Trypan Blue Dye Exclusion Assay Research Articles

Suppression of p53R2 gene expression with specific siRNA sensitizes HepG2 cells to doxorubicin

Introductionp53R2 is a p53-inducible protein that contributes to DNA repair by providing dNTPs in response to DNA damage. The roles of p53R2 in cancer cells and malignancies still remain controversial. Herein, we examined the effects of p53R2 silencing on HepG2 human hepatocellular carcinoma (HHC) cell line (wild-type p53) viability, apoptosis and cell cycle arrest in the presence and absence of doxorubicin. MethodsCell transfection was performed using a liposomal approach. Gene silencing was determined by quantitative real-time PCR and western blot analysis. To evaluate the cell growth rate after transfection, trypan blue dye exclusion assay was employed. The cytotoxicity of the doxorubicin and p53R2 siRNA as single agents or in combination against HepG2 cell was analyzed by MTT assay and the drug combination effects was evaluated by calculating the combination index. The effects of treatments on different stages of cell cycle were analyzed by flow cytometry using propidium iodide (PI) and induction of apoptosis was assessed using DNA-histone ELISA. ResultsWe found that silencing of p53R2 alone had a strong effect on growth inhibition and spontaneous apoptosis in HepG2 cells. p53R2 siRNA synergistically enhanced the cytotoxic effect of doxorubicin. Furthermore, when used in combination with doxorubicin (0.4μM), a significant increase in the rate of apoptosis was observed (P<0.05). Moreover, cell cycle at S and G2/M phases progressed at a lower rate after p53R2 combination treatment compared with doxorubicin mono-therapy. ConclusionThese findings suggest that siRNA-mediated silencing of p53R2 has great potential as a therapeutic tool and adjuvant in chemotherapy.

Hypoxia negates hyperglycaemia-induced chemo-resistance in breast cancer cells: the role of insulin-like growth factor binding protein 2.

BackgroundWomen who suffer from breast cancer and type II diabetes with associated hyperglycaemia respond less well to chemotherapy. We have shown that hyperglycaemia induces resistance to chemotherapy through upregulation of fatty acid synthase (FASN) in breast cancer cells and increased insulin-like binding protein 2 (IGFBP-2) in prostate cancer cells. As a tumour develops the tumour mass can outgrow the blood supply resulting in the cancer cells being exposed to hypoxia that stimulates many tumorigenic signalling pathways.MethodsWe used MCF-7 and T47D breast cancer cell lines. Trypan blue dye exclusion assay was employed to assess cell death and Western immunoblotting was used to determine changes in protein abundance. Hypoxia was induced both chemically by the addition of cobalt chloride (CoCl2) and using a hypoxia chamber.ResultsIGFBP-2 abundance increased with increasing concentrations of glucose (0-25 mM) that contributed to hyperglycaemia-induced chemo-resistance as it was abrogated by downregulating IGFBP-2 using siRNA. Production of IGFBP-2 is ER dependent: pre-treatment of MCF-7 cells with β-estradiol increased IGFBP-2 and induced chemo-resistance to doxorubicin. The hyperglycaemia-induced chemo-resistance and increases in FASN and IGFBP-2 were negated in a hypoxic environment, with levels of cell death unaffected by glucose concentrations.ConclusionsThe sensitivity of breast cancer cells to chemotherapy is reduced in hyperglycaemic conditions but this effect is negated by hypoxia. These effects appear to be mediated via regulation of IGFBP-2 and FASN. Understanding the role of FASN and IGFBP-2 in chemo-resistance could provide a novel target for improving the effectiveness of breast cancer treatment.

Abstract 3476: Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence

Abstract Telomerase, the enzyme responsible for the length maintenance of telomeres is expressed in about 85% of cancer cells, being a key to tumorigenesis. Telomerase inhibitor drugs have been studied as a promising cancer therapy due to its specificity, with less side effects than conventional approach. However, the effects expected from these therapy require many cell divisions, and the consequences of these long-term therapies are poorly understood. This work evaluated the effects of a long term treatment in cancer cells (HeLa) using BRACO-19, a G-quadruplex-interactive molecule, evaluating the consequences of its use in cell viability and its potential to induce senescence. Telomerase positive HeLa cells were grown in DMEM medium supplemented with 10% fetal bovine serum at pattern conditions. Culture medium was changed every 2 days. For preliminary citotoxicity assay, cells were seeded in 96 wells plate at 3.2x104 cells/cm2, incubated for 24 hours and treated for 24 hours with increasing concentrarions of BRACO-19 and vehicle (0.1% H2O). Cell viability was measured by MTT assay. For the long-term treatment, a subtoxic concentration was chosen. The cells (5.5x102 cells/cm2) were grown under continuous presence of the drug or 0.02% H2O for 11 weeks. During the long-term exposure to BRACO-19, the cells were splitted every 7 - 20 days, depending on its proliferation rate, viable and unviable cells were counted at these points through trypan blue dye exclusion assay. β-Galactosidase (β-gal) activity was used as cell senescence marker to evaluate the senescence after 24 hours treatment and after a long exposure to the telomerase inhibitor. Changes in cell morphology were accessed by phase contrast microscopy. The short-term (24h) treatment with BRACO-19 showed a dose-dependent effect on cell viability. The IC50% for HeLa was 5.25 µM. The subtoxic concentration of 1µM was used for long-term treatment, showing effect on cell proliferation in a way that HeLa cells exposed to the drug showed their proliferative profile decreased when compared to control. Furthemore, no senescent cells were found in control group, even after many passages; on the other hand, 24 hours and long-term exposure to BRACO-19 led to senescence. Long-term treatment was also associated with visual changes on cell morphology. The telomerase inhibitor BRACO-19 showed an expected dose-dependent effect on HeLa cells at short-term exposition, since cytotoxic effect of this compound has been demonstrated in several cell lines, but the impact of the long-term treatment on cell proliferation pattern points BRACO-19 as a potential prototype of a new telomere related drug for cancer therapy. Also, it was demonstrated the senescence inducing action of this drug, perhaps due to erosion of HeLa telomeres. These results show that HeLa is sensitive do BRACO-19 even under subtoxic concentrations. Its effectiveness against other cell lines and tumors is under investigation. Citation Format: Ana Flavia Reis Guimaraes, Diêgo M. de Oliveira. Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2017-3476

Abstract 2568: Mutant p53 regulates HSP70 expression and nuclear localization

Abstract Pancreatic cancer is the 3rd leading cause of cancer related deaths in the US with a median survival of less than one year. No effective therapies or early detection tests are available for these patients. Scientists have been studying cellular origins of pancreatic cancer to aid in the development of novel treatments and biomarkers. Several studies have demonstrated that acinar cells have a high propensity to undergo acinar to ductal metaplasia and form precursor lesions of pancreatic ductal adenocarcinoma. We have recently shown that simultaneous expression of Kras and mutant TP53 can generate invasive ductal adenocarcinoma from ductal cells. We hypothesized specific mutations in TP53 have different mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells into invasive ductal adenocarcinoma, we used a lentiviral system to express mutant TP53R175H and TP53R273H, two of the most frequently mutated TP53 alleles in pancreatic cancer patients, in immortalized pancreatic ductal epithelial cells carrying a Kras mutation (HPNE-KrasG12D). Trypan blue dye exclusion assay and Spheroid formation assays were used to study cellular proliferation and cancer stem cell (CSC) populations. Mutant TP53 over expression enhanced CSC populations without altering cellular proliferation in HPNE-KrasG12D cells. Reverse phase protein array (RPPA) was carried out to detect gene expression changes in HPNE-KrasG12D cells upon mutant TP53 over expression. RPPA assay results suggested that TP53R175H uniquely induced HSP70 expression in HPNE-KrasG12D cells, as cells expressing either vector control or TP53R273H failed to do so. HSP70 expression was further validated by transiently overexpressing TP53R175H and TP53R273H. Surprisingly, TP53R175H specifically promoted nuclear localization of HSP70 without altering the expression of a recently identified HSP70 nuclear transporter, Hikeshi. Future studies will determine 1) whether HSP70 is required for p53-mediated stemness in HPNE-KrasG12D cells and 2) the function of nuclear HSP70. In summary, over expression of mutant p53 enhanced cancer stem cell properties of HPNE-KrasG12D cells, through upregulation HSP70. The exact mechanism behind HSP70 nuclear localization and increased cancer stem cell properties is being more rigorously explored. Citation Format: Kishore Polireddy, Kanchan Singh, Melissa Pruski, Wasim Dar, John S. Bynon, Jennifer M. Bailey. Mutant p53 regulates HSP70 expression and nuclear localization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2017-2568

Abstract 5484: Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer

Abstract BACKGROUND: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and elevated βIII-tubulin expression is associated with chemotherapy resistance and tumor aggressiveness1. βIII-tubulin modulates glucose metabolism and stress response signaling to support cancer cell survival and proliferation in glucose starvation conditions in the tumor microenvironment2. βIII-Tubulin shares sequence similarity with other β-tubulin isotypes, differing in the C-terminal tail region of the protein, however the function of βIII-tubulin structural domains remain uncharacterized. This study investigated the importance of the βIII-tubulin body and tail structural domains in conferring survival advantage to glucose-starved NSCLC cells. METHODS: H460 NSCLC cells were gene-edited at the endogenous βIII-tubulin gene using zinc finger nuclease technology to replace the endogenous βIII-tubulin protein with either the full-length or truncated βIII-tubulin protein, or replacement of the βIII-tubulin body or tail with the βI-tubulin sequence, each fused to a GFP tag. Clones expressing the modified βIII-tubulin proteins were cultured in glucose-free conditions for 48h to 10 days and cell growth and viability were measured by Trypan Blue Dye Exclusion and Annexin-V apoptosis assay. The effect of the βIII-tubulin modifications on the endoplasmic reticulum (ER) stress response, autophagy and Akt signaling were examined by western blotting. RESULTS: Characterization of the gene-edited clones revealed that loss of the βIII-tubulin C-terminal tail or substitution of the βIII-tubulin body or tail sequence with that of the βI-tubulin sequence did not alter the microtubule architecture, cellular proliferation rate or viability in normal growth conditions. However, loss of the βIII-tubulin C-terminal tail significantly reduced the viability of H460 cells by 37 ± 0.05% in acute glucose starvation. Substitution of the βIII-tubulin body with the βI-tubulin body significantly reduced cell number by 53 ± 0.01% during prolonged glucose starvation. Loss of the βIII-tubulin C-terminal tail increased Akt activation and LC3B-II expression in glucose-rich conditions and potentiated the increase in ER stress response markers GRP78, ATF4 and CHOP in response to glucose starvation. These results indicate that the βIII-tubulin body and tail differentially confer a survival advantage to glucose-starved H460 cells and modulate multiple stress response signaling pathways in an isotype-dependent and independent manner. CONCLUSION: This study provides insight into the importance of βIII-tubulin structural domains in conferring βIII-tubulin function in NSCLC, thereby advancing our understanding of the role of tubulin isotypes in cell biology and the mechanisms contributing to poor patient outcome in this disease. 1 Kavallaris, Nat Rev Cancer 2010 2Parker et al., Carcinogenesis 2016 Citation Format: Amelia L. Parker, Wee S. Teo, Joshua A. McCarroll, Maria Kavallaris. Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2017-5484

Non-homologous End Joining Inhibitor SCR-7 to Exacerbate Low-dose Doxorubicin Cytotoxicity in HeLa Cells.

Among the genotoxic drug regimens, doxorubicin (DOX) is known for its high-dose side effects in several carcinomas, including cervical cancer. This study reports on testing the combined use of a DOX genotoxic drug and SCR-7 non-homologous end joining (NHEJ) inhibitor for HeLa cells. An in vitro DNA damaging assay of DOX was performed on plasmid and genomic DNA substrate. In vitro cytotoxicity was investigated using trypan blue dye exclusion, DNA metabolizing, and propidium iodide-based flow cytometric assays. DOX (between 20–100 μM) displayed clear DNA binding and interaction, such as the shearing and smearing of plasmid and genomic DNA. DNA metabolizing assay data indicate that HeLa lysate with DOX and SCR-7 treatment exhibited better in vitro plasmid DNA stability compared with DOX treatment alone. SCR-7 augmented the effects of low-dose DOX by demonstrating enhanced cell death from 15% to 50%. The flow cytometric data also supported that the combination of SCR-7 with DOX lead to a 23% increase in propidium iodide-based HeLa staining, thus indicating enhanced death. In summary, the inhibition of NHEJ DNA repair pathway can potentiate low-dose DOX to produce appreciable cytotoxicity in HeLa cells.

Synthesis and characterization of chitosan-alginate scaffolds for seeding human umbilical cord derived mesenchymal stem cells.

Chitosan and alginate are two natural and accessible polymers that are known to be biocompatible, biodegradable and possesses good antimicrobial activity. When combined, they exhibit desirable characteristics and can be created into a scaffold for cell culture. In this study interaction of chitosan-alginate scaffolds with mesenchymal stem cells are studied. Mesenchymal stem cells were derived from human umbilical cord tissues, characterized by flow cytometry and other growth parameters studied as well. Proliferation and viability of cultured cells were studied by MTT Assay and Trypan Blue dye exclusion assay. Besides chitosan-alginate scaffold was prepared by freeze-drying method and characterized by FTIR, SEM and Rheological properties. The obtained 3D porous structure allowed very efficient seeding of hUMSCs that are able to inhabit the whole volume of the scaffold, showing good adhesion and proliferation. These materials showed desirable rheological properties for facile injection as tissue scaffolds. The results of this study demonstrated that chitosan-alginate scaffold may be promising biomaterial in the field of tissue engineering, which is currently under a great deal of examination for the development and/or restoration of tissue and organs. It combines the stem cell therapy and biomaterials.

Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis

BackgroundPlant mediated green synthesis of nanoparticles is an eco-friendly and efficacious approach which finds immense application in the field of medicine. This study aimed to evaluate the cytotoxicity of platinum nanoparticles (ptNPs) synthesized through green technology against normal and different cancer cell lines. MethodsPlatinum nanoparticles were synthesized by green technology and characterized earlier. In this study we examined the cytotoxic effect of platinum nanoparticles (ptNPs) on human lung adenocarcinoma (A549), ovarian teratocarcinoma (PA-1), pancreatic cancer (Mia-Pa-Ca-2) cells and normal peripheral blood mononucleocyte (PBMC) cells and evaluate anticancer potential through induction of apoptosis on PA-1 cells if any. Cytotoxicity was evaluated using MTT assay, trypan blue dye exclusion assay and anticancer potential assessed through clonogenic assay, apoptosis assay, cell cycle analysis. ResultsWe found that ptNPs exerted cytotoxic effect on cancer cell lines, whereas no cytotoxic effect was observed at highest dose on normal cells. The results showed that ptNPs had potent anticancer activities against PA-1 cell line via induction of apoptosis and cell cycle arrest. ConclusionOverall, these findings have proved that biosynthesized ptNPs could be potent anti-ovarian cancer drugs. Further studies are required to elucidate the molecular mechanism of ptNPs induced anti-tumor effect in vivo.

Promising anticancer activities of Justicia simplex D. Don. in cellular and animal models

Ethnopharmacological relevanceJusticia simplex D. Don. belonging to the family of Acanthaceae has been traditionally used for treatment of rheumatism, inflammation and bronchitis. The plant is traditionally considered as an anticancer medicine and is used by healers of Karnataka to treat various types of cancers. Aim of the studyThe present study aims at the elucidation of anticancer activity of various extracts of J. simplex, isolation of its active constituents and assessment of the role in growth inhibition and angiogenesis both in vitro and in vivo. Materials and methodsExtracts of J. simplex was evaluated for the in vitro cytotoxic effect by Brine Shrimp Lethality assay, Trypan Blue dye exclusion assay and antiproliferative assay. In vivo cytotoxicity of the extracts were determined by liquid tumor model in Swiss albino mice. Tumor prognosis, metastasis and angiogenesis were assessed by VEGF expression of the solid tumor. Phytochemical analysis afforded the isolation of a compound, the chemical structure of which was established using IR, NMR and TOF-MS spectral method. The compound was also evaluated for the growth inhibitory and angiogenic effects. Results and conclusionThe petroleum ether extract revealed potent anticancer activity in in vitro and in vivo studies. The anti-angiogenic effect is due to the down regulation of VEGF expression. The growth inhibitory assay revealed that the isolated compound namely triacontanoic ester of 5ʹʹ-hydroxyjustisolin is responsible for the anticancer activity.

Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells.

Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations. Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy. We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells. From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.

Synthesis and Cytotoxicity of Novel Analogues of Podophyllotoxin

We have synthesized four novel derivatives of podophyllotoxin, the chalcone derivatives namely 2-benzylidene-6,7-dimethoxy-4-phenyl-3,4-dihydro-2H-naphthalen-1-one (4a), 2-benzylidene-5,7-dichloro-6-hydroxy-4-phenyl-3,4-dihydro-2H-naphthalen-1-one(4b), and the amino-thiazolyl derivatives namely7,8-dimethoxy-5-phenyl-4,5-dihydro-naphtha[1,2-d] thiazol-2-yl amine (5a) and 2-amino-6,8-dichloro-5-phenyl-4,5-dihydro-naphtho[1,2-d] thiazol-7-ol (5b). The aryl tetralin ring system in these compounds were attached to a fused aminothiazolyl ring or a chalcone ring. Synthesis of the tetralin ring in these podophyllotoxin derivatives was accomplished by the cyclization of the γ-hydroxyketones. These derivatives were evaluated for the induction of cytotoxicity in mouse mammary carcinoma cells in vitro. All the four compounds exhibited time dependent cytotoxicity at a concentration of 100μM when assessed by Trypan blue dye exclusion assay on Ehrlich Ascites Tumor (EAT) cells in vitro.

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

Background:To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin-MMPs system.Methods:MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values.Results:All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis.Conclusions:EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’.

Taurine Attenuates Doxorubicin-Induced Toxicity on B16F10 Cells.

This study aimed to investigate the effect of doxorubicin co-treatment with taurine on B16F10 melanoma cells. Frequently, Doxorubicin is used in the treatments of many different kinds of cancers, some of which are soft tissue sarcomas, hematological malignancies and carcinomas. However, the clinical application of doxorubicin is compromised by its severe adverse effects, including cardiotoxicity. In the present study, the efficacy of doxorubicin co-treatment with taurine was investigated. B16F10 cell viability was evaluated using MTT assays, trypan blue dye exclusion assays, and fluorescent staining technique. Apoptotic cells were detected by flow cytometry and the proteins associated with apoptosis and cellular differentiations were assessed by immunoblotting. Doxorubicin inhibited cell growth and induced cell death in B16F10 cells. Interestingly, doxorubicin co-treatment with taurine inhibited apoptosis in B16F10 cells. These results indicate that doxorubicin co-treatment with taurine attenuates doxorubicin-induced cytotoxicity and reduces ROS production in B16F10 cells.

Effect of isobutyl methacrylate and methacrylic acid eluted from chairside denture hard reliners on enzymatic cellular antioxidants: An in vitro study in human primary buccal mucosal fibroblasts.

Aim:This study was conducted with the objective to evaluate the cytotoxicity of monomers isobutyl methacrylate (IBMA) and methacrylic acid (MA) in human buccal mucosal fibroblast primary cell culture and to study their effect on cellular enzymatic antioxidants-glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT).Materials and Methods:The tissue for fibroblast cell culture was harvested from oral buccal mucosa of a healthy donor. Fibroblast cells were plated at a density of 1 × 104 cells per well in 96-well tissue culture plates. Cells were exposed to various concentrations of IBMA and MA. The cell viability and various enzyme activities were evaluated 24 h after exposure to the above treatments. All tests were done in triplicate. Cell viability was assessed by trypan blue dye exclusion assay and all enzyme activities were done using assay kits from Cayman Chemicals, Ann Arbor, USA.Results:At all concentrations tested a statistically significant decrease in viability was observed in IBMA- and MA-treated cells. Around 42% cells were viable at the highest test concentration of IBMA (80 μmol/L) and only 20% cells were viable at the highest dose (144 μmol/L) of MA exposure (P < 0.05). Dose-dependent decrease in the GPx and SOD activities was observed in cells treated with IBMA and MA (P < 0.05). CAT activity was not detectable in the controls. However, a fall in CAT activity was detected in cells exposed to IBMA and MA at all concentrations tested (P < 0.05).Conclusion:IBMA and MA leaching out from the chairside denture hard reliners are cytotoxic on human buccal fibroblast primary cell cultures. This could be due to the oxidative stress caused by the generation of reactive oxygen species which is evidenced by the fall in activities of antioxidant enzymes (GPx, SOD, and CAT) and cytotoxicity.

Soybean peptide fractions inhibit human blood, breast and prostate cancer cell proliferation.

In this study, we examined in vitro the bio-activity of peptide fractions obtained from soybeans against blood (CCRF-CEM and Kasumi-3), breast (MCF-7), and prostate (PC-3) cancer cell proliferation. Gastro-intestinal treated peptide fractions (<5, 5-10 and 10-50kDa) prepared from seed proteins of two high oleic acid soybean lines-N98-4445A, S03-543CR and one high protein line-R95-1705, were tested for anticancer activity against human breast, blood and prostate cancer cell lines. Anti-proliferative cell titer assay was conducted to assess the inhibitory effects of the peptide fractions, while trypan blue dye exclusion assay was used to determine the dose response of most effective fractions. Results showed that the peptide fractions inhibited the cancer cell lines up to 68.0% and the minimum concentration to get 50% inhibitory activity (IC50) ranged between 608 and 678µg/mL. This multiple site in vitro cancer inhibition by GI friendly peptides could have the potential use as food ingredients or nutritional supplements in an alternative cancer therapy.

Influence of Arsenic (III), Cadmium (II), Chromium (VI), Mercury (II), and Lead (II) Ions on Human Triple Negative Breast Cancer (HCC1806) Cell Cytotoxicity and Cell Viability

The hazardous consequences of heavy metal ions (HMIs) on human health necessitate the immediate need to probe fundamentally the interactions and cytotoxic effects of HMIs on humans. This study investigated the influence of five toxic HMIs (arsenic (As (III)), cadmium (Cd (II)), chromium (Cr (VI)), mercury (Hg (II)), and lead (Pb (II))) on human TNBC (HCC 1806) cell viability using optical microscopy, trypan blue dye-exclusion assays, and flow cytometry. The TNBC cells were exposed to varying concentrations of HMIs for 24 and 48 hours. We evaluated the influence of the concentrations and duration of HMIs exposure on TNBC cell viability. Light microscopy, cell viability assays, revealed that after 48-hour treatment of TNBC cells with 1 x 10 -5 M of As (III), Cd (II), Hg (II), Cr (IV), and Pb (II) resulted in cell viabilities of 23%, 34%, 35%, 56%, 91% respectively, suggesting that As (III) has the greatest cytotoxicity (77% cell death) while Pb (II) showed the least (9% cell death). Furthermore, flow cytometry revealed that while Pb (II), As (III) and Cr (IV) had significant increases in cell death, Hg (II) caused a G1 arrest. Together, this study revealed that HMIs cause a differential cytotoxic effect on TNBC cells and suggest that they may have very different genotoxic targets and implications in their mutagenic potential.

Beta radiation induces apoptosis in human histiocytic lymphoma cells

Objective: Lu-177 has a great potential for use as theranostics radiopharmaceuticals for management of cancer, evidenced by its increasing use over the past decade in nuclear medicine. The detail mechanisms of cell toxicity induced by the beta-radiation emitted from Lu-177 are not well understood. Hence, to explore the lurking mechanism of cell death, different parameters were assessed after treatment of human histiocytic lymphoma cells (U937) with Lu-177. Materials and Methods: U937 cells (1 × 106) were exposed to 3.7 and 37 MBq of Lu-177 and incubated for 24 and 48 h in humidified CO2incubator. The cell viability and apoptosis were estimated in treated and control cells by trypan blue dye exclusion and electrophoretic DNA ladder assay, respectively. Reverse transcriptase polymerase chain reaction was carried out to study the expression of anti-apoptotic genes. Results: It was found that the cell death and apoptosis were high in the cells which were exposed for a longer duration of time with 37MBq of Lu-177. These results were further confirmed by observation of downregulation of anti-apoptotic genes such as BCL-2 and BCLXL. Conclusion: It is concluded from the study that the Lu-177 induced apoptotic cell death in human histiocytic lymphoma cells by downregulation of anti-apoptotic genes.

Inhibitory Effect of Mesenchymal Stem Cell Co-Culture on Erythroid Differentiation of K562 Cells Compared to The Control Group.

Bone marrow mesenchymal stem cells (BMMSCs) reside in the bone marrow and control the process of hematopoiesis. They are an excellent instrument for regenerative treatment and co-culture with hematopoietic stem cells (HSCs). In this experimental study, K562 cell lines were either treated with butyric acid and co-cultured with MSCs, or cultivated in a conditioned medium from MSCs plus butyric acid for erythroid differentiation. We used the trypan blue dye exclusion assay to determine cell counts and viability in each group. For each group, we separately assessed erythroid differentiation of the K562 cell line with Giemsa stain under light microscopy, expression of specific markers of erythroid cells by flowcytometry, and erythroidspecific gene expressions by real-time polymerase chain reaction (RT-PCR). There was enhandced erythroid differentiation of K562 cells with butyric acid compared to the K562 cell line co-cultured with MSCs and butyric acid. Erythroid differentiation of the K562 cell line cultivated in conditioned medium with butyric acid was higher than the K562 cell line co-cultured with MSCs and butyric acid, but less than K562 cell line treated with butyric acid only. Our results showed that MSCs significantly suppressed erythropoiesis. Therefore, MSCs would not be a suitable optimal treatment strategy for patients with erythroid leukemia.

Effect of ethyl acetate aroma on viability of human breast cancer and normal kidney epithelial cells in vitro

BackgroundAromatherapy is used in clinical settings for patients suffering from several chronic and critical diseases such as cancer. Ethyl acetate (EA) is a colorless liquid with a characteristic fruity smell and is naturally present in fruits and wines. MethodsIn the present study, the effect of the aroma of EA was evaluated on human breast cancer cell line MDA-MB-231 and normal cell line, Vero. Cell line viability and mechanism of EA cytotoxicity were determined by Trypan blue dye exclusion assay and phase contrast microscopy. ResultsIt was found that EA at a concentration of 0.026M was effective in causing considerable cytotoxicity in breast cancer cells (without even coming in contact with the culture medium and cells), while showing no effect on normal cells. Mechanism of action of EA on cancer and Vero cells was investigated by DNA fragmentation and dye binding assays using agarose gel electrophoresis (AGE) and fluorescence microscopy/cytometry, respectively. It was found that EA aroma induced predominantly necrosis in the cancer cells exposed to it. ConclusionA study such as this has not been attempted before and results need further investigation before EA aroma can be used as a complementary therapy.

Hyperglycaemia-induced resistance to Docetaxel is negated by metformin: a role for IGFBP-2.

The incidence of many common cancers varies between different populations and appears to be affected by a Western lifestyle. Highly proliferative malignant cells require sufficient levels of nutrients for their anabolic activity. Therefore, targeting genes and pathways involved in metabolic pathways could yield future therapeutics. A common pathway implicated in energetic and nutritional requirements of a cell is the LKB1/AMPK pathway. Metformin is a widely studied anti-diabetic drug, which improves glycaemia in patients with type 2 diabetes by targeting this pathway. We investigated the effect of metformin on prostate cancer cell lines and evaluated its mechanism of action using DU145, LNCaP, PC3 and VCaP prostate cancer cell lines. Trypan blue dye-exclusion assay was used to assess levels of cell death. Western immunoblotting was used to determine the abundance of proteins. Insulin-like growth factor-binding protein-2 (IGFBP-2) and AMPK genes were silenced using siRNA. Effects on cell morphology were visualised using microscopy. IGFBP-2 gene expression was assessed using real-time RT-PCR. With DU145 and LNCaP cells metformin alone induced cell death, but this was reduced in hyperglycaemic conditions. Hyperglycaemia also reduced the sensitivity to Docetaxel, but this was countered by co-treatment with metformin. LKB1 was required for the activation of AMPK but was not essential to mediate the induction of cell death. An alternative pathway by which metformin exerted its action was through downregulation of IGFBP-2 in DU145 and LNCaP cells, independently of AMPK. This finding could have important implications in relation to therapeutic strategies in prostate cancer patients presenting with diabetes.