Trypan Blue Exclusion Method Research Articles

Angiosuppressive effects of bio-fabricated silver nanoparticles synthesis using Clitoria ternatea flower: an in vitro and in vivo approach.

Biosynthesis of silver nanoparticles (CTNP's) by Clitoria ternatea flower in the aqueous extract was investigated. Synthesized nanoparticles were characterized by using UV-Visible spectroscopy, followed by DLS, Zeta potential, XRD, FTIR, SEM, and AFM. The biocompatibility nature of CTNP's was determined using erythrocytes model system. Cytotoxicity of CTNP's against MCF-7 and EAC cells were determined by using MTT and Trypan blue exclusion method and their IC50 was found to be 19.37µg/mL and 24µg/mL. Cytotoxic potential of CTNP's was further confirmed by clonogenic assay. Further in vivo studies using EAC mice model supports the anti-cancer potential of silver nanoparticles. Results found that the CTNP's effectively control the proliferation rate by inhibiting the ascites secretion and cellular density. Further quantification of VEGF, microvessel density counts and CAM assays show the anti-angiogenic potential of the CTNP's. The apoptotic inducing activity of CTNP's was confirmed by DNA fragmentation, fluorescent staining studies. More interestingly, EAC treated mice exhibit significant increase in lifespan (~ 2.25 fold) compared to control EAC mice. Interestingly CTNP's did not exhibit any secondary complications against normal mice. The present findings give an experimental proof that the CTNP's could serve as a promising candidate to overcome limitations of existing conventional cancer therapeutics.

IN VITRO CYTOTOXICITY ACTIVITY OF METHANOLIC LEAF AND FRUIT EXTRACT OF ZANTHOXYLUM OVALIFOLIUM WIGHT AGAINST DALTON’S LYMPHOMA ASCITES AND EHRLICH ASCITES CARCINOMA CANCER CELL LINES

Objectives: The objective of the present study, the methanolic leaf and fruit extraction of Zanthoxylum ovalifolium, was examined for in vitro cytotoxicity using trypan blue dye exclusion technique.
 Methodology: In vitro cytotoxicity activity of methanolic leaf and fruit extracts of Z. ovalifolium. Against Dalton’s lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cell lines evaluated using trypan blue exclusion method. Non-viable cell absorbs blue color of trypan blue while viable cell does not absorb the dye. The percentage of total number of viable and non-viable cells is calculated using % viability=(live cell count/total cell count)×100.
 Results: Cytotoxicity activity of methanolic leaf and fruit extract against chosen tumor cell line at different concentrations of 10, 20, 50,100, and 200 μg/ml, respectively. The result confirmed that the fruit extract at 200 showed less toxic effect against DLA and EAC cell line showed 72% and 75% of inhibition growth of cell line and the most potent 80% and 82% of cell death were found in leaf extract against the chosen tumor cell line. The present study indicates that both leaf and fruit extract of Z. ovalifolium were appreciable antitumor activity against DLA and EAC cell lines may be due to the presence of phytochemicals such as flavonoids and alkaloids present in the plant.
 Conclusion: Zanthoxylum ovalifolium wight fruit and leaf methanolic extracts showed satisfying result of cytotoxicity activity against DLA and EAC cell line. Hence, Z.ovalifolium could be suggested as an important source of anticancer agent for pharmacological industries.

Synthesis, Spectroscopic Characterization, Antibacterial and Short Term in vitro Cytotoxicity Studies of Copper(II) Complexes of Novel Tridentate N,N,S Donor Ligand 2-Benzoylpyridine-N(4),N(4)-(N,N-diethyl-N-methylamine-2,2'-diyl)thiosemicarbazone

A tridentate N,N,S-donor ligand, 2-benzoylpyridine-N(4),N(4)-(N,N-diethyl-N-methylamine-2,2'-diyl)thiosemicarbazone (Hbptsc) has been synthesized and characterized by elemental CHN analysis, UV-visible, FT-IR and 1H NMR spectroscopy. Copper(II) complexes of the ligand, Hbptsc synthesized have been characterized by elemental analysis, UV-visible spectra, FTIR spectra and EPR spectroscopic simulation. The complexes hold the stoichiometry of the type [CuLX] where X= Cl (1), NO3 (2), SO4 (3), N3 (4), SCN (5) confirmed by the molar conductivity studies of 10-3 M solutions in DMF at room temperature. The EPR spectra of the complexes recorded in DMF at 77 K shows an axial type spectra with two distinct g-values, g|| and g⊥ indicating a four coordinated planar geometry. The antimicrobial studies of the copper(II) complexes shows an appreciable activity against both gram positive and gram negative bacteria using streptomycin as positive control. The short term in vitro cytotoxicity studies following trypan blue dye exclusion method exhibits pronounced activity against the Dalton’s Lymphoma Ascites tumour cells extruded from the peritoneal cavity of mice.

A label-free approach to detect cell viability/cytotoxicity based on intracellular xanthine/guanine by electrochemical method

IntroductionCell viability and cytotoxicity is one of the most important toxicology indicators. In this study, an electrochemical method for detecting cell viability and cytotoxicity was discussed with the intracellular small molecule metabolite purines as indexes. MethodsThe electrochemical behaviors of Balb/c 3T3, CHO, PC-12 and V79 cell suspensions were studies by cyclic voltammetry, and cell viability and cytotoxicity of four cell lines were compared by electrochemical, cell counting, 3-(4,5-dimethyl-2-Thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) and trypan blue exclusion methods. ResultsFour cell lines all showed an oxidation peak derived from mixture of xanthine and guanine at about 0.7 V. Using intracellular xanthine and guanine as index, the electrochemical method could not only describe the cell growth curves of four cell lines, but also reflect the changes of cell viability at various phases of the cell growth prior to the counting method. Compared with MTT, cell counting and trypan blue staining methods, the electrochemical method could detect the cytotoxicity of carcinogen earlier and more sensitively. DiscussionThe electrochemical method could track the change of intracellular xanthine and guanine contents, and used it as index to detect cell viability and cytotoxicity at the molecular level without markers, showing greater advantages over the method with apparent cell proliferation as the endpoint.

Influence of LBP alone or Combined with TRAIL on Apoptosis of MLL Rearranged Leukemic Cells

To investigate the effect of lycium barbarum polysaccharide (LBP) alone or combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemia cell lines with MLL gene-rearrangement, and to explore the cell apoptotic pathway after the combined action. MLL-ALL cell line KOCL44 and KOCL45 were selected as the research object, then the control and experimental groups were set up. The cell survival rate was measured by the trypan blue dye exclusion method, the cell early apoptosis and expression of death receptors on the cell surface were detected by flow cytometry with Annexin-V/PI double staining. The protein level of caspase-8, BID, caspase-3, caspase-9, BAD, BCL-2, as well as mitochondrial and cytosol Cyto-C were detected by Western blot. LBF combined with TRAIL inhibited the growth of KOCL44 and KOCL-45 cells and showed the synergistic effect, the results of flow cytometry with Amnexiu V/PI double staining were consistent with above-mentioned results. After treatment of KOCL44 and KOCL45 cells with LBF plus TRAIL, the significant expression of DR4 on cell surface was not found, while the expression of DR4 receptor was enhanced significantly, the pro-apoptotic proteins including caspase-8, BID, caspase-3, caspase-9 and BAD were activated significantly and BCL-2 was suppressed significantly with time-dependent manner. The expression of mitochondria cyto-C in KOCL44 and KOCL45 decreased along with prolonging of treatment time (r=-0.95, r=-0.866), while the expression of cytosol cyto-C in KOCL44 and KOCL45 increased along with prolonging of treatment time (r=0.883, r=0.903). The combination of LBP and TRAIL significantly increases the apoptosis of KOCL44 and KOCL45, and the LBP and TRAIL can up-regulate the expression of TRAIL death receptor-DR5 on the cell surface, activate the pathway of caspase and mito-chrondia mitachondria, thus enhance the sensitivity of KOCL44 and KOCL45 to TRAIL induced apoptosis through both mitochondrial and apoptotic pathway.

Effect of single and multiple doses of low-level laser therapy on viability and proliferation of stem cells from human exfoliated deciduous teeth (SHED).

The present study aimed to evaluate in vitro whether the low-level laser (LLL) delivering fractionated total energy (multiple irradiation) or single irradiation stimulates regeneration-associated events (viability and proliferation) in stem cells from human exfoliated deciduous teeth (SHED). Cells received LLL irradiation (InGaAlP-660nm), according to the following experimental groups: G1 (single irradiation 2.5J/cm2, 10mW, 10s, 0.10J), G2 (single irradiation 5.0J/cm2, 10mW, 20s, 0.20J), G3 (single irradiation 7.5J/cm2, 10mW, 30s, 0.30J), G4 (twoirradiations 2.5J/cm2, 10mW, 10s; total energy 0.20J), G5 (threeirradiations 2.5J/cm2, 10mW, 10s; total energy 0.30J), and G6 (non-irradiated). Cell viability was assessed by MTT and trypan blue exclusion (TBE) methods, while cell proliferation was evaluated by crystal violet (CV) and sulforhodamine B (SRB) assays after 24, 48, and 72h after the first irradiation. By MTT, there was no difference between groups at 24 and 72h. At 48h, the groups subjected to multiple irradiation (G4 and G5) presented higher cell viability rates. The average percentages of viable cells for all groups by TBE method were 91.04%, 96.63%, and 97.48% at 24, 48, and 72h, respectively. By CV, there was no significant difference between groups at 24 and 48h; at 72h, G2, G3, and G4 presented higher cell proliferation. By SRB, G1 and G4 presented lower proliferation rates in all the periods. When the groups presenting the same total energy were compared, G2 (0.20J) presented lower cell viability rates and higher cell proliferation rates in comparison with G4; G3 (0.30J) presented similar results to those of G5, with higher cell viability and proliferation. The application of laser delivering fractionated total energy (two or three applications of 2.5J/cm2) induced higher cell viability at 48h, while the single irradiation with 2.5J/cm2 did not stimulate metabolic activity in such period and the proliferation over time. The 5.0 and 7.5J/cm2 single doses and the threeapplications of 2.5J/cm2 maintained cell viability and stimulated proliferation of SHED at 72h.

A polyherbal formulation, HC9 regulated cell growth and expression of cell cycle and chromatin modulatory proteins in breast cancer cell lines

Ethnopharmacological relevanceHC9, a polyherbal formulation, is based upon a traditional Ayurvedic formulation, Stanya Shodhana Kashaya (SSK, having 10 plant materials), formulated on Stanyashodhana gana, explained by Charaka in Charakasaṃhita Sutrasthana IV and mentioned in other texts as well. Stanyasodhana is the Sanskrit name for a group of medicinal plants, classified for “improving the quality of milk”. SSK is used by Ayurvedic practitioners for the cleansing and detoxification of breast milk in lactating mothers as well as for the management of various clinical conditions. HC9 is composed of equal ratios of nine different medicinal plants that include Picrorhiza kurroa Royle ex Benth., Cyperus rotundus L., Zingiber officinale Roscoe, Cedrus deodara (Roxb. ex D.Don) G.Don, Tinospora cordifolia (Willd.) Miers, Holarrhena antidysenterica (Roth) Wall. ex A.DC., Swertia chirata Buch.-Ham. ex Wall., Cissampelos pareira L. and Hemidesmus indicus (L.) R. Br. ex Schult.. It differs from the SSK formulation by having one ingredient [Marsdenia tenacissima (Roxb.)Moon (Murva)] less, due to its unavailability since it is mostly found in tropical hilly tracts of peninsular India and Vindhya ranges as well as in lower Himalayan tracts. All the medicinal plants in the formulation have reported activity against different types of cancers. Aim of the studyThe present study is aimed at evaluating the anticancer activity of the polyherbal formulation (HC9) and its mechanism of action against breast cancer cell lines. Materials and MethodsThe effect of HC9 on the viability of breast cancer (MCF-7 and MDAMB231) and non-cancerous (MCF-10A) cell lines was evaluated by MTT assay. The effect on cell growth and colony formation potential of cancer cells was determined by trypan blue dye exclusion method and soft agar assay, respectively. Cell cycle arrest was determined by propidium iodide (PI) staining and analyzed by flowcytometer. Scratch wound assay was used for studying cell migration. Cell invasion was determined by using BD BioCoat Matrigel invasion chambers. The gene expression of HIF-1α was examined by RT-PCR. The expression of p53, SMAR1, p16, MMP-2, CDP/Cux, p21, Rb, phospo-Rb (ppRb), VEGF, NFқB and COX-2 proteins was determined by western blotting. ResultsHC9 significantly altered growth of breast cancer cell lines, MCF-7 and MDA MB-231. It blocked the cell cycle progression at S phase in MCF-7 by up regulating the expression of p53, p21 and p16 proteins. In MDA MB-231, HC9 induced G1 phase arrest by up regulating the expression of p53, p21 and pRb proteins with simultaneous decrease in ppRb. It significantly reduced migration and invasion in both the cell lines, accompanied by decrease in the expression of MMP-2/9, HIF-1α and VEGF. HC9 decreased the expression of inflammatory markers (NF-қB, COX-2), and modulated the expression of chromatin modulators (SMAR1 and CDP/Cux) in both MCF-7 and MDA MB-231. ConclusionsHC9 exhibited potent anticancer activity against breast cancer cells, thereby warranting further pre-clinical and clinical studies in future.

Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells

BackgroundLactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated.MethodsPostbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses.ResultsUL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis.ConclusionIn conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.

PF161 THIOREDOXIN SYSTEM INHIBITORS ARE EFFECTIVE AGAINST B CELL ACUTE LYMPHOBLASTIC LEUKEMIA IN VITRO AND IN VIVO

Background:B‐cell acute lymphoblastic leukemia (B‐ALL) is a heterogeneous hematological disorder characterized by accumulation of B cell precursors. Although undeniable improvement has been made in the therapy ofB‐ALL (>90% of pediatric patients experience remission), there are still specific subtypes associated with poor outcome. Among such subtypes are B‐ALL with mixed lineage leukemia rearrangements (MLLr ALL) or BCR‐ABL1 fusion protein (BCR‐ABL1+ ALL). Importantly, MLLr ALL does not respond to novel immunotherapies, thus novel treatment options are urgently needed. More and more data highlight disturbed redox homeostasis as a novel target in cancer treatment. Cancer cells upregulate different antioxidant machineries to overcome excessive production of reactive oxygen species (ROS) in result of increased metabolism and uncontrolled proliferation. One of such ROS‐defending systems is the thioredoxin (TXN) system, which is composed of three independent enzymes: thioredoxin reductase (TXNRD), thioredoxin (TXN), and peroxiredoxin (PRDX).Aims:To investigate the efficacy of TXN‐family enzymes inhibitors – auranofin (AUR) and adenanthin (ADE) in B‐ALL in vitro and in vivo.Methods:Primary material was isolated from pediatric and adult B‐ALL patients at diagnosis. Primograft B‐ALL samples representing BRC‐ABL1+ and MLLr ALL were generated in NOD scid gamma (NSG) mice by tail vein injections of primary B‐ALL cells. The engraftment of leukemic cells was assessed by whole blood staining and flow cytometry. Primary bone marrow derived mesenchymal stem cells (BM‐MSC) were isolated from non‐leukemic donors who underwent hip replacement surgery. Cytostatic/cytotoxic effects of TXN‐family enzymes inhibitors, AUR and ADE were assessed in mono‐ and co‐culture with BM‐MSC by MTT viability assay or by trypan blue exclusion method. The efficacy of AUR in vivo was evaluated in MLLr ALL patient derived xenograft model in NSG mice. For mechanistic studies we used SEM cell line representing MLLr ALL.Results:We have previously found increased mRNA and protein levels of particular enzymes of TXN system in B‐ALL cell lines and primary cells, thus we evaluated efficacy of two thioredoxin system inhibitors, AUR and ADE in B‐ALL primary cells. We found that both inhibitors decreased the viability of various B‐ALL cells grown not only in monoculture, but also in a co‐culture with bone marrow derived mesenchymal stem cells (BM‐MSC) of confirmed characteristics (Fig. 1A), which were shown to protect leukemic cells from drugs toxic effects. Next, we compared the effects of both inhibitors against normal peripheral blood mononuclear cells (PBMC) and found that ADE is significantly more toxic than AUR, thus we chose the latter for further studies. Accordingly, we observed that in patient derived xenograft model of MLLr ALL, AUR delayed the progression and prolonged the survival of NSG mice (Fig. 1B). Elucidating the mechanism standing behind AUR toxic effects, we found increased oxidative stress, as we observed elevated ROS levels which were abrogated by ROS scavengers, pyruvate and catalase. In addition, we observed that AUR induced oxidative stress biomarker, heme oxygenase 1 and moderate DNA oxidative damage in primograft B‐ALL cells (Fig. 1C).Summary/Conclusion:Above results show that targeting TXN system with their inhibitors can be considered as novel approach in the treatment of B‐ALL. Considering that AUR is an FDA approved drug, it could be repurposed and tested in combination with other targeted drugs in B‐ALL.image

ANTIOXIDATIVE ACTIVITY OF ALPHA-MANGOSTIN IN ACETALDEHYDE-INDUCED HEPATIC STELLATE CELLS: AN IN VITRO STUDY

Objective: Alcohol accumulation in the liver can cause pathological disorders such as liver fibrosis that can develop into hepatocellular carcinoma,one of the main causes of mortality associated with liver disease. The previous studies have shown that a plant compound, alpha-mangostin, has anantioxidant effect in the inhibition of pancreatic tumor growth in vitro. This study aimed to analyze the antioxidative properties of alpha-mangostin inacetaldehyde-induced liver fibrosis in vitro.Methods: Immortalized hepatic stellate cells (HSCs), of the LX-2 cell line, were incubated with acetaldehyde in the presence or absence of alphamangostin(10 and 20 μM). The cells were then counted and lysed, and LX-2 cell viability was determined with the trypan blue exclusion method. Themalondialdehyde levels, superoxide dismutase activity, and glutathione (GSH) levels were also determined using the cell lysates.Results: Acetaldehyde treatment resulted in a significant increase in HSC cell viability and decreased the production of GSH. Alpha-mangostintreatment resulted in reduced cell viability of the HSCs and prevention of the loss of intracellular GSH.Conclusion: Alpha-mangostin reduced acetaldehyde-induced cell proliferation, and this was affected at least in part by its antioxidative properties

THE EFFECT OF ALPHA-MANGOSTIN ON TRANSFORMING GROWTH FACTOR BETA 1 (TGF-β1) AND MATRIX METALLOPROTEINASE-3 EXPRESSION IN TGF-β-INDUCED HEPATIC STELLATE CELLS

Objective: Alpha-mangostin (α-MG) has been shown to possess antifibrotic effects. However, the specific mechanism of action of this compoundremains poorly understood. Therefore, the aim of this study was to investigate the effect of α-MG on the expression levels of transforming growthfactor (TGF)-β1 and matrix metalloproteinase-3 (MMP3) in hepatic stellate cells (HSCs) induced by TGF-β.Methods: Immortalized HSCs and LX-2 cells were incubated with TGF-β with or without α-MG (5 and 10 μM). The viability of LX-2 cells was assessedusing the Trypan Blue Exclusion Method. The effect of α-MG on cell morphology and the mRNA expression levels of TGF-β1, TGF-β receptor, and MMP3was then evaluated.Results: TGF-β enhanced the proliferation of HSCs and caused significant increases in the expression levels of TGF-β1, TGF-β receptor, and MMP3.α-MG treatment reduced the proliferation of HSCs and decreased the expression levels of TGF-β1, TGF-β1 receptor, and MMP3.Conclusion: α-MG is a potential antifibrotic agent due to its antiproliferative and antifibrogenic effects, mainly by suppressing the expression of TGF-βand MMP3 on the surfaces of activated HSCs.

Effect of omega‐3 and omega‐6 fatty acids on nitric oxide and IL‐6 production by RAW 264.7 macrophages

Chronic low‐grade inflammation, or meta‐inflammation, which may occur in obese subjects, is related to the infiltration of macrophages into adipose tissue (AT) as adipocyte number and size increase. The increased count in fat cells is correlated with an increase in adipose tissue macrophages (ATMs). These ATMs can secrete pro‐inflammatory cytokines which can have effects on the surrounding adipocytes. Omega‐3 fatty acids (FAs) reduce inflammation through direct competition with omega‐6 FAs, the precursors of the inflammatory eicosanoids, as well as through changes in gene expression of inflammatory molecules. Less is known about the effects of omega FAs on pro‐inflammatory cytokine production by immune cells, such as macrophages. The objective of this research was to determine the effects of omega‐3 and omega‐6 FAs on the production of nitric oxide (NO), a signaling molecule secreted by activated macrophages which is involved in mediating inflammation and production of the pro‐inflammatory cytokine interleukin‐6 (IL‐6), in lipopolysaccharide (LPS)‐activated macrophages. RAW 264.7 (murine macrophages) were incubated with 100 μM of sodium salts of linoleic acid (LA; C18:2, omega‐6), arachidonic acid (ARA; C20:4, omega‐6), or eicosapentaenoic acid (EPA; C20:5, omega‐3) for 24 h. After pre‐incubation with FAs macrophages were stimulated with 0.01 μg/ml of LPS for 6 h. Conditioned media was collected for NO and IL‐6 quantification via Griess assay and ELISA, respectively. FA treatments were cytoprotective, resulting in higher percentages of live cells than both control and LPS‐treated cells (Trypan blue exclusion method; p<0.0001, two‐way ANOVA). LPS‐stimulated macrophages pre‐incubated with LA, ARA, or EPA produced less NO than LPS‐stimulated macrophages without FA pre‐incubation (p=0.0001, two‐way ANOVA). Pre‐incubation of macrophages with LA, ARA, or EPA decreased IL‐6 production in comparison to LPS‐stimulated macrophages, with omega‐6 FAs having a more pronounced effect than omega‐3 FAs (p<0.0001, two‐way ANOVA). In conclusion, both omega‐6 and ‐3 FAs decreased activation of murine macrophages and decreased production of the inflammatory molecules NO and IL‐6, with omega‐6 FAs having a more marked effect than omega‐3 FAs. The stronger anti‐inflammatory effect of omega‐6 FAs on murine macrophages, compared to omega‐3 FAs, warrants further investigation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of SAHA and RG7388 on p21WAF1/CIP1 and p27KIP1 mediated Pathways in Cancer Cells

Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21WAF1/CIP1 and p27Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genotoxic potential of selected medicinal plant extracts in human whole blood cultures

Introduction: Many plant-derived products despite wide usage are not scientifically evaluated for their safety and efficacy. Therefore, in the present study, we aimed to evaluate the cytotoxic and genotoxic activities of Polygonum aviculare L., Equisetum arvense L., Plantago lanceolata L. and Artemisia absinthium L. ethanolic extracts in human white blood cells. Methods: Cell viability was assayed by trypan blue exclusion method, while the genotoxicity was tested by cytokinesis-block micronucleus (CBMN) assay upon cells stimulation with noncytotoxic concentrations of the plant extracts. Results: None of the plant extracts showed high cytotoxic activity. At the same time, only extract of P. lanceolata did not present any mutagenic activity, while E. arvense, P. aviculare and A. absinthium were clearly genotoxic. Conclusion: Caution is advice in the case of long-term use of E. arvense, P. aviculare and A. absinthium herbal medicines by population.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis.

AIMTo investigate the effect of adipose-derived mesenchymal stem cells (ADMSCs) and their conditioned media (CM) on hepatocellular carcinoma (HCC) cell tumorigenesis.METHODSThe proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit 8 (commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTSOur data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSIONThese findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells.

PurposeModulated electro-hyperthermia (mEHT) stands to be a significant technological advancement in the hyperthermia field, utilizing autofocusing electromagnetic power on the cell membrane to create massive apoptosis. Since mEHT possesses the unique ability to excite cell membranes, we hypothesized that mEHT could enhance the uptake of liposomal drugs by enhancing phagocytic activity.Materials and methodsWater bath control and mEHT were used to compare the enhancement of liposome-encapsulated doxorubicin (Lipodox®) uptake by cancer cells. Cancer cells were made visible by doxorubicin fluorescence to investigate drug uptake. Viable cell yield was determined via the Trypan Blue exclusion method. Various substrates were used to investigate the mechanism of drug-uptake enhancement. The murine colon carcinoma model, CT26, was used to confirm the tissue infiltration of Lipodox® and its therapeutic effect.ResultsmEHT treatment showed a significant enhancement of Lipodox® uptake of doxorubicin fluorescence compared with 37°C or 42°C water bath treatment. Tumor tissue sections also confirmed that mEHT treatment achieved the highest doxorubicin concentration in vivo (1.44±0.32 µg/g in mEHT group and 0.79±0.32 µg/g in 42°C water bath). Wortmannin was used to inhibit the macropinocytosis effect and 70 kDa dextran-FITC served as uptake substance. The uptake of dextran-FITC by cancer cells significantly increased after mEHT treatment whereas such enhancement was significantly inhibited by wortmannin.ConclusionThe result showed mEHT-induced particle-uptake through macropinocytosis. mEHT-enhanced uptake of Lipodox® may amplify the therapeutic effect of liposomal drugs. This novel finding warrants further clinical investigation.

Cell Cycle Arrest and Cytotoxic Effects of SAHA and RG7388 Mediated through p21WAF1/CIP1 and p27KIP1 in Cancer Cells.

Background and Objective: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. Materials and Methods: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. Results: The RG7388 treatment was able to induce cell death by elevating p21WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21WAF1/CIP1 in MCF-7 cells. Conclusion: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi.

Recombinant asialoerythropoetin protects HL-1 cardiomyocytes from injury via suppression of Mst1 activation

BackgroundRecombinant human erythropoietin (rhuEPO) and asialoerythropoietin (asialo-rhuEPO) are cardioprotective. However, the protective effects of rhuEPO could not be translated into clinical practice because of its hematopoiesis-associated side effects while non-erythropoietic asialo-rhuEPO is unavailable in large quantities for clinical studies. This study was designed to investigate the cardiomyocyte protective potential of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine (STS)-induced injury in HL-1 murine cardiomyocytes and identify cellular pathway(s) responsible for its cardioprotection. MethodsHL-1 cardiomyocytes were simultaneously treated with STS and asialo-rhuEPOP. Cellular injury, apoptosis, and cell viabilities were measured by LDH assay, Hoechst staining and trypan blue exclusion method, respectively while western blotting was used to study its effects on apoptosis and autophagy hallmarks. ResultsOur results showed that 20 IU/ml asialo-rhuEPOP provided 39% protection to cardiomyocytes compared to STS-treated cells, which is 2-fold better than that of mammalian cell-produce rhuEPO (rhuEPOM). Asialo-rhuEPOP was found to suppress activation of proapoptotic kinase Mst1 (mammalian Sterile-20-like kinase 1) and FOXO3, leading to inhibition of apoptotic pathway and restoration of autophagy as indicated by the reduction of fragmented/condensed nuclei, altered ratios of Bax/Bcl2, p-Bad/Bad, cytosol/mitochondrial cyt c and caspase-3 activation, and the restored levels of autophagy markers Beclin1, p62 and LC3B-II. Additionally, Akt was found to be activated and FOXO3 was phosphorylated on Ser253, suggesting inhibition of FOXO3 transcriptional function. ConclusionsAsialo-rhuEPOP-mediated cardioprotection occurs through activation of PI3K/Akt pathway leading to suppression of Mst1 activation and promoting cardiomyocyte survival. General significanceAsialo-rhuEPOP could be used to modulate Mst1 activity elevated under numerous pathological states.

HPLC Analysis of Phenolic Acids, Antioxidant Activity and in vitro Effectiveness of Green and Roasted Caffea arabica Bean Extracts: A Comparative Study.

Coffee is a popular drink; it is one of the most commercialized food products and a rich source of biologically active compounds that are important for human health. This study aimed to prove the anticancer activity of Green Coffee (GC) and Roasted Coffee (RC) bean aqueous extracts (Coffea arabica) on breast cancer adenocarcinoma cell line (MCF-7) and the safety of both extracts on normal Human Peripheral Blood Lymphocytes culture (HPBL). Total phenolic content for GC and RC extracts was measured and result of both extracts were (0.308±0.016 & 0.233±0.013mg/g) respectively. The phenolic acids were screened by HPLC at the wavelength of 254& 278 and 300 nm and 5-caffeoylquinic acids (Chlorogenic acid), the predominant form of phenolic acids, was identified in GC and RC samples. Ferric Reducing Antioxidant Power (FRAP) as well as the free radical scavenging activity (DPPH) proved the antioxidant properties of both extracts. The DPPH IC50 mean values of GC and RC extracts were (2.4±0.08, 2.3±0.16 µg/ml) respectively. Cytotoxicity of both extracts on MCF-7 cells were evaluated by neutral red uptake assay which showed the IC50 mean values (377±5.7,500±8.1 µg/ml) for GC and RC extracts respectively. The safety of both extracts (0, 125, 250, 500 µg/ml) on HPBL was evaluated in vitro using trypan blue exclusion method and DNA single strand breaks (alkaline comet assay). Result revealed non-significant cytotoxic difference (P<0.001) between cultures especially at lower doses of GC and RC extracts except the highest dose of GC and RC extract which showed slightly significant damage (P<0.001). This study proved that GC and RC aqueous extracts were found to be selectively cytotoxic in vitro to cancerous cells (MCF-7 cell line) causing cell death with no cytotoxicity on normal human lymphocytes especially at lower doses.

CELL growth and viability analysis in poli membranes (L-lactic acid-CO-glycolic acid): an in vitro study

ABSTRACT Objective: Performing an in vitro evaluation of the biological effects on cell growth and viability of fibroblasts in PLGA membranes with and without simvastatin. Methods: Two groups of resorbable synthetic polymeric membranes were used: PLGA, with and without simvastatin, cut into a suitable format to fit to 24 thermometric wells. Fibroblasts were grown on resorbable membranes and evaluated for proliferation and viability at 24, 48 and 72 hours after the beginning of cultivation, being the tests performed in triplicate. For the cell growth analysis, the Trypan blue exclusion method was applied, while cell viability was observed by the MTT test. The results were statistically analyzed applying the Two-Way ANOVA, followed by the Bonferroni test, with 95% confidence interval and P value smaller than 0.05 was accepted as statistically significant. Results: Statistical difference (p &lt;0.01) was seen between the control group (2.16x104 ± 0.51 cells) and the PLGA group with simvastatin (1.58x104 ± 0.36 cells) in the 48-hour period. After 72 hours, statistical differences (p &lt;0.001) were observed between the PLGA group with simvastatin (1.66x104 ± 0.49 cells) and the PLGA group without simvastatin (2.25x104 ± 0.2 cells) when compared to the control group (2.81x104 ± 0.33 cells) for cell proliferation. Statistical differences (p &lt;0.05) were observed between the control group (0.27 ± 0.05) and the PLGA group with simvastatin (0.21 ± 0.03). Likewise, a statistical difference (p &lt;0.001) was seen between the PLGA group without simvastatin (0.19 ± 0.02) and the control group after 24 hours. In the 48 – 72-hour period, statistical differences (p &lt;0.001) were observed between the control group (0.36 ± 0.09 and 0.55 ± 0.05, after 48 and 72 hours respectively) and the PLGA group without simvastatin (0.26 ± 0.05 and 0.34 ± 0.07, after 48 and 72 hours respectively), as well as in the PLGA group with simvastatin (0.27 ± 0.04 and 0.31 ± 0, 04, after 48 and 72 hours respectively) for the cell viability test. Conclusion: The association of simvastatin to PLGA membranes had an inhibitory effect on fibroblast proliferation, as well as induced a reduction in cell viability. Thus, the use of PLGA along with simvastatin may assist in guided bone regeneration.