Trypan Blue Dye Exclusion Test Research Articles

Investigation of the Cytotoxic, Genotoxic, and Apoptosis-Inducing Effects of Estragole Isolated from Fennel (Foeniculum vulgare)

The present study was undertaken to evaluate, in the HepG2 human hepatoma cell line, the in vitro cytotoxic, genotoxic, and apoptotic activities of estragole (1), contained in the essential oil of Foeniculum vulgare (fennel) and suspected to induce hepatic tumors in susceptible strains of mice. Toward this end, an MTT cytotoxicity assay, a trypan blue dye exclusion test, a double-staining (acridine orange and DAPI) fluorescence viability assay, a single-cell microgel-electrophoresis (comet) assay, a mitochondrial membrane potential (Δψm) assay, and a DNA fragmentation analysis were conducted. In terms of potential genotoxic effects, the comet assay indicated that estragole (1) was not able to induce DNA damage nor apoptosis under the experimental conditions used.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

Aim:To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells.Materials and Methods:HTM cells were cultured and treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II.Results:Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 μg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 μg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 μg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S.Conclusions:Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 μg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.

Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair

Background:In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair.Methods:Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay.Results:Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency.Conclusion:These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

BackgroundComplementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcerea carbonica-induced tumor regression.MethodsTo investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells.ResultsInterestingly, although calcarea carbonica administration to Ehrlich’s ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. Knocking out of p53 by RNA-interference inhibited calcarea carbonica-induced apoptosis thereby confirming the contribution of p53.ConclusionThese observations delineate the significance of immuno-modulatory circuit during calcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.

Japanese Black Vinegar “Izumi” Inhibits the Proliferation of Human Squamous Cell Carcinoma Cells Via Necroptosis

Kurozu (Japanese black vinegar), a traditional product made from unpolished rice, contains beneficial organic materials and minerals. Improved manufacturing processes yielded a new vinegar, Izumi, that contains large amounts of these constituents. Because the antioxidative effects of Kurozu are well understood, we examined Izumi for its anticancer activity against the human squamous cell carcinoma (SCC) cell line HSC-5. HSC-5 cells were treated with Izumi or ordinary grain vinegar adjusted to 4.2% acidicity. MTT assay and the trypan blue dye exclusion test showed that Izumi significantly inhibited the proliferation of HSC-5 cells compared to ordinary grain vinegar. Propidium iodide (PI) flow cytometry and annexin V/PI staining revealed that among cells treated or untreated with Izumi or ordinary grain vinegar there was no difference in the number of apoptotic cells. A new form of necrosis, programmed necrosis or necroptosis, has been proposed. It is mediated by receptor-interacting serine-threonine kinase 3 (RIPK3), key signaling molecule, and results in the release of cellular danger-associated molecular patterns (DAMPs). When HSC-5 cells were treated with Izumi, the cellular level of RIPK3 protein and the amount of high-mobility group protein B1, one of the DAMPs, released into culture media were remarkably increased. These findings indicate that Izumi inhibits the proliferation of human SCC cells via programmed necrosis (necroptosis).

Rac1 modulates the vitreous‐induced plasticity of mesenchymal movement in retinal pigment epithelial cells

The vitreous has been shown to induce epithelial-mesenchymal transdifferentiation because it induces fibroblast-like morphology, enhanced migration and invasion in retinal pigment epithelial cells in proliferative vitreoretinopathy. Rac1 is the principal mediator of cell migration. In the current study, the relationship between Rac1 and cell migration, and invasion in vitreous-transformed retinal pigment epithelial cells was investigated using NSC23766, a specific inhibitor of Rac guanosine-5'-triphosphatase activity, and the involvement of a Rac1 guanosine-5'-triphosphatase-dependent pathway was detected. One-way design with multiple levels and repeated measurement design. The vitreous humor was collected from 20 healthy donor eyes and the retinal pigment epithelial cells were obtained from 9 healthy donor eyes. Human low-passage retinal pigment epithelial cells were treated with normal medium or 25% vitreous medium. Rac1 activity was measured using a pull-down assay. The cytotoxicity of NSC23766 was measured using the trypan blue dye exclusion test. Cell migration was measured using a wound healing assay. Cell invasion was determined using a transwell invasion assay. Protein expression of Rac1 and phosphorylation of LIM kinase 1 and cofilin were detected by Western blot analysis. Cell migration, invasion, Rac1 activity and phosphorylation of LIM kinase 1 and cofilin. Rac1guanosine-5'-triphosphatase was activated in vitreous-transformed retinal pigment epithelial cells. A Rac inhibitor suppressed vitreous-induced migration and invasion in retinal pigment epithelial cells. Cofilin phosphorylation was activated by vitreous treatment but blocked by NSC23766. Rac1 mediates vitreous-transformed retinal pigment epithelial cells' plasticity of mesenchymal movement via Rac1 guanosine-5'-triphosphatase-dependent pathways that modulate LIM kinase 1 and cofilin activity. Rac inhibition may be considered a novel treatment for proliferative vitreoretinopathy.

Genotoxicity Testing of Two Anticaking Agents: Sodium and Potassium Ferrocyanide in vitro

Anticaking agents are mainly used in food to prevent agglomeration in certain solids, permitting a free-flowing condition. Sodium and potassium ferrocyanide are among the popular anticaking agents which are used in table salt. The present study focuses on the genotoxic effect of sodium and potassium ferrocyanide on human lymphocyte. Human lymphocytes were exposed to the sodium and potassium ferrocyanide at concentrations ranging from 0 to 10 mM for 3 h at 37oC. Cytotoxicity was evaluated by trypan blue dye exclusion test and resazurin test was carried for cell viability. To assess the extent of DNA damage comet assay was performed. The results show that potassium ferrocyanide was cytotoxic and genotoxic at the concentrations tested, whereas, sodium ferrocyanide was non genotoxic.

Combination Effect of Paclitaxel and Hyaluronic Acid on Cancer Stem-Like Side Population Cells

Cancer recurrence is the main cause of chemotherapeutic treatment failure. The mechanisms driving cancer recurrence may be due to very rare subpopulation cells, cancer stem-like cells (CSCs). Therefore, the early detection and better treatment of cancer stem-like cells are of great interest. In this study, we investigated how to eliminate the side population cells (SP), which have the characteristics of cancer stem-like cells, and also show chemotherapy resistance. Fluorescence-activated cell sorting (FACS) were used to sort SP and non-SP cells from human liver cancers, Huh-7 Hyaluronic acid (HA), which is an abundant component in the extracellular matrix, is known to involve in proliferation of normal and cancer cells. Herein, we investigated the effect of HA component on chemotherapy against SP cells. Cell growth inhibitory effects of the paclitaxel (PTX) chemotherapy combined with the HA component on SP cells of Huh-7 was determined using the trypan blue dye exclusion test. PTX combined with HA was found to show more increased inhibition of cell growth in both SP and non-SP cells, compared to free PTX treatment. In conclusion, SP cells of Huh-7 shows chemotherapeutic drug resistance due to the over-expressed efflux pumps. HA proposed one of possibilities to overcome the limitation of chemotherapy against cancer stem-like cells.

Protein Profiling of Hematopoietic Progenitor Cells and Leukemia Cells Identifies Novel Biomarkers Associated with Exposure of Polycyclic Aromatic Hydrocarbons

AbstractAbstract 4690 Background:Polycyclic aromatic hydrocarbons (PAHs) are one of the major environmental hazardous compounds in living environment generated by combustion of fossil fuels. PAHs are classified as carcinogens by International Agency for Research on Cancer. However, the study on biomarkers and biological effect of PAHs has not been thoroughly studied in peripheral blood stem cells (PBSC) and leukemia cell lines. Therefore, the present study investigated biological characteristics and biomarkers in hematopoietic cells and leukemia cell lines after exposure of PAHs. Materials and Methods:Cytokine stimulation (10 mg/kg/d) was used in a 28-year-old healthy male donor from 5 d before to 2 d after. One to three apheresis procedures were planned for on day 0, day 1, and day 2, using a Baxter CS-3000 PLUS machine (Baxter Healthcare) and a COBE-Spectra (Gambro). PBSC and three leukemia cell lines (THP-1, Molt-4 and K562) were cultured in RPMI media containing 10% fetal bovine serum. In this study, four types of PAHs- Benzopyrene (BaP), Pentacene, Fluoranthene and Pyrene -were added in the cell culture media with 100μM concentration (Sigma-Aldrich, USA). Cell count was performed using an automated blood cell analyzer. Viability and apoptosis were assessed by trypan blue dye exclusion test and flowcytometry based on annexin V staining protocol. Cytotoxicity was determined by the water-soluble tetrazolium salts -1 assay. Proteomics in mitochondrial-rich cytoplasmic fraction was performed using nano-LC-ESI-MS/MS analysis. The acquired fragment spectra were searched in the BioWorksBrowser (version Rev. 3.3.1 SP1, Thermo Fisher Scientific Inc., CA) with the SEQUEST search engines against National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov/) non-redundant human database. Results:Depending on the type of PAHs, each cell count showed different aspects. In comparison to control group, Fluoranthene displayed profound significant reduction in cell count, especially in PBSC and THP-1 cells. Viability decreased significantly after two days of exposuring into Fluoranthene. On the third day of PAHs exposure, viability reduced remarkably in all the cells. Each type of cell lines displayed different proportionality of apoptosis. Several hundreds of PAHs exposure biomarkers were identified in comparison to control group. The notable biomarkers for PAHs exposure were displayed as following: prelamin-A/C isoform 3 and annexin A1 for BaP; prelamin-A/C isoform 3 and DNA topoisomerase 2-alpha for Pentacene; poly [ADP-ribose] polymerase 1 for Fluoranthene; talin-1 and DNA topoisomerase 2-alpha for Pyrene. By using Gene Ontology tool, we further identified protein candidate biomarkers for PAH exposure. Conclusion:This study shows new biomarkers and biological characteristics of polycyclic aromatic hydrocarbons exposure in hematopoietic tissue. Specifically, talin-1 was identified as universal biomarker for PAHs exposure. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Antineoplastic activity of a nutrient mixture in Y-79 malignant retinoblastoma cells

Retinoblastoma is one of the most common ocular malignancies in children under the age of six. Occasionally, retinoblastoma metastasizes to extraocular organs including the bone, lung and brain. Left untreated, retinoblastoma is fatal. At present, there is no effective treatment for metastatic retinoblastoma. We investigated the antineoplastic activity of a nutrient mixture (NM) (lysine, proline, ascorbic acid and green tea extract) at concentrations of 10, 50, 100, 500 and 1,000 μg/ml in triplicate at each dose in the human malignant retinoblastoma Y-79 cell line. The parameters used were cell proliferation, expression of matrix metalloproteinases (MMPs), invasion through Matrigel, morphology and apoptosis. Cell viability was assessed by trypan blue dye exclusion test. Invasion was evaluated through Matrigel and MMP activity by gelatinase zymography. H&E staining for morphological cell alterations and apoptotic studies using the Live Green Poly Caspase Detection kit were also conducted. The nutrient mixture at 10–100 μg/ml demonstrated approximately 25% toxicity towards Y-79 retinoblastoma cells and significant toxicity at 500 and 1,000 μg/ml. The Y-79 cells secreted only MMP-2 as demonstrated by zymography; the nutrient mixture had no effect on MMP-2 expression up to 100 μg/ml, but completely blocked it at 500 μg/ml. Importantly, Y-79 retinoblastoma cells were not invasive through Matrigel. H&E staining showed cell morphological changes related to apoptosis, which was confirmed using the Live Green Poly Caspase Detection kit. Our results suggest that this nutrient mixture, which inhibited cell proliferation, expression of MMP-2 and induced apoptosis, may be a candidate for further exploration for its therapeutic potential in metastatic retinoblastoma.

Craniopharyngioma cell growth is promoted by growth hormone (GH) and is inhibited by tamoxifen: Involvement of growth hormone receptor (GHR) and IGF-1 receptor (IGF-1R)

Hormone receptors are related to the biological behavior and recurrence of craniopharyngioma (CP). The effect of therapeutic growth hormone (GH) replacement on CP growth remains largely unclear. We intended to study expression patterns of GH-related receptors in CP, and to study the effect of GH and its mediator, insulin-like growth factor-1 (IGF-1), as well as tamoxifen, on primary CP cell cultures. Primary cell cultures were established from fresh tumor specimens. The expression of GH receptor (GHR) and IGF-1 receptor (IGF-1R) in tumor specimens was studied using immunohistochemistry. Cell cultures were treated with various concentrations of recombinant GH, IGF-1 and tamoxifen. Cell growth promotion or inhibition was assayed using the Trypan blue dye exclusion test of cell viability. Expression of GHR, IGF-1R, phosphorylated-Akt and Akt after treatment was studied using Western blot assay. Twenty-nine primary cultures from 36 patients were established. GHR and IGF-1R were expressed in tumor tissue. The promotion of cell growth by GH compared to control was most prominent at 100ng/mL, while inhibition by tamoxifen was concentration dependent. IGF-1 was more effective in promoting growth in CP cell cultures with high IGF-1R expression, and it increased phosphorylation of Akt protein. Primary cell cultures can be established in more than 80% of fresh CP specimens. GH and its endogenous mediator, IGF-1, promotes CP cell growth in vitro, while tamoxifen inhibits growth.

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24 h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC(50) doses within the range of 1-100 nM. As the results were evaluated, IC(50) doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50 nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as -1.48, 6.59 and -13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25 × 10(-5), 2.20 × 10(-8) and 6.20 × 10(-9), respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.

Sentulic acid: A cytotoxic ring A-seco triterpenoid from Sandoricum koetjape Merr

A new ring A-seco triterpenoid, sentulic acid, along with a known oleanane-type triterpenoid, 3-oxoolean-12-en-27-oic acid, were isolated from the Indonesian plant Sandoricum koetjape Merr. Their chemical structures were elucidated by spectroscopic analysis. In addition, the cytotoxic effects of these compounds on human promyelocytic leukemia HL-60 cells were studied. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and trypan blue dye exclusion tests showed that sentulic acid and 3-oxoolean-12-en-27-oic acid were able to induce cytotoxicity in these cells. Furthermore, morphological examination and DNA fragmentation analysis indicated that these cytotoxic effects were mediated by apoptosis.

Abstract 1170: Anti-tumor effects of p53-pathway restoring compound Prodigiosin in colorectal cancer involve effects on apoptotic signaling, angiogenesis and cancer stem cells

Abstract Colorectal cancer (CRC) is the third leading cause of death due to cancer in USA. Restoration of wild-type (wt) p53 function alone has been shown to cause tumor regression in mice with minimal toxicity. p53-restoration compounds may provide a tumor-specific therapeutic regimen to overcome the resistance and toxicity associated with current CRC therapy. Utilizing a p53-responsive luciferase reporter-based screen we identified the fluorescent red bacterial pigment Prodigiosin as a potent p53 family transcriptional activator. Anti-tumoral properties of Prodigiosin have been reported for various types of cancer both in vitro and in vivo with minimal toxicity to normal tissues but the p53 mechanism has not been previously recognized. Prodigiosin has been shown to induce mitochondria-dependent apoptosis in cancer cells, but the role of the pro-apoptotic protein Bax in anti-tumor efficacy of Prodigiosin in CRC has not been clearly defined. Using fluorescence microscopy we determined that Prodigiosin localizes to the mitochondria in CRC cells. Results of trypan-blue dye exclusion tests reveal that HCT116 wild-type and Bax-null cells were equally sensitive to Prodigiosin-induced cell death. Thus, Bax played a minor role in Prodigiosin-induced cell death. The p53 family transcriptional activation by Prodigiosin is independent of the p53 status of the CRC cells. In vivo Prodigiosin induced p53 transcriptional activity and reduced tumor volume in subcutaneous xenografts of CRC cells. Prodigiosin induced apoptosis, inhibited cell proliferation and suppressed angiogenesis in vivo in CRC cells. Emerging evidence suggests that CRC tumors contain a rare population of CRC stem cells (CSCs) capable of self-renewal that are responsible for long-term sustenance of the tumor, local tumor recurrence and metastatic relapse. CSCs are therapy resistant since they are dormant and possess various efflux mechanisms. We hypothesize that anti-tumor effects of Prodigiosin involve targeting of the CSC population by p53 restoration in CSCs. With flow cytometric analysis for Prodigiosin fluorescence we determined that Prodigiosin is not effluxed from CRC cells. We have previously demonstrated that efflux of calcein AM is a marker for the cancer stem cell population. Here, we report that Prodigiosin is retained by the calcein low population (CloP) and induced apoptosis in CloP, as determined by fluorescence microscopy. Prodigiosin suppressed the ability of CRC cells to be serially passaged in vitro. Ongoing work involves determination of: mechanism of anti-angiogenic effect of Prodigiosin; effect of Prodigiosin on CSC frequency, self renewal and tumorigenicity; synergy of Prodigiosin with chemotherapeutic drugs for CRC in targeting CSCs. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1170. doi:1538-7445.AM2012-1170

Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

Differential immunotoxic effects of ethanol on murine EL-4 lymphoma and normal lymphocytes is mediated through increased ROS production and activation of p38MAPK

Ethanol has been used to achieve thymic depletion in myasthenia gravis patients. Ethanol (95%) has also been used widely in the therapy of many tumors including hepatocellular carcinoma. In light of these findings, we delineated the differential immunotoxic behavior and mechanism of lower concentration of ethanol towards murine EL-4 lymphoma and its normal counterpart lymphocytes. EL-4 lymphoma and normal lymphocytes were cultured with ethanol (0%–5%) for 6 h and cytotoxicity was measured by various methods. EL-4 cells treated with ethanol showed concentration-dependent loss of viability at 2%–5% ethanol concentration and exhibit proliferative arrest at preG1 stage. Acridine-orange and ethidium-bromide staining indicated that ethanol induced death in EL-4 cells, by induction of both apoptosis and necrosis which was further supported by findings of DNA-fragmentation and trypan blue dye exclusion test. However, treatment of lymphocytes with similar concentration of ethanol did not show any death-associated parameters. Furthermore, ethanol induced significantly higher ROS generation in EL-4 cells as compared to lymphocytes and caused PARP cleavage and activation of apoptotic proteins like p53 and Bax, in EL-4 cells and not in normal lymphocytes. In addition, ethanol exposure to EL-4 cells led to phosphorylation of p38MAPK, and upregulation of death receptor Fas (CD95). Taken together, these results suggest that ethanol upto a concentration of 5% caused no significant immunotoxicity towards normal lymphocytes and induced cell death in EL-4 cells via phosphorylation of p38MAPK and regulation of p53 leading to further activation of both extrinsic (Fas) and intrinsic (Bax) apoptotic markers.

Modulatory Effect of Phytoproteins Isolated from Roots of Boerhaavia diffusa and Leaves of Clerodendrum aculeatum on Neoplastic Growth of Human Breast Cancer Cell Line

The anticarcinogenic potential of phytoproteins isolated from B.diffusa and C. aculeatum were evaluated in MCF-7 cell lines obtained from human breast adenocarcinoma having the membrane receptor for estrogens and androgen. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 0.1% antibiotic-antimycotic in a humidified atmosphere in 5% CO 2 at 37°C. Monolayered batches of MCF7 cells showing cell viability more than 95% in Trypan Blue Dye exclusion test were used in all the experiments. Independent exposure of proteins of Boerhaavia diffusa showed a dose dependent reduction in cell growth at 0.1X, 1.0X and 10.0X concentrations in all the time intervals i.e., from 24 hr to 96 hr. However, 20.0X concentration were found to be cytotoxic. Isolated proteins of Clerodenrum aculeatum were found almost equally effective under experimental conditions. Interestingly, a synergistic effect on the growth inhibition (36.5 ± 3.1%) was observed on co-exposure of proteins of both the plants. However, mixture of proteins was also found to be cytotoxic at 20.0X concentration. The data of cell proliferation assay were also corresponding to the growth inhibition. Findings are suggestive that the combination of isolated proteins from Boerhaavia diffusa and Clerodendrum aculeatum have the potential of growth inhibition in MCF-7 cells under experimental conditions without having any cytotoxicity up to 10.0X concentrations and thus could be a useful anticancerous drug of future and will also be helpful for rural health care.

Modified Field stain - rapid viability test for Trichomonas vaginalis

We previously reported that Modified Field Stain (MF) can be used as a rapid stain for diagnosis. In the present study we extend the observation to include the stain as an alternative method to assess viability of the cells. Six isolates of Trichomonas vaginalis were used to assess the utility of the Modified Field stain as a rapid viability test for T. vaginalis and to compare with 0·4% Trypan Blue dye exclusion test in three conditions; normal in vitro culture growth using Hollander medium, lysed in distilled water and treated with metronidazole. MF stain showed similar growth profile pattern as Trypan Blue dye exclusion for identifying viable cells of T. vaginalis. Although, Trypan Blue dye exclusion test is ready made, rapid and widely used in laboratory as reliable viability assay, however, the limitation using Trypan Blue is the dye was unable to show internal morphological changes during the parasite's transition from being viable to non-viable. On day 3 where cultures peaked the correlation factor of both assays done to assess the viability of parasites harvested from the controls, metronidazole and distilled water treated parasites were more than 0·9 respectively. This confirms that MF staining does not only record permanently the morphological changes and retain internal structural details but also provides a reliable and rapid viability assay for the parasites. Therefore, in our study, Modified Field's stain may offer the researchers and laboratory technologists the opportunity to get the result on the same day and the most important thing is the ability to differentiate between viable and non-viable of T. vaginalis under three different conditions (normal culture, drug and distilled water condition). Modified Field's staining method enhanced the morphological identification of T. vaginalis compared to Trypan Blue dye exclusion.

Src Kinases Act Downstream Akt and Are Required for the Transforming Properties of Constitutively Activated Forms of STAT5A and STAT5B

Abstract 2378 Background:Stat5A and Stat5B transcription factors play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological neoplasia. Moreover, expression of constitutively active Stat5 (caStat5) mutants in primary hematopoietic cells can lead to leukaemia. The transforming properties of CaStat5 have been previously shown to require Akt activation. However, the mechanisms by which CaStat5 mutants and Akt are activated remain enigmatic. In this study we aimed to determine the roles of src kinases in these processes, Methods:For this purpose, we used murine Ba/F3 cells transfected or not with CaSta5A (Stat5A1*6) or CaStat5B (Stat51*B6) or with the oncogenic fusion proteins Tel-JAK2 and Tel-Abl as controls. Primary hematopoietic progenitors obtained from mouse bone marrow were also used in this sutdy. Cells were treated or not with the pan-src inhibitors PP1, PP2, and SU 6656 (and with PP3 an inactive analogue of PP1 as a control), imatinib (Abl inhibitor) and AG490 (JAK inhibitor). In some experiments cells were treated with U0126 (MEK inhibitor) or with the highly transducible TaT-dominant negative (dn) Akt fusion protein. Cell proliferation was assessed by cell counting. Cell death was evaluated using the trypan blue dye exclusion test. Cell apoptosis was assessed by analysing annexin V staining using flow cytometry and PARP cleavage using western blot. Expression of kinases and their phosphotylated forms were examined by western blot. Results:CaStat5A and CaStat5B cells were shown to express constitutively phosphorylated Lyn, Hck, Src kinases as well as Akt, IKK and ERK1/2. Stat1, Stat3, and JAK family members (TYK2 and JAK1-3) were not phosphorylated in these cells. Use of pan-Src inhibitors inhibited proliferation of CaStat5 cells and induced apoptosis of a fraction of these cells, in a dose-dependent manner. By contrast, imatinib and AG490 had no effect on the growth and survival of CaStat5 cells. Moreover, pan-src inhibitors prevented Erk but not Akt, IKK or STAT5 phosphorylation. By contrast, inhibition of Akt by means of TaT-dnAkt abolished Lyn, Src and ERK1/2 phosphorylation. Importantly, inhibition of MAPK did not interfere with Akt phosphorylation and did not interfere with CaStat5 cell survival. Conclusion:Altogether our data indicate that Akt acts upstream in the CaStat5-associated signalling cascade, then activates src kinases and thereby promotes MAPK-dependent cell proliferation and MAPK-independent cell survival. These results shed light onto a hitherto undescribed role of Src kinases acting downstream Akt in the mechanisms of action of CaStat5. Disclosures:No relevant conflicts of interest to declare.

Natural polyphenols may ameliorate damage induced by copper overload

The effect of the simultaneous exposure to transition metals and natural antioxidants frequently present in food is a question that needs further investigation. We aimed to explore the possible use of the natural polyphenols caffeic acid (CA), resveratrol (RES) and curcumin (CUR) to prevent damages induced by copper-overload on cellular molecules in HepG2 and A-549 human cells in culture. Exposure to 100μM/24h copper (Cu) caused extensive pro-oxidative damage evidenced by increased TBARS, protein carbonyls and nitrite productions in both cell types. Damage was aggravated by simultaneous incubation with 100μM of CA or RES, and it was also reflected in a decrease on cellular viability explored by trypan blue dye exclusion test and LDH leakage. Co-incubation with CUR produced opposite effects demonstrating a protective action which restored the level of biomarkers and cellular viability almost to control values. Thus, while CA and RES might aggravate the oxidative/nitrative damage of Cu, CUR should be considered as a putative protective agent. These results could stimulate further research on the possible use of natural polyphenols as neutralizing substances against the transition metal over-exposure in specific populations such as professional agrochemical sprayers and women using Cu-intrauterine devices.