Trypan Blue Exclusion Method Research Articles

Age-related changes in cell yield and viability of feline Adipose Tissue-Derived Mesenchymal Stem (fAD-MSCs)

In the present study, omental adipose tissue was collected from, the animals that underwent ovariectomy and ovariohysterectomy, surgical procedures, at the age of seven months to 11/2 years of age groups. The sample was digested with 0.1% (W/V) collagenase type I and transferred to a beaker with a magnetic stirrer and kept in a stirrer with 600 rpm at 37 °C for 30 minutes. The viability of the cell was evaluated by the trypan blue exclusion method using a hemocytometer. Trypan blue had a high affinity to nuclear DNA, which traverse the member in a dead cell and dye it blue. In the present study, the cell yield of fAD-MSCs was 8.15 ± 0.68, 6.55 ± 0.26, 4.85 ± 0.42, 3.90 ± 0.34, and 3.51 ± 0.43 in different age groups viz., 7,8,9 month 1 and 1½ year respectively. In younger age groups, cell yield and viability percentage were more than in animals of higher age groups. In the younger age group, stem cells proliferation status is considered potent for therapeutic application.

Genotoxic effects of the ochratoxin A (OTA), its main metabolite (OTα) per se and in combination with fumonisin B1 in HepG2 cells and human lymphocytes

Ochratoxin A (OTA) and fumonisin B1 (FB1) are mycotoxins distributed in a wide variety of foods for human or animal consumption and are classified as possible carcinogens for humans. This study aimed to evaluate the cytotoxic, cytostatic and genotoxic effects of OTA and its main metabolite, ochratoxin α (OTα), FB1 and three combinations of OTA and FB1 at moderate and environmental doses. Cell viability was evaluated through MTT assay and the trypan blue exclusion method. The cytostatic and genotoxic effects were evaluated through the cytokinesis-block micronucleus assay. The results showed synergistic time- and concentration-dependent cytotoxic effects of one of the combinations of OTA and FB1. In contrast, significant differences were observed in the micronuclei (MN) frequency from OTA, OTα and coexposure of OTA + FB1. Some of these combinations increased the frequency of nuclear buds, nucleoplasmic bridges, donut-shaped nuclei, necrotic and apoptotic cells and MN in mononucleated cells. In conclusion, OTA and its main metabolite OTα, as well as the co-exposure of OTA and FB1, cause stable DNA damage at environmentally relevant concentrations, which was greater in metabolically competent cells. More studies are needed to understand the chemical interactions that occur due to the joint presence of mycotoxins, which occurs commonly.

Validation of automated fluorescent-based technology for measuring total nucleated cell viability of hematopoietic progenitor cell products.

A reliable rapid method for measuring total nucleated cell (TNC) viability is essential for cell-based products manufacturing. The trypan blue (TB) exclusion method, commonly used to measure TNC viability of hematopoietic progenitor cell (HPC) products, is a subjective assay, typically uses a microscope, and includes a limited number of cells. The NucleoCounter NC-200 is an automated fluorescent-based cell counter that uses pre-calibrated cartridges with acridine orange and DAPI dyes to measure cell count and viability. This study describes the validation of the NC-200 for testing HPC's viability. Samples from 189 fresh and 60 cryopreserved HPC products were included. Fresh products were tested for viability after collection by both TB and NC-200. 7-aminoactinomycin D (7AAD) CD45+ cell viability results were obtained from a flow cytometry test. Cryopreserved products thawed specimens were tested for viability by both TB and NC-200. The NC-200 viability results were compared with the other methods. Acceptability criteria were defined as ≤10% difference between the NC-200 method and the other methods for at least 95% of the samples. Fresh products' mean viability difference between NC-200 and TB or 7AAD CD45+ method was 4.9% (95%CI 4.6-5.4) and 2.8% (95%CI 2.2-3.4), respectively. Thawed products' mean viability difference between NC-200 and TB was 3.0% (95%CI 0.4-5.6). The NC-200 automated fluorescent-based method can be used effectively to determine HPC's viability for both fresh and cryopreserved products. It can help eliminate human bias and provide consistent data and operational ease.

In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells. Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated. Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.

Effect Of Retinol In The Vitrification Medium On Viability Of Vitrified Ovine Preantral Follicles And Expression Of Key Developmental And Apoptosis Related Genes

BACKGROUND: Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production. OBJECTIVE: To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles. MATERIALS AND METHODS: Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 μM retinol; Group 4, vitrification + 5 μM retinol ; Group 5, vitrification + 10 μM retinol . Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression. RESULTS: Vitrification with 5 μM retinol positively affected viability in comparison with vitrification without retinol (P < 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 μM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i. e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 μM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in the Group vitrified with 5 μM retinol. CONCLUSION: The supplementation of 5 μM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.

Recycled Pequi (Caryocar brasiliense, Camb.) Shell Ethanolic Extract Induces Apoptosis in Canine Osteosarcoma Cells

Abstract Osteosarcoma is a mesenchymal cancer associated with high mortality in dogs and in humans. The biodiversity-rich Cerrado, the predominant biome in the Midwest region of Brazil, is native to locally appreciated fruits such as pequi (Caryocar brasiliense, Camb). Although this plant has been frequently used in folk medicine, the pharmacological properties of pequi fruit shells have not been fully evaluated. Thus, this study aimed to determine the cytotoxic activity of ethanol extract of recycled pequi shells on canine osteosarcoma cells in vitro. Cells were cultured and treated with final extract concentrations of 0, 0.029 µg/µL, 0.29 µg/µL, and 2.91 µg/µL for 24, 48, or 72 hours. Cell viability assay using trypan blue exclusion method and tetrazolium reduction method, cell survival assay, and double labeling with annexin V and propidium iodide were performed in the treated osteosarcoma cells. These allowed the determination of IC50, survival fraction, and type of cell death, respectively. Pequi shell ethanol extract at a concentration of 2.91 µg/µL showed the greatest inhibition of osteosarcoma cell growth in vitro, resulting in a 71.80% decrease in growth compared to the control. The mean IC50 was 155.2 μg/mL at 72 hours. The calculated survival fractions showed that cell growth at 72 hours was 3.33% lower in cells treated with 2.91 µg/µL extract. Results from the double labeling experiment suggest that apoptosis was the predominant type of cell death in cells treated with 2.91 µg/µL extract. These results demonstrate that ethanol extract of recycled pequi shells promotes apoptosis in canine osteosarcoma cells.

Immunocytotoxic Effect of Aqueous Leaf Extract of Cassia occidentalis on Human Peripheral Blood Mononuclear Cells and Neutrophils

The study evaluates the potential immunocytotoxic effect of aqueous leaf extract of Cassia occidentalis on human peripheral blood mononuclear cells (PBMCs) and Neutrophils. Different concentrations (25 µg/ml, 50 µg/ml, 100 µg/ml) of the extract were prepared. Six millilitres (6ml) of peripheral blood from consented healthy volunteers was collected and PBMCs and neutrophils were isolated on Histopaque media. The viability of PBMCs and neutrophils was determined using trypan blue dye exclusion methods. One-way analysis of variance was used in analysing the results. Total viable cell count (TVCC) for PBMCs and neutrophils yielded 298.9 x 104 cells/ml and 327.9 x 104 cells/ml respectively. The TVCC of the highest concentration (100 µg/ml) of the extract used for the treatment of PBMCs was 192.7 x104 cells/ml and that of neutrophil was 50.47 x104 cells/ml equivalent to 82.51% and 61.71% mean viability percentages respectively. The PBMCs and neutrophils treated with 25 µg /ml of the extract have the highest mean percentage viability scores of 94.88% and 74.61% respectively. There was a significant difference in the mean percentage viability when control PBMCs was compared with those treated with 100 µg/ml (p<0.0001) and also when control neutrophils was compared with those treated with 25 µg /ml (p=0 .02), 50 µg /ml and 100 µg /ml (p<0.0001) respectively. The cell viability tends to decrease in a dose-dependent manner. The aqueous leaf extract of C. occidentalis has a potent cytotoxic effect on both cells, especially at a higher dose. The study recommends an in-depth study to improve the credence of the present study findings.

Olaparib-induced Apoptosis Through EBNA1-ATR-p38 MAPK Signaling Pathway in Epstein-Barr Virus-positive Gastric Cancer Cells.

Epstein-Barr virus (EBV)-associated gastric cancer has been identified as a cancer subtype with definitive clinical and molecular characteristics. Although olaparib, a poly ADP ribose polymerase (PARP) inhibitor, is considered a potential effective agent for gastric cancer, the effect and underlying mechanism of olaparib on gastric cancer depending on EBV infection is not fully understood. EBV-positive SNU719 and EBV-negative SNU638 gastric cancer cell lines were used to identify the effects of olaparib using the trypan blue exclusion method and annexin V staining assay. To observe the underlying cellular signaling mechanisms of olaparib-induced cell death, Epstein-Barr virus nuclear antigen 1 (EBNA1) and signaling related molecule expression were assessed using transfection, silencing of specific genes using small interfering RNA (siRNA), western blotting and signaling inhibition assay. Olaparib decreased the cell viability of EBV-positive SNU719 gastric cancer cells through caspase-3-dependent apoptosis in a dose dependent manner, whereas EBV-negative SNU638 gastric cancer cells showed drug resistance to olaparib. EBNA1 was expressed in SUN719 gastric cancer cells; however, ataxia telangiectasia and Rad3 related (ATR) and phosphorylated ATR kinase were expressed in SNU638 gastric cancer cells. EBNA1 transfection decreased ATR phosphorylation through p38 mitogen-activated protein kinase (MAPK) phosphorylation in SUN638 gastric cancer cells, and silencing of ATR kinase increased the susceptibility of these cells to olaparib treatment. Moreover, VE-821, an ATR kinase specific inhibitor, also increased the sensitivity of SNU638 cells to olaparib. In contrast, SB203580, a p38 MAPK inhibitor, inhibited this increase in sensitivity to olaparib by EBNA1 transfection. Olaparib treatment led to different cellular responses depending on EBV infection in gastric cancer cell lines. These results provide new insights into the mechanism of olaparib-induced apoptosis in gastric cancer cells and suggest that EBV infection should be considered when developing new potential therapeutic agents for gastric cancer.

Evaluation of cytotoxic activity of Fe doped cobalt oxide nanoparticles

BackgroundThis contribution of work describes a new strategy for manufacturing cobalt oxide nanoparticles and the results assured that, its efficiency was increased by adding Fe ions. The anticancer drugs usually have a limited medical value owing to their nonspecific cytotoxicity. It has been proven that by using the nanosystems to deliver tablets to tumour cells reduces the toxic quality. Because of these qualities, nanoparticles can be used as a stronger rival for potent cancer treatment. MethodThis study investigated the cytotoxicity of iron doped cobalt oxide nanoparticles through trypan blue exclusion method. ResultThe newly generated Fe doped Co3O4 nanoparticles had proved its biocompatibility from the report of reduced toxicity below 200 μg/mL on malignant cell lines. ConclusionThe observed findings may encourage the development of anticancer drugs based on the inorganic particles, especially Fe doped Co3O4 nanoparticles, that could be serve as an excellent framework for the drug delivery and provide a new perspective for interpreting and targeting various therapeutic methodologies to tumours.

Highly diluted compounds effects on B16-F10 melanogenesis, reactive oxygen species (ROS) production and tumorigenesis.

Background: Cutaneous melanoma is a highly malignant tumor derived from pigment-producing (melanin) melanocytes of skin epidermis. Cutaneous pigmentation is described as the major physiologic defense against UV radiation. During melanin biosynthesis and other tumorigenic process, reactive oxygen species (ROS) are produced and might be critically involved in several melanomagenesis stages. ROS play key roles on regulation of many types cell proliferation, including melanoma cells. Aims: In this work we evaluated the effects of highly diluted compounds on melanogenesis and changes in reactive oxygen species after 96 hours of treatment and possible involvement in tumorigenesis. Methodology: Melanin content was measured in B16-F10 cells after 96 hours of treatment with highly diluted compounds, as well as the superoxide anion, hydrogen peroxide and nitric oxide. Furthermore, the effects of highly diluted compounds on cell proliferation were investigated by trypan blue exclusion method after 48 hours of treatment. Results: Treatment led to an increase in B16-F10 melanin content and a decrease in nitrite concentration, an intermediate product of nitric oxide. We also observed a decrease in cell proliferation after treatment. It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Several articles show that NO treated B16-F10 cells exhibited higher metastatic capacity. Thereby, reduction in cell proliferation can be due to low NO levels. It is speculated that melanocytes are programmed to survive in order to preserve their photoprotective role, thus in a compensatory manner the cell may be synthesizing melanin in response to cell proliferation reduction. Conclusions: These results suggest that treatment may be reducing tumorigenic capacity via ROS reduction. However further studies are need to better understand highly diluted compounds mechanisms of action.

In Vitro and In Vivo Anti-Toxoplasma Activities of Dracocephalum kotschyi Extract in Experimental Models of Acute Toxoplasmosis.

The problem of resistance to antiparasitic drugs, associated with their side effects, suggest exploring other alternatives, including medicinal plants. Dracocephalum kotschyi (D. kotschyi), for example, from Lamiaceae family, is a plant widely used in Iran and in many countries, and to which interesting pharmacological properties have been attributed. This study aimed to investigate in vitro and in vivo anti-Toxoplasma activities of D. kotschyi extract in experimental models of acute toxoplasmosis. Anti-Toxoplasma activity of the extracts in vitro was performed on Vero Cell, using the MTT test. Vero cell were infected with (3 × 105 tachyzoites/well) following treatment with Dichloromethane (F1), dichloromethane: methanol (F2), methanol (F3), methanol: water (F4), and deionized water (F5) extracts of D. kotschyi, and pyrimethamine and sulfadiazine (positive control). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure cell viability. Effects of extracts on tachyzoites viability were evaluated by trypan blue exclusion method, followed by light microscopy. For in vivo test, 18 groups of 8-10-week-old Inbred Balb/c mice weighing 18-20g, were intraperitoneally infected with 2 × 103 tachyzoites and then treated with sterile PBS (negative control), pyrimethamine (25mg/kg) and sulfadiazine (500mg/kg) as positive controls, and F1 to F5 extracts (at doses 50, 100 and 200mg/kg). The 50% Inhibitory Concentration of F1 extract, F2 extract, Sulfadiazine (Positive control) and Pyrimethamine (Positive control) were 8.77µg, 7.1µg 391.18µg, and 84.20µg, respectively, while the selectivity indices were 15.667, 30.197, 1.552 and 4.064, respectively. In vivo anti-Toxoplasma activity test showed that Methanol: water (F-4) 50 extract was more active than the positive control. Indeed, the extract allowed a survival rate of 10% of the mice, compared to 0% for all the other groups. D. kotschyi has the potential to be valorized in the management of toxoplasmosis.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200–300 μm) were randomly assigned into four groups. Group1 – Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2– FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2– FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (−196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Anticancer, in vitro cytotoxicity and antimicrobial properties of a Schiff base ligand, 3-((2-(-(1-(2-hydroxyphenyl)ethylidene)amino)ethyl)imino)-N-(p-tolyl)butanamide, and its metal complexes

A Schiff base ligand, 3-((2-(-(1-(2-hydroxyphenyl)ethylidene)amino)ethyl)imino)-N-(p-tolyl)butanamide, was synthesized by the condensation of o-hydroxylacetophenone, p-methylacetoacetanilide and 1,2-ethylenediamine and its V(IV)O, Cr(III), Mn(II), Fe(III), Co(II), Ni(II) and Cu(II) complexes have been prepared. The ligand and metal complexes were characterized analytically and spectroscopically by elemental analysis, HRMS, IR, 1H NMR, 13C NMR and UV-vis spectroscopies and molar conductance and magnetic susceptibility measurements. The ligand and its metal complexes have been evaluated for their cytotoxic activity against Dalton’s Lymphoma Ascites (DLA) cell line by Trypan blue exclusion method. In vivo anticancer studies using the Cu(II) complex in Swiss Albino female mice showed its potential efficiency against EAC-induced ascites tumor and DLA-induced solid tumor with an IC50 value of 46 µg/mL. Also, the complexes were tested for antibacterial and antifungal properties against some Gram-positive bacteria, Staphylococcus aureus, Bacillus cereus and Streptococcus criceti, and Gram-negative bacteria, Escherichia coli, Klebsilla pneumoniae and Pseudomonas aeruginosa and some fungi, Pencillium expansum, Fusarium oxysporum and Aspergillus niger. The complexes exhibited potential antibacterial and antifungal properties than the ligand, H2L, and in some cases even more than those of standard antibacterial and antifungal drugs.

Ethanolic extract of gabirobeira (Campomanesia adamantium) leaves reduces proliferation of osteosarcoma cells in vitro

Osteosarcoma is the most commonly diagnosed malignant bone tumor in humans, with a higher incidence in children and young people. It is highly aggressive and has a high metastatic potential. Its treatment is based on both chemotherapy and surgical intervention. However, currently used chemotherapeutic agents, such as doxorubicin, have several adverse effects on the patient. Therefore, there is a growing demand for new chemotherapeutic agents that stimulate new researches, such as those involving compounds extracted from plants, such as the gabirobeira. In this study, we aimed to evaluate the cytotoxic effects of ethanolic extract, both crude and ethyl acetate, of gabirobeira leaves on osteosarcoma cells in vitro. Cytotoxicity was evaluated using the Trypan blue exclusion method and the IC50 values were calculated using the tetrazolium reduction method. The ethanolic extract of gabirobeira leaves showed a cytotoxic effect on osteosarcoma cells in vitro. The group treated with the crude extract at 1. 0μL mL-1 concentration for 48 hours showed higher cytotoxicity and the lowest IC50 value for this extract was found in the 24 to 48 hours interval. The ethanolic extract of gabirobeira leaves is cytotoxic for osteosarcoma cells.

Protective Effect of Coleus Aromaticus Plant Protein against T-Booh Induced Cell Damage

Coleus aromaticus plant is having potential medicinal properties. In this study, we have purified a protein and characterized it by investigating the active protein was purified by G-50 column chromatography and its homogeneity was confirmed with SDS PAGE and RP HPLC. The protein molecular weight was confirmed with MS MALDI. The Phosphomolybdenum assay was used to evaluate the antioxidant capacity, where α- tocopherol was used as standard. The protective effect of purified Coleus aromaticus leaves protein CALP against t-BOOH induced DNA damage was studied using submarine gel electrophoresis, where ascorbic acid is used as the positive control. The trypan blue dye exclusion method was used to find the protective and non toxic nature of protein, where antioxidants like Butylated Hydroxylnisole (BHA) and Ascorbic acid were used as the positive control. The results showed that the molecular weight of Coleus aromaticus plant leaves protein (CALP) is 36 kDa. The purified CALP antioxidant capacity is compared to α- tocopheroland showed maximum protection at a concentration of 25ug.The cell viability study showed that lymphocytes treated with t-BOOH in presence of CALP (25µg) exhibited 69 ± 1% viability, whereas BHA (400μM) had 70 ± 2% viability and ascorbic acid (400µM) had 75 ± 2 % viability when compared to untreated lymphocytes (81 ± 3%) as they are not affected by any external factor like t-BOOH. The purified 36kDa CALP is a nontoxic antioxidant which provides protection against DNA damage induced by t-BOOH.

In vitro Toxicity Evaluation of Sterol Isolate from Echinoderm Stellaster equestris against Human Peripheral Blood

Background: Ocean acts as a wealthy resource of biological diversity producing prospective novel metabolites from the organisms present in the environment. Novel bioactive compounds from Echinoderms mainly from sea stars have been extensively studied procuring rich diverse compounds which exhibits in vitro cytotoxicity comparable to or better than those of the potent anticancer drug. These sea stars which reside in the benthic region serves as their habitat and they are therefore predators and persistently being attacked by various organisms subsequently they acclimatize the survival approach to defend themselves from the external pathogens. Since these metabolites are obscured as a metabolic product during their endurance an initial evaluation of these compounds is necessary to assess human risk. Objectives: The present study focuses in vitro toxicity evaluation of isolated sterol like isolate from echinoderm sea star Stellaster equestris by incorporating hemolytic, chromosomal aberration assay and the cell viability tryphan blue exclusion assay against human peripheral blood. Methods: The in vitro toxicity evaluation was studied against the human peripheral blood collected from the healthy donors with the defined concentration of the sterol like compound from the sea star Stellaster equestris. The hemolytic, cell viability trypan blue exclusion method and chromosomal aberration assay were performed to check the hemolytic, mutagenic and genotoxic effect against the lymphocytes and the red blood cells. Results and Discussion: The result suggested that the hemolytic assay and the cell viability assay even in a dose dependent manner were non-hemolytic and percentage of the viability was not affected due to the exposure of the compound. The absence of genotoxicity was evident for the chromosomal aberration assay indicated that the isolated compound from the sea star Stellaster equestris might be considered as effective novel compound.

A Novel Antibody-Toxin Congujate to Treat Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL), while sensitive to several chemotherapeutic regimens including rituximab, cure rates remain low and new treatments are needed.We have identified a membrane bound “activated” matriptase, as an attractive antigen target for the highly selective delivery of toxins to epithelial tumors and MCL. Matriptase, a member of type II transmembrane serine proteases, is a glycoprotein (80-90 kDa) synthesized as a latent single-chain structure. Activation generates a disulfide-linked-two-chain fully active enzyme through an auto-activation step. Reactive oxygen species (ROS), as well as acidic environments present in tumors can activate matriptase. In addition to roles in initiating carcinogenesis, the enzyme also plays important roles in tumor progression, including invasiveness and metastasis.Importantly, while matriptase is present in a latent form on epithelial cells and B-cells, activated matriptase expression is restricted to the membranes of epithelial tumors and some B-cell malignancies .MethodsMaterials: The M-69 antibody was generated by CY. Bortezomib was from the CINJ pharmacy.Cell Culture: The MCL cells (JeKo-1, Mino, Maver and Z138) were cultured in 1X RPMI Media 1640 (Life Technologies) containing 10% fetal bovine serum (FBS) at 37ºC.Western blotting: The M-69 antibody was used for western analysis and Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) purchased from Millipore was used as a control.Cytotoxicity assay: Cell viability was measured by the trypan blue dye exclusion method using a Vi-Cell XR© cell viability analyzer (Beckman Coulter). The cytotoxicity data were analyzed using GraphPad Prism 4 software (GraphPad Software Inc., CA).Animal studies: NOD/SCID/IL2 receptor gamma chain null (NOD/SCID/IL2gnull, NSG) mice were obtained from the Jackson Laboratory. JeKo-1 MCL cells were used for the xenograft studies. Once tumors were palpable, the mice were treated i.p. with anti-matriptase antibody (M-69) conjugated with MMAE (ADC). Controls used were M-69 antibody alone and saline. In a second experiment mice were treated with the ADC alone, bortezomib alone or the combination. Tumor size and body weights were measured twice a week.ResultsThe linkage of monomethyl auristatin-E (MMAE), a potent tubulin-inhibitor, to M69 used the releasable linker technology which is based on Seattle Genetics' valine-citrulline-PABA linker that is cleavable by cathepsin B in the lysosome, while showing stability in circulation. MALDI-TOF mass spectrometry of the ADC showed a 7000 Da increase of the average M.W. of the antibody that corresponds to an average of 3.5 drug molecules linked to each mAb molecule.MCL (JeKo-1, Mino, Maver and Z138) cell lines were found to overexpress activated matriptase using the M-69 antibody. These cell lines were sensitive to the ADC with IC50s at single digit µg/ml of the conjugate.We elected to use the JeKo-1 cell line for the initial xenograft studies. We found that the 5 mg/kg dose of the ADC weekly produced a better anti-tumor effect (90% growth inhibition) than a 1 mg/kg dose, without any body weight loss. In combination with bortezomib, treatment with the ADC led to some complete responses.ConclusionsThe mouse antibody to human matriptase, M-69, conjugated to MMAE potently inhibited growth of four MCL cell lines and inhibited xenografts of the JeKo-1 MCL in NSG mice alone and produced enhanced effects in combination with bortezomib without significant side effects. DisclosuresRather:Phosplatin Therapeutics: Research Funding. Bertino:Phosplatin Therapeutics: Research Funding.

Effect of betulinic acid on proliferation of human gastric cancer MGC-803 cell line

Objective: To investigate the effect of betulinic acid on the proliferation of human gastric cancer MGC-803 cells in vitro. Methods: Human gastric cancer MGC-803 cells were divided into 4 groups, each with 3 multiple holes. Control cells add betulinic acid at a concentration of 0 μg /ml, and the other three experimental groups were added with final concentration of 10, 20, 30 μg/ml Betulinic acid respectively. Cells in each group were incubated in a 5% CO2 incubator for 48 hours, and the Giemsa staining method and trypan blue exclusion method were used to detect the effect of betulinic acid on the cell clone formation rate and growth inhibition rate; EdU method and flow cytometry were used to detect cell proliferation and cell cycle changes; qRT-PCR and Western blot were used to detect the expressions of cell cycle regulators CCNB1 and CCND1. Results: Compared with the control group, the clone formation rate of human gastric cancer MGC-803 cells was significantly reduced (P＜0.01), the growth inhibition rate was significantly increased, and the cell proliferation ability was significantly decreased (P＜0.01); with the increase of betulinic acid concentration in each experimental group the proportion of cells in the G1 phase was gradually decreased, and the number of cells in S phase was increased significantly (P＜0.01); the mRNA and protein expression levels of cell cycle regulators CCNB1 and CCND1 were decreased significantly, and the 30 μg/ml betulinic acid treatment group performed best. Conclusion: At a final concentration of 10～30 μg/ml, betulinic acid can reduce the proliferation of human gastric cancer MGC-803 cells, inhibit cell growth, and down-regulate the expression of CCNB1 and CCND1 to block human gastric cancer MGC-803 cells in the S phase.

Photo-Mediated Facile Synthesis of Silver Nanoparticles Using Curcuma zanthorrhiza Rhizome Extract and Their In Vitro Antimicrobial and Anticancer Activity

Plants possess unique bioactive principles justifying their multifunctional properties. In this work silver nanoparticles (AgNPs) were prepared via a photo-mediated green approach for enhanced antimicrobial and anticancer activity. The aqueous rhizome extract of Curcuma zanthorrhiza Roxb. (Cz) was employed as a source of reducing and capping agent for the synthesis of colloidal Cz-AgNPs. The materialization of Cz-AgNPs was identified from the surface plasmon resonance observed at 416 nm using UV–Visible spectrophotometer. The FTIR analysis of both Cz-AgNPs and plant rhizome extract revealed the as-synthesized Cz-AgNPs were capped with plant derived phytoconstituents. The crystallinity, surface morphology, hydrodynamic size and surface area of Cz-AgNPs were characterized by PXRD, HR-TEM, FESEM, DLS and BET analysis. The as-synthesized Cz-AgNPs exhibited strong antimicrobial activities against bacterial strains and plant pathogenic fungi in a dose-dependent manner. The antimicrobial activity of Cz-AgNPs was assessed using standard disc diffusion, broth microdilution assay for bacterial strains and poisoned food technique for fungal strains. The Cz-AgNPs were inhibitory to all the microbes even at the lowest concentration. The biocompatible nature of Cz-AgNPs was analyzed for hemolytic properties with human erythrocytes and promising results were observed. In addition, the cytotoxicity activity (Trypan Blue Exclusion Method) on the cell lines of Dalton’s Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC) showed Cz-AgNPs exhibited potential anticancer activity with concentration as low as 1.67 µg/mL and 1.84 µg/mL respectively (IC50 value). This work attempted at optimizing a safe dosage of silver nanoparticles using hemolysis assay and thus for application in therapy.

Apoptotic regulator BCL-2 blockade as a potential therapy in classical Hodgkin Lymphoma

The challenge in classical Hodgkin Lymphoma (cHL) management is the 30–40% of refractory/relapsed cases. AimsThe aim of this work was to determine whether NIK and BCL-2 could be useful as prognosis biomarkers in cHL. In addition, we evaluated BCL-2 as a directed-therapy in cHL cell lines using venetoclax. Main methodsWe evaluated NIK and BCL-2 expression in 112 untreated cHL patients' lymph-node biopsies by immunohistochemistry. cHL cell lines were treated with venetoclax alone or combined with vincristine or doxorubicin. Cell viability, metabolic activity and cell death were analyzed by trypan-blue exclusion method, MTS assay and FDA/IP staining respectively. Key findingsNo correlation between NIK or BCL-2 expression and the majority of the clinical parameters was found. Patients with ≥60% BCL-2+ HRS-cells had a shorter disease-free survival (DFS) and overall survival (OS) (p = 0.002, p = 0.02 respectively). A decision tree analysis, in a 30 patients subgroup, showed that patients with <60% NIK+ HRS-cells but with ≥60% BCL-2+ HRS-cells had a worse outcome in terms of DFS and OS. These parameters performed better as prognosis indicators as compared to the diagnosis bone marrow status. Human cHL cell lines U-H01, KM-H2, L1236, SUPHD1, L540 showed sensitivity to venetoclax. The co-treatment effect of venetoclax and vincristine or doxorubicin on cell viability was diverse depending on the cell line evaluated. SignificanceBCL-2 should be considered as a prognosis biomarker as well as a potential new therapeutic target in cHL. We report for the first time the cytotoxic effect of venetoclax in human cHL cell lines.