Trypan Blue Dye Exclusion Assay Research Articles

Studies on Genotoxic Effects of Mobile Phone Radiation on A375 Cells

Introduction: Radiation from cell phones has been associated with an increased risk of cancer. The literature has reported evidence of certain biological effects resulting from exposure to various wavelengths, doses, and intensities of radiofrequency radiation. The present study aimed to evaluate the possible adverse effects of radiation from a GSM mobile phone operating at 900 MHz on human melanoma A375 cells. Material and Methods: Cellular morphology was observed under an inverted phase contrast microscope. Cell viability was determined through trypan blue dye exclusion and clonogenic assay. Moreover, flow cytometry was applied to detect DNA damage, cell cycle arrest, and reactive oxygen species (ROS) production. Cellular reduced glutathione (GSH) content was estimated by measuring the total soluble thiol. In addition, the physico-chemical changes were assessed using spectrophotometer and viscometer. Results: This study revealed that there was no change in cellular morphology and necrotic cell killing; although a small effect was observed on delayed cell death. Depletion in GSH content was noted, but ROS generation was not significantly different from that of the control group. No DNA damage was found during such exposure and there was no alteration in cell cycle distribution. In vitro evaluation of radiation effect on calf thymus DNA showed a slight perturbation in absorption spectra that was completely reversible with time. On the other hand, viscometric analysis showed no changes. Conclusion: From the findings, it can be concluded that this range of mobile phone radiation for 60 min of continuous exposure has no genotoxic impact on A375 cells.

Contraction Dynamics of Rod Microtissues of Gingiva-Derived and Periodontal Ligament-Derived Cells.

Tissue engineering strategies using microtissues as “building blocks” have high potential in regenerative medicine. Cognition of contraction dynamics involved in the in vitro self-assembly of these microtissues can be conceived as the bedrock of an effective periodontal tissue regenerative therapy. Our study was directed at evaluating the shrinkage in the rod-shaped structure of a directed self-assembly of human gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) and developing insights into the potential mechanisms responsible for the shrinkage. GC and PDLC were seeded in non-adherent agarose molds to form rod microtissues. Cells used for the experiments were characterized using fluorescence-activated cell sorting (FACS). To assess the viability, resazurin-based cytotoxicity assays, trypan blue dye exclusion assay, MTT and live/dead staining, and histological evaluation of rods based on hematoxylin and eosin staining were performed. Rod contraction was evaluated and measured at 0, 2, 6, and 24 h and compared to L-929 cells. The role of transforming growth factor (TGF)-β signaling, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen activated protein kinase (MAPK) signaling was analyzed. Our results show that the rod microtissues were vital after 24 h. A reduction in the length of rods was seen in the 24 h period. While the recombinant TGF-β slightly reduced contraction, inhibition of TGF-β signaling did not interfere with the contraction of the rods. Interestingly, inhibition of phosphoinositide 3-kinase by LY294002 significantly delayed contraction in GC and PDLC rods. Overall, GC and PDLC have the ability to form rod microtissues which contract over time. Thus, approaches for application of these structures as “building blocks” for periodontal tissue regeneration should consider that rods have the capacity to contract substantially. Further investigation will be needed to unravel the mechanisms behind the dynamics of contraction.

APRICOXIB

Background: Breast cancer is most frequently diagnosed cancer globally but there is not any ideal economical and safer agent that not only decreases the progression but also resolve complexities associated with breast cancer such as inflammatory conditions. There was strong link between inflammation and cancer specially breast cancer. Thus by inhibiting the COX enzyme may inhibit the progression of cancer beside of its role in inflammatory conditions of breast. Study Design: Interventional In Vitro trial. Setting: Department of Pharmacology in alliance with PCMD. Period: The duration of study from April 2016 to February 2017. Methodology: For this purpose we used five cancerous lines MCF-7, MDA-MB-231, MCF-10, HT-29 and Hela cell lines. For demonstrating the cytotoxic effects of Apricoxib we used MTT assay (for all cell Lines) and Trypan blue dye exclusion assay (Primarily for MCF-7 cell lines). For calculation of minimum dose required for exert cytotoxic effects of Apricoxib and its selectivitytowards cancerous cells of breast tissue we calculated its IC50 value and Selectivity Index (SI) by MTT assay. Results: Apricoxib significantly reduce the viability of MCF-7, MDA-MB-231, Hela, HT-29 as assessed by MTT assay in dose dependent manner (χ2 (2) = 26.483, p<0.001), (χ2 (2) = 26.49, p<0.001), (χ2 (2) = 26.062, p<0.001) and (χ2 (2) = 26.062, p<0.001) respectively. However Apricoxib had non-significant effects on % viability of MCF-10 cell line (χ2 (2) = 4.167, p=0.654) as assessed by MTT assay. Furthermore Apricoxib had lowest IC50 value against MCF-7 cell line. Conclusion: This study demonstrated that beside of primarily anti-inflammatory effects Apricoxib have additional benefits in term of exerting the cytotoxic effects (in vitro) on cancerous cell lines as indicated by reducing the % viability and reducing the Absorbance value of test sample as compare to control. This opens the newer path for researcher to evaluate different aspects of Apricoxib in field of chemotherapy.

The Impact of Hyperthermia in Radiosensitivity of Human Cervical Carcinoma Cell Line HeLa

Introduction: The objective of this work was to investigate the therapeutic effect of a combination of hyperthermia at 43oC exposured for 20, 40, 60 min with 6MeV electron beams at different radiation doses (2, 4, 6 Gy) compared to radiation alone. Materials and Methods: In this in vitro study, HeLa cells were exposed to either: 6MeV electron beams at different doses (2, 4, 6 Gy) and hyperthermia at 43oC for 20, 40, 60 min alone or electron beam followed by hyperthermia with 20-minute interval. The effect of hyperthermia and radiation alone as well as a combination of radiation with hyperthermia on cell viability and cell survival were estimated using the trypan blue dye exclusion assay and MTT assay immediately and 48 h after treatments, respectively. Results: The data obtained by trypan blue exclusion test showed that no significant reduction on cell viability when cells were treated RT (6 MeV, 2, 4, 6 Gy) and HT (43oC, 20, 40, 60 min) alone as well as RT followed by HT in comparison with control group (cells without treatment with the RT and HT). In addition, the data obtained by MTT assay showed significant cytotoxicity in the radiated groups compared to the control group. Also, our results showed HT alone did not effect on cytotoxicity. Moreover, our results showed that RT in combination with HT at 43oC for 20 min did not create significance cytotoxicity on HeLa cells in comparison with control group (group treated with radiation) (p>0.05). In contrast, RT in combination with HT at 43oC for 40,60 min had significant cytotoxicity on HeLa cells in comparison with control group (group treated with radiation) (p<0.05). Conclusion: Our data suggest that combined treatment of 6MeV electron beams (RT) and hyperthermia (HT) can result in radiosensitization of cervical cancer cells.

Alternative Overexpression of NRF2 or MYC Defines a Subgroup of Poor Prognosis Acute Myeloid Leukemia and Suggests a Novel Therapeutic Strategy By Combined Bromodomain Inhibition and Forced NRF2 Pathway Activation

Introduction. Inhibition of Bromodomain and extraterminal (BET) proteins was effective against different acute myeloid leukemia (AML) subtypes in preclinical studies (Dawson et al. Nature 2011; Zuber et al. Nature 2011; Dawson et al. Leukemia 2013; Chen et al. Cancer Cell 2014; Gröschel et al. Cell 2014; Zhao et al. Cell Reports 2016). However, the drug had limited clinical activity, suggesting the need of ad hoc combination therapies able to target leukemia stem cells (LSCs) in their microenvironment. Hypoxia is an integral component of the bone marrow microenvironment and plays a crucial role in survival and chemoresistance of LSCs.Aims. The study aims to elucidate the consequences of BETi treatment in AML under hypoxic conditions and identify novel potential combination strategies.Methods. AML cell lines (OCI-AML3: NPM1- and DNMT3A-mutated, Kasumi-1: t(8;21), HL60: MYC-amplified, MOLM-13, NOMO-1: MLL-driven, KG-1: TP53-mutated) were treated with the BET inhibitor (i) GSK1215101A (250/500 nM, 48h) or the NRF2 activator omaveloxolone (NRF2a, 0.2-1 mM, 48h) and with the drug combination (72h) at 1% or 20% O2 concentration. Cell viability, apoptosis and cell cycle were evaluated by trypan blue dye exclusion assay, AnnexinV and PI staining, respectively. Gene expression profiling (HTA2.0, Affymetrix) was carried out on actively translated mRNAs isolated by polysome profiling after 16h of BETi treatment and on 61 primary AML. The TCGA AML dataset was analyzed on the cBioPortal. Gene expression correlation and enrichment analysis were performed by Pearson coefficient and GSEA, respectively. Kaplan-Meier survival curves were compared by Logrank test. Glutathione was quantified by mass spectrometry (Metabolon).Results. BETi induced a dose-dependent reduction of cell viability in AML cells lines under hypoxia (25%-65% decrease at 500 nM) except for HL-60. Under the same conditions, the treatment caused a significant arrest in the G0/G1 phase of the cell cycle in OCI-AML3, Kasumi-1, HL-60 and KG-1 models (p<0.05) and induction of apoptosis in NOMO-1 and Kasumi-1 (40% and 50% AnnexinV+ cells, respectively, p<0.05). BETi reduced the translational rate of Kasumi-1 and OCI-AML3 cells, as determined by a decrease of disome-polysome peaks height. The treatment exacerbated hypoxia-mediated MYC suppression and associated with downregulation of a MYC signature at translational level. Moreover, it induced upregulation of the NRF2 regulator ARNT (p=0.02) and the NRF2 targets CAT, EPHX1, FTH1, GSTM1, MGST1, PRDX1 (p<0.05) under normoxia and/or hypoxia, with reduced KEAP1 mRNA and protein specifically at 1% O2 (p=0.01). These results suggest stabilization of NRF2 protein and activation of the pathway, as strengthened by increased levels of reduced and oxidized glutathione in OCI-AML3 cells (p<0.01). Based on this alternative activation of MYC and NRF2 pathway in AML, we analyzed gene expression and mutation in non-M3 AML from an internal cohort and the TCGA dataset. Upregulation of NRF2 expression and deregulation of MYC (overexpression/driver mutation) occurred in 4% and 9% of cases, respectively (independent of genomic amplification), with mutual exclusivity and an inverse correlation (p=0.03). MYC or NRF2 alterations defined a subgroup of patients with poor overall survival (10 vs. 18.1 months, p=0.04) and progression-free survival (7.2 vs. 17 months, p=0.0006). We then asked whether antioxidant gene expression was a defense response under BETi pressure. However, pharmacological inhibition of NRF2 or glutathione biosynthesis failed to potentiate the anti-leukemic effects of BETi. Conversely, activation of NRF2 pathway, which is effective as single agent on AML cells, potentiates the effects of BETi treatment in non-M3 AML, with reduced cell viability and increased apoptosis.Conclusions. BET protein activity drives alternative NRF2 or MYC overexpression in AML, which defines a subgroup of patients with poor prognosis. NRF2 activation is finely tuned in AML, as both inhibition and activation of the pathway induce cell death. However, NRF2 activation specifically potentiates BETi treatment under hypoxia and normoxia, suggesting a novel combination therapy against AML LSCs.Supported by: EHA Non-Clinical Junior Research Fellowship, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, Fondazione del Monte BO e RA project. [Display omitted] DisclosuresCavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Abbvie: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau.

Polyphyllin I Inhibits Proliferation and Induces Apoptosis by Downregulating AML1-ETO and Suppressing C-KIT/Akt Signaling in t(8;21) Acute Myeloid Leukemia.

Polyphyllin I (PPI), a bioactive constituent extracted from traditional medicinal herbs, is cytotoxic to several cancer types. However, whether PPI can be used to treat t(8;21) acute myeloid leukemia (AML) cells requires further investigation. Here, we determined the inhibitory effects of PPI on t(8;21) AML cells by Cell Counting Kit-8 (CCK-8) and the trypan blue dye exclusion assay. DAPI staining and Wright-Giemsa staining were performed to check for apoptosis. Detection of apoptotic protein and AML1-ETO signaling protein expression were conducted by Western blot analysis. Our results suggested that PPI decreased growth and induced apoptosis in a dosage-dependent manner in the t(8;21) AML cell line Kasumi-1. PPI significantly downregulated AML1-ETO expression in a dosage- and time-dependent manner. PPI also upregulated P21 and downregulated survivin expression by reducing AML1-ETO. Mechanistically, PPI significantly reduced the expression of C-KIT, another therapeutic target for AML with t(8;21), followed by inhibition of Akt signaling. These results suggest that PPI can suppress growth and induce apoptosis of t(8;21) AML by suppressing the AML1-ETO and C-KIT/Akt signaling pathways. Therefore, PPI may be an anticancer therapeutic to treat t(8;21) AML.

Overexpressed CYP450 mediated apoptosis evaluates cytotoxicity and teratotoxicity of Cucumis callosus

Fruits of Cucumis callosus (Rottl.) Cogn. (Family: Cucurbitaceae) plant, are commonly known as “bitter cucumber” (English) and “Kachri” (Hindi) in India and have been traditionally used for antioxidant, rich source of vitamin C, antidiabetic and anticancer actions. Tribal peoples of Odissa and West-Bengal are using these fruits during worship and as a vegetable. In vitro cytotoxicity of methanolic seed extract of C. callosus (MSCC) at different concentrations (25, 50, 100, 200, 400) µg/ml and methanolic pericarp extract of C. callosus (MPCC) at (30, 60, 120, 240, 360) µg/ml on Ehrlich Ascites Carcinoma (EAC) cell line, has been evaluated by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and Trypan blue dye exclusion assay for a period of 3 h treatment. Acute toxicity and in vivo teratotoxicity of both extracts were also evaluated using the Zebrafish embryo developmental assay. The MSCC and MPCC showed the direct cytotoxic effect on EAC cells in a dose-dependent manner with IG50 value 273.17 and 235.08 µg/ml respectively. The MPCC in 240 and 360 µg/ml showed a strong teratotoxic effect on Zebrafish embryo in a dose-dependent manner. The cytotoxicity and teratotoxicity of MPCC were due to overexpressed CYP450 mediated apoptosis in the tissue.

Abstract 5767: Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy

Abstract Background Photoimmunotherapy (PIT) is a new class of molecular targeted cancer theranostics, which employs monoclonal antibody (mAb) conjugated to a photosensitizer, IR700, that is activated by focal near-infrared (NIR) light irradiation leading to necrotic cell death by cell membrane rapture where mAb-IR700 conjugates binds to target membrane proteins specifically. We have previously demonstrated HER2-targeted PIT employing trastuzumab-IR700 conjugates for HER2 expressing cancers. However, some cancer cells were survived partly because of tumor heterogeneity and inhomogeneous micro-distribution of mAb-IR700 conjugates. In this study, we developed a new type of PIT agents targeting VEGFR-2 expressed on vascular endothelium in a tumor and evaluated the feasibility by comparing conventional PIT in vitro and in vivo. Methods HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. HER2-targeting trastuzumab and VEGFR-2-targeting ramucirumab were conjugated to IR700. Cells were treated with mAb-IR700 conjugates followed by NIR light irradiation after washing the cells. LIVE/DEAD assay was performed to assess short-term phototoxicity and trypan blue dye exclusion assay to assess long-term phototoxicity. Next, mouse tumor xenograft models were created for in vivo PIT. Tumor-bearing mice were randomized and treated with mAb-IR700 conjugates followed by NIR light irradiation under anesthesia. Antitumor effects were monitored by measuring tumor diameters with a caliper. Results PIT utilizing ramucirumab-IR700 conjugates did not induce phototoxic effect in vitro because of the absence of membranous expression of VEGFR-2 in NCI-N87 cells, while PIT utilizing trastuzumab-IR700 conjugates induced rapid phototoxic effect because of the strong membranous expression of HER2 in NCI-N87 cells. By contrast, antitumor effects were observed in NCI-N87 xenograft tumors in vivo utilizing both ramucirumab- and trastuzumab-IR700 conjugates followed by NIR light irradiation. Microscopic analysis of the harvested tumors showed decreasing tumor micro-vessel densities only when tumors were treated with PIT utilizing ramucirumab-IR700 conjugates but not trastuzumab-IR700, which represents a different mechanism than that of conventional PIT targeting antigens expressed on the tumor cell membrane. Conclusion We demonstrated a new mechanism of PIT utilizing ramucirumab-IR700 conjugates. As VEGFR-2 is upregulated in many types of solid cancers, this method may be considered as being applicable to various types of cancers in future clinical settings. Citation Format: Makoto Mitsunaga, Takashi Nishimura, Kimihiro Ito. Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5767.

Level of hydrogen peroxide affects expression and sub-cellular localization of Pax6.

The Pax6 is a multifunctional pairedbox and homeobox containing transcription factor which is involved in several functions of brain, eyes, and pancreas. It regulates expression of genes involved in cell proliferation, differentiation, inflammation, oxidative stress management, and neuropathy. Dynamic changes in the sub-cellular localization of Pax6 are proposed to regulate its activity, however, the underlying mechanism remains poorly understood. The oxidative stress mediated changes were studied in sub-cellular localization of Pax6 in cultured cells derived from the eye (cornea) and pancreas. The impact of induced oxidative stress was investigated on reactive oxygen species scavenger molecules, Superoxide dismutase1 (SOD1) and Catalase, and a critical cell signalling molecule Transforming growth factor-beta (TGF-β1). The cells were treated with three different concentrations of H2O2, viz., 0.3, 1.5, and 3.0mM. The cell viability was analysed through Trypan blue dye exclusion assay. The localization of Pax6 was observed by immunofluorescence labeling, and alterations in levels of Pax6, SOD1, Catalase, and TGF-β1 were investigated by semi-quantitative RT-PCR. Nucleo-cytoplasmic shuttling of Pax6 was observed in cells of corneal epithelial (SIRC) and pancreatic origins (MIA-PaCa2). The percentage distribution of Pax6 in nuclear and cytoplasmic compartments of SIRC and MIA-PaCa2 cells was analyzed through ImageJ software. Level of hydrogen peroxide affects expression and sub-cellular localization of Pax6. Expression of Pax6 and TGF-β1 are directly associated with changes in sub-cellular localization of Pax6 and modulation in expression of Catalase. This may be the result of a cellular protective mechanism against peroxide-dependent cellular stress.

IN VITRO ANTICANCER POTENTIAL OF BIOSYNTHESIZED ZINC OXIDE NANOPARTICLES FROM THE SEAWEED TURBINARIA CONOIDES

Objective: The objective of this study is to investigate the anticancer potential of zinc oxide nanoparticles (ZnO-NPs) and hydroethanolic extract of Turbinaria conoides (HETC) against Dalton’s lymphoma ascites (DLA) cell line. Methods: Nanoparticles were synthesized from the HETC. An ultraviolet-visible spectrophotometric analysis was performed to confirm the formation of ZnO-NPs. Size, morphology, and elemental composition of ZnO-NPs were also analyzed using scanning electron microscope-energy dispersive X-ray diffraction. The cytotoxic activity of ZnO-NPs and HETC was evaluated by 3-(4,5-dimethythiazol-. 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue dye exclusion assays on DLA cells. The apoptosis inducing the effect was observed through acridine orange staining method (AO and EB) and DNA ladder assay. Results: The results of in vitro cytotoxic studies by MTT and trypan blue dye exclusion assays on DLA cell line in the presence of ZnO-NPs showed an IC50 value of 23.13 and 25.81 μg/ml, respectively. The DNA ladder assay and AO/EB staining clearly demonstrated that the ZnO-NPs at 50 μg/ml concentration induced a maximum apoptosis in DLA cells when compared with HETC. Conclusion: In the present study, the cytotoxic and apoptotic inducing effect of the synthesized ZnO-NPs and HETC were assessed, and it was found that ZnO-NPs possessed potent anticancer effect against DLA cells.

Lipid mediator Leukotriene D4-induces airway epithelial cells proliferation through EGFR/ERK1/2 pathway

BackgroundCysteinyl leukotrienes (CysLTs), the potent lipid inflammatory mediators, are elevated in many pathological conditions and implicated in various inflammatory diseases including asthma, however their role in airway epithelial cells modulation is not clearly understood. We have investigated the effects of a CysLT, Leukotriene D4 (LTD4) on human airway epithelial cells, and assessed its role and mode of action in these cells. MethodologyHuman small airway epithelial cells (SAECs) and A549 cells were incubated with different concentrations of LTD4 for different time intervals. Subsequently trypan blue dye exclusion assay, MTT assay, Western blotting, RT-PCR and immunofluorescence experiments were performed to examine the effects of LTD4 on proliferation and related molecular changes in the airway epithelial cells. ResultsThe treatment of human airway epithelial cells with LTD4 resulted in a significant increase in cell proliferation and modulation in the expression of receptors, CysLT1R and CysLT2R in SAECs as well as A549 cells. In both types of cells, LTD4 increased the expression levels of PCNA and c-myc, and trans-activated EGF receptor and increased the activation of ERK1/2. When treated along with epidermal growth factor (EGF), LTD4 showed a marginal additive effect in ERK1/2 and EGFR phosphorylation compared to LTD4 alone in both types of airway epithelial cells. ConclusionIn conclusion, these results suggest that sustained presence of lipid inflammatory mediator LTD4 could induce human airway epithelial cell proliferation through ERK1/2 phosphorylation, either directly via CysLT1 receptor or by transactivating EGFR.

Genotoxicity and Cytotoxicity Assessment of Graphene Oxide Nanosheets on HT29 Cells

Background: Graphene and graphene-related materials are broadly applied for biomedical purposes due to their unique properties. However, little information is available on their toxicity and biocompability, thus it is necessary to assess their safety. We investigated the cytotoxic and genotoxic effects of graphene oxide nanosheets (GOs) on HT29 cells. Methods: GOs were synthesized by oxidation of natural graphite flakes according to the modified Hummers method. GOs were characterized using IR spectroscopy and TEM. Cytotoxicity and genotoxicity of GOs on HT29 cells were evaluated in the range of 10 - 250 µg/mL after 24, 48 and 72 hours of treatment using MTT assay and in the range of 10 - 50 µg/mL after 48h by micronucleous assay. Results: The results of IR indicate that the graphite is successfully oxidized. The GOs were found to be 3 - 6 µm in length and ≤ 1 nm in diameter by TEM. The half maximal inhibitory concentration (IC50) in different doses were calculated at 50 µg/mLby trypan blue dye exclusion assay. The results of MN assay showed a significant increase in DNA damage and MN formation in GOs treated samples as compared with the control sample (P < 0.05). Conclusions: Concentration, shape, time and physicochemical properties GOs like size, shape, agglomeration rate and surface chemistry play important roles in inducing cytotoxicity and genotoxicity of treated cells.

Synthesis and Biological Evaluation of Novel Thiazol-2yl-amine Derivatives as Potential Anticancer Agents

Background: Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that can occur in any age group but often seen in adults and contributing for about 20% of adult leukemias and it may contribute up to 15% of all types of leukemias threatening the globe. Therefore, treatment of CML remains as a major challenge in cancer therapeutics. Methods: We synthesized a library of novel 2-amino-4-(4-substituted phenyl) thiazole derivatives and evaluated their anti-leukemic activity by trypan blue and MTT assay. 4-(4'-phenoxybiphenyl-4-yl) thiazol- 2-amine (compound 3m) was identified as a lead anticancer agent and further, the effect of 3m on CML cells (K562) was investigated by flow cytometry, annexin V-FITC-propidium iodide staining, measuring the mitochondrial membrane potential (JC-1 staining) and DNA fragmentation assay. Results: MTT and trypan blue dye exclusion assay results presented 3m as the lead anticancer agent. Flow cytometric analysis revealed the accumulation of K562 cells in subG1phase in a time- and dosedependent manner. Annexin-V-FITC-PI staining demonstrated the increase in percentage of apoptotic cells on treatment with 3m. Furthermore, 3m also induced DNA fragmentation and disrupted mitochondrial membrane potential in K562 cells in dose-dependent manner. In addition, apoptosis inducing effect of 3m was reconfirmed by live-dead assay and confocal microscopic studies. Conclusion: The present study suggests that compound 3m has the potential to be a promising candidate for the treatment of CML. Keywords: Anticancer agent, chemotherapeutics, hantsch reaction, K562, suzuki coupling, thiazole.

Anti-proliferative effect of isorhamnetin on HeLa cells through inducing G2/M cell cycle arrest.

As a major cancer type in females, cervical cancer has been explored in depth by researchers. HeLa is a cervical cancer cell line. Isorhamnetin is an O-methylated flavonol that is primarily extracted from sea buckthorn. In the present study, the anti-proliferative effect of isorhamnetin on HeLa cells was evaluated using a Trypan blue dye exclusion assay. Isorhamnetin inhibited the cell proliferation in a time- and dose-dependent manner. Flow cytometric analysis of the cell cycle distribution revealed that isorhamnetin inhibited the cell cycle progression of HeLa by causing G2/M phase arrest and decreasing the proportion of cells in G1 phase. In addition, western blot analysis was performed to evaluate the presence of certain cell cycle-associated proteins. It was demonstrated that isorhamnetin inhibited the protein expression of cyclin B1, cell division cycle 25C (Cdc25C) and Cdc2, but enhanced checkpoint kinase 2 (Chk2), Cdc25C and Cdc2 phosphorylation. In addition, tubulin depolymerization participated in the isorhamnetin-induced cell cycle arrest in G2/M phase. In conclusion, the present results indicated that the anti-proliferative action of isorhamnetin is associated with arrest of the cell cycle in G2/M phase, which is a consequence of activation of the ataxia telangiectasia mutated Chk2 pathway and disruption of microtubule function.

Kolaviron and selenium reduce hydrogen peroxide-induced alterations of the inflammatory response.

The abilities of kolaviron and selenium (either separately or in combination) to prevent hydrogen peroxide-induced alterations in cell viability and activation were investigated. The cell line U937 was incubated with the antioxidants (i.e. kolaviron or selenium) for 24 h before exposure to hydrogen peroxide and cell viability was assessed via trypan blue dye exclusion assay. The U937 cells were also transformed to the macrophage form, incubated with the antioxidants before exposure to hydrogen peroxide. Subsequently, production of nitric oxide and pro-inflammatory cytokines were assessed as indices of macrophage activation. The myoblast cell line H9c2 was also incubated with Se and kolaviron for 24 h before exposure to hydrogen peroxide. Cell viability and generation of reactive oxygen species (ROS) were assessed via MTT and DCHF assays. The results revealed that hydrogen peroxide significantly reduced (p < 0.05) the viability of U937 cells which was ameliorated by kolaviron and selenium. Kolaviron and selenium also reduced hydrogen peroxide-induced secretion of nitric oxide, TNF-α, IL-1 and IL-6 by transformed U937 cells. Hydrogen peroxide also significantly reduced (p < 0.05) the viability of H9c2 cells which was significantly restored by kolaviron. Though selenium had no effect on the proliferation of H9c2 cells, co-treatment with kolaviron significantly reduced hydrogen peroxide-induced alterations. Both kolaviron and selenium also reduced hydrogen peroxide-mediated ROS production by H9c2 cells. In all cases, the combined action of kolaviron and selenium offered greater amelioration of the hydrogen peroxide-induced alterations than their separate effects (p < 0.05) but may not be synergistic or additive.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (β-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 μg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 μg/mL Bulldock®125 SC (β-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

Comparative evaluation of cytotoxic effects of MTAD and sodium hypochlorite using lactate dehydrogenase and trypan blue assays: An in vitro study

Aim: This study aims to examine the cytotoxic effects of BioPure MTAD and three clinical concentrations of sodium hypochlorite (NaOCl) solutions (1%, 3%, and 5%) on cultured human periodontal ligament (PDL) cells. Materials and Methods: Human PDL cells were grown on cell culture plates and were placed in contact with various concentrations of tested irrigants. Cytotoxicity of both BioPure MTAD and different concentrations of NaOCl was assessed for immediate short-term intervals (1, 5, 10, and 15 min) and midterm intervals as well (24, 48, 72, and 96 h). Different parameters of toxicity such as lactate dehydrogenase assay and trypan blue dye exclusion assay were studied along with morphological changes at all the different time points. The mean zones of inhibition were calculated for each group and were statistically analyzed. Results: BioPure MTAD was least toxic of all test irrigants in short-term and midterm time intervals. 5% NaOCl showed the maximum cytotoxicity at short-term time intervals. All cells died within the first 24 h for all three NaOCl concentrations. Conclusions: BioPure MTAD showed minimal toxicity and the three concentrations (1%, 3%, and 5%) of NaOCl showed detrimental effects on the PDL cells and its toxicity was dose dependent.

ILKAP Binding to and Dephosphorylating HIF-1α is Essential for Apoptosis Induced by Severe Hypoxia

Background/Aims: Integrin-linked kinase-associated phosphatase (ILKAP), a serine/threonine phosphatase that belongs to the protein phosphatase 2C family, has a role in cell survival and apoptosis. Hypoxia-inducible factor 1α (HIF-1α) is the key transcription factor in the response to oxygen deficiency in mammals. Direct phosphorylation and dephosphorylation of HIF-1α affect its function. The present study investigated the role of ILKAP on HIF-1α dephosphorylation and cell behavior. Methods: HIF-1α was induced by hypoxia. Physical binding between ILKAP and HIF-1α was demonstrated by a co-immunoprecipitation assay. HIF-1α transcriptional activity was investigated using a hypoxia-response element-containing luciferase reporter plasmid. Cell viability was evaluated by a trypan blue dye exclusion assay. ILKAP function was explored by a gain and loss assay with an overexpression plasmid and shRNA infection. Results: ILKAP physically interacted with HIF-1α and induced its dephosphorylation. Both the HIF-1α-p53 interaction and apoptosis relied on ILKAP. Conclusion: The results indicated that the ILKAP directly binds and dephosphorylates HIF-1α and responsible for severe hypoxia-induced cell apoptosis.

Evaluation on hot/cold drug property of Açaí (Euterpe oleracea Mart.) by cytological study methods

Objective: To determine the hot/cold of South American Acai (Euterpe oleracea Mart.). Materials and Methods: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to compare the influence of Acai and phellodendron bark (Cortex Phellodendri Chinensis) on the growth and proliferation of HepG2 and Hep3B cell strains. The morphological changes of the two cell strains treated with Acai and C. Phellodendri were observed using an inverted phase contrast microscope. Trypan blue dye exclusion assay was used to compare and analyze the toxicity effects of Acai. The content of nicotinamide adenine dinucleotide (NADH) and the ratio of NADH/NAD+ of the treated cells were detected using a spectrophotometer to determine the influence of Acai to the energy metabolism of the two cell strains. Results: Within the range of measured concentrations, both Acai and C. Phellodendri displayed separately the stronger inhibitory effects on cell growth, proliferation, and energy metabolism of the two cell strains. By MTT assay, Acai showed significant (P

Protective Effects of 17β-Estradiol on Benzo(e) pyrene[B(e)P]-induced Toxicity in ARPE-19 cells.

Purpose:The aim of this study was to examine the effect of 17β-estradiol on Benzo(e)pyrene [B(e)P]-induced toxicity in ARPE-19 cells.Methods:We pretreated ARPE-19 cells with 20 nM and 40 nM 17β-estradiol for 6 hours, followed by addition of 300 μM B(e)P for additional 24 hours. Cell viability was measured using Trypan blue dye-exclusion assay. JC-1 assay was performed to measure mitochondrial membrane potential (ΔΨm). For a quantitative estimation of cell death, apoptotic markers such as caspase-3/7, caspase-9, and caspase-12 were measured.Results:Our results demonstrated that when treated with B(e)P, the viability and ΔΨm of ARPE-19 cells declined by 25% and 63%, respectively (P < 0.05). However, pretreating with 17β-estradiol increased the viability of ARPE-19 cells by 21% (20 nM) and 10% (40 nM) (P < 0.05). Furthermore, the significantly reduced ΔΨm in βE+B(e)P treated cells ARPE-19 cells was restored by pre-treatment with 17β-estradiol- ΔΨm was increased by 177% (20 nM) and 158% (40 nM) (P < 0.05). We further observed a significant up-regulation in the activity of Caspases-3/7, -9, and -12 in B(e)P-treated ARPE-19 cells. However, 17β-estradiol treatment significantly reduced the activity of all apoptotic markers (P < 0.05).Conclusion:In conclusion, our results demonstrate that 17β-estradiol protects ARPE-19 cells against B(e)P-induced toxicity by decreasing apoptosis, preventing cell death, and restoring mitochondrial membrane potential.