and apoptosis. Migration and invasion were explored by wound healing and transwell assays. Genes related to multiple pathways were assessed by a western blot. It was found that the PI3K pathway was significantly activated in paclitaxel-resistant HeLa and ME180 cells compared to parental cells. PTX + PI3K inhibitor combined therapy showed a synergistic effect by strengthening paclitaxel-induced S and G2M arrest in PTX-R cell sublines by the inactivation of cyclin A1, cyclin B1, cyclin E, and Cdc2 expression. Moreover, combination therapy significantly enhanced drug sensitivity and apoptosis through the activation of Bax, and cleavage of poly-(ADP-ribose) polymerase compared with paclitaxel alone. In addition, PI3K inhibition also suppressed tumor migration and invasion by targeting β-catenin and matrix metalloproteinase-2/9. The authors suggest that the combination of a PI3K inhibitor with paclitaxel may enhance antitumor activity through a cascade of PI3K signaling events.

Abstract 338: Inhibition of phosphatidylinositol 3-kinase signaling antagonizes paclitaxel-mediated resistance in cervical cancer

Abstract Paclitaxel is used as an effective treatment against advanced cervical cancer. However, acquired resistance limits its success and results in cancer progression. We investigated whether the inhibition of phosphatidylinositol 3-kinase (PI3K) signaling overcomes paclitaxel resistance in cervical cancer. Two human cervical cancer cell lines (Me180 and HeLa) were used. We established paclitaxel-resistant cell lines (PTX-R Me180/PTX-R HeLa) and confirmed inhibitory concentrations (IC)50 and cell death rates by tetrazolium dye (MTT) and apoptosis assays, respectively, after exposure to a combination of paclitaxel with PI3K inhibitors (LY294002 and BYL-719). Migration and invasive abilities were explored by wound healing and transwell assays, respectively. Western blots were performed to determine the mechanism underlying the antitumor effects of PI3K inhibitors.PTX-R Me180 and PTX-R HeLa cells were gradually established from parental cell lines by stepwise exposure to paclitaxel over 7 months and with stable resistance at under 20 nM. We found that the activation of the PI3K pathway in paclitaxel-resistant lines increased tumor growth and invasiveness, and impeded cell apoptosis. The combination of LY294002 or BYL-719 with paclitaxel significantly enhanced drug sensitivity and synergistic apoptosis through the activation of Bax, caspase-3, and cleaved poly ADP ribose polymerase compared with paclitaxel alone. In addition, PI3K inhibition also suppressed tumor migration and invasive ability by targeting vimentin, matrix metalloproteinase-9 and vascular endothelial cell growth factor.We suggest that a combination of a PI3K inhibitor with paclitaxel may enhance antitumor activity through a cascade of PI3K signaling events. Note: This abstract was not presented at the meeting. Citation Format: Youn Jin Choi, Yoo Yeon Jung, Jing Jing Liu. Inhibition of phosphatidylinositol 3-kinase signaling antagonizes paclitaxel-mediated resistance in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 338.

Design, Synthesis and Anticancer Evaluation of Carbazole Fused Aminopyrimidine Derivatives

Carbazole is an important type of tricyclic nitrogen containing compound and isolated first from coal tar in 1872 by Graebe and Glazer. Carbazole alkaloids have received considerable attention since their discovery in the 1960’s and their derivatives are well known for their pharmacological activities. Many recent studies have reported that carbazole derivatives exhibit a variety of biological activities. Toxicity of test compound in cells was determined by MTT assay based on mitochondrial reduction of yellow MTT tetrazolium dye to a highly blue colored formazan product. Among the ten synthesized compounds, 12c, 12d, 12e, 12f, 12g and 12i were showed good activity. In which 12c, 12d and 12e showed moderate activity on MCF-7 cancer cell line. Compounds 12f, 12g and 12i showed more potent activity than control. Compounds 12f, 12g and 12i showed more potent activity than control adriamycin. In future, we will plan further mechanistic studies on most potent active compounds 12f, 12g and 12i.

Antimicrobial peptides prevent bacterial biofilm formation on the surface of polymethylmethacrylate bone cement

Antibiotic-loaded polymethylmethacrylate-based bone cement has been implemented in orthopaedics to cope with implant-related infections associated with the formation of bacterial biofilms. In the context of emerging bacterial resistance to current antibiotics, we examined the efficacy of short antimicrobial peptide-loaded bone cement in inhibiting bacterial adhesion and consequent biofilm formation on its surface. The ability of α-helical antimicrobial peptides composed of 12 amino acid residues to prevent bacterial biofilm [methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli] formation on the surface of model implants made from polymethylmethacrylate-based bone cement was evaluated by colony-forming unit (c.f.u.) counting of bacteria released by sonication from the biofilms formed on their surfaces. The biofilms on model implant surfaces were also visualized by light microscopy after staining with tetrazolium dye (MTT) and by scanning electron microscopy. When incorporated in the implants, these peptides caused a mean reduction in the number of bacterial cells attached to implants' surfaces (by five orders of magnitude), and 88 % of these implants showed no bacterial adhesion after being exposed to growth media containing various bacteria. The results showed that the antibiofilm activity of these peptides was comparable to that of the antibiotics, but the peptides exhibited broader specificity than the antibiotics. Given the rapid development of antibiotic resistance, antimicrobial peptides show promise as a substitute for antibiotics for loading into bone cements.