Trypan Blue Cell Counting Research Articles

Immunomodulatory role of Rhaphidophora korthalsii methanol extract on human peripheral blood mononuclear cell proliferation, cytokine secretion and cytolytic activity.

Rhaphidophora korthalsii methanol extract induce the immunomodulatory effect on human peripheral blood mononuclear cell (PBMC). The present study was performed to investigate the immunomodulatory potential of the extract through different tests: MTT assay, BrdU proliferation assay, Trypan blue direct cell counting, cytokine (IL-2, IL-12 and IFN-γ) ELISA assay and Annexin V cytotoxicity assay. Concanavalin A (Con A) and lipopolysaccharide (LPS) were used as positive control. The observed results from all MTT assay, BrdU proliferation assay and Trypan blue direct cell counting showed that the plant extract stimulated the PBMC at 48 and 72 h where 25 μg/ml was the optimum concentration. On the other hand, the results of the cytokines study showed that 25 μg/ml induced the secretion of IL-2 (433 ± 25 pg/ml) and IFN-γ (7336 ± 131.79 pg/ml) but not IL-12 at 72 h only. The activated PBMC also directed the effector cell (K562) in mix culture to undergo apoptosis (12.96 ± 0.57% of early apoptosis and 10.60 ± 0.21% of late apoptosis) at the ratio of 2/1 of PBMC/K562. The present results indicate that R. korthalsii extract showed evidences of immunomodulating activity which may be potential to use as an immunotherapy agent.

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms. The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis. Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 μmol/L. Genipin 200-400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L. These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

A possible cannabinergic neuroprotective mechanism of action of Lepidium meyenii (Maca)

The pentane extract of the plant Lepidium meyenii (Maca), has been shown to possess neuroprotective activity. This study was designed to examine the involvement of cannabinoid CB1 receptors in the action of some of the lipophilic constituents of Maca, the macamides, in neuronal cell lines. Cells were pretreated with Maca pentane extract (MPX), isolated macamides, anandamide or tetrahydrocannabinol (THC). After 1 hr incubation, cells were subjected to oxidative damage with H2O2. Cell viability was determined 24 hrs later using the trypan blue exclusion cell count method and the spectrophotometric dimethylthiazolyl‐carboxymethoxyphenyl‐sulfophenyl‐tetrazolium (MTS) method. Significant neuroprotective activity was observed with the MPX, macamide‐2, anandamide and THC. O‐2050 (a cannabinoid CB1 receptor silent antagonist) significantly (p<0.05) antagonized the neuroprotective activities. Additionally, the antioxidant activity of the MPX, anandamide and THC was determined using the 2,2′‐azino‐di‐3‐ethylbenzthiazoline sulfonate (ABTS) assay, and found to have no measurable anti‐oxidant capacity. These results demonstrate that some of the lipophilic compounds from Maca may act through the cannabinoid system to protect neurons against oxidative damage. Further studies are required to determine their detailed molecular mechanism.

Fibronectin confers survival against chemotherapeutic agents but not against radiotherapy in DU145 prostate cancer cells: Involvement of the insulin like growth factor‐1 receptor

Tumor growth is influenced by an increase in cell proliferation and a reduction in apoptosis; both of which are affected by alterations in extracellular matrix (ECM). Our aim was to assess if the susceptibility of prostate cancer cells to apoptosis induced by either chemotherapeutics or radiotherapy was altered by changes in the ECM. Prostate cancer cell lines LNCaP and DU145 (androgen independent) cells were treated with chemotherapeutics (ceramide and docetaxel) or radiotherapy in the presence or absence of fibronectin, laminin, or vitronectin. Cell death was assessed using Trypan blue cell counting and apoptosis was confirmed by measuring PARP cleavage by Western immunoblotting (WIB). To identify a mechanism of action, changes in the abundance (WIB) or association (immunoprecipitation followed by WIB) of key proteins was also assessed. We found that fibronectin, but not laminin or vitronectin activated a survival pathway that protected DU145 but not LNCaP prostate cancer cells against ceramide and docetaxel-induced apoptosis but not that induced by radiotherapy. This survival effect involved the insulin-like growth factor (IGF-I) and beta1 integrin receptors and was associated with an increase in the recruitment of the beta1 integrin to a complex containing the IGF-IR and protein receptor for activated C kinase (RACK-1) and an increase in the abundance of a MAPK-phosphatase-1 (MKP-1). Changes in the ECM associated with disease progression may contribute to resistance to chemotherapeutic drugs but not to radiation therapy. The susceptibility to chemotherapy may be improved by targeting either the IGF-I or beta1 integrin receptors.

CEBPalpha and a Critical Circadian Clock Downstream Target Gene, PER2, Are Highly Dysregulated in Diffuse Large B-Cell Lymphoma.

Abstract 3454Poster Board III-342Non-Hodgkin's lymphoma (NHL) is a heterogeneous clinicopathologic entity characterized by distinct cells of origin and unique cytogenetic and molecular aberrations. Recently, we found by transcription profiling of CCAAT/enhancer-binding proteins (CEBPs) that they could regulate expression of the core circadian clock gene Period (PER) 2. We now report that C/EBPalpha and PER2 are highly dysregulated specifically in diffuse large B-cell lymphoma (DLBCL), one of the commonest type of high-grade NHL. Real time RT-PCR analysis of human samples of diffuse large B-cell lymphoma (DLBCL; n = 37), mantle cell lymphoma (MCL; n = 12), follicular lymphoma (FL; n = 12), and Burkitt's lymphoma (BL; n = 6) compared to normal tonsil samples (n = 10) revealed a markedly downregulated expression of both CEPBalpha and PER2 specifically in the DLBCL samples. The expression of these two genes was also significantly decreased in the 7 human DLBCL cell lines, OCI-Ly1, -Ly4, -Ly7, -LY10, and SUDHL-4, -6, -16. In contrast, mRNA levels of CEBPalpha and PER2 in samples of MCL, FL or BL showed no significant change compared to the normal tonsils, which were confirmed in cell lines of MCL (SP-49, Jeko-1, NCEB-1), FL (FLK-1) and BL (Daudi). Moreover, PER1, another important circadian rhythm gene of the Period family, showed no differences of expression in any of the NHL samples compared to control lymph nodes. The murine pro-B lymphoid cell line Ba/F3 had low levels C/EBPalpha, and forced expression of the transcription factor resulted in decreased growth and increased apoptosis as measured by trypan blue cell count and Annexin V (FACS), suggesting that this transcription factor may induce apoptosis in lymphoma. Moreover, transfection of Ba/F3 cells with a zinc-inducible CEBPalpha gene increased PER2 expression levels by 7-fold. The analysis of the PER2 promoter region showed several potential CEBPalpha binding sites, and chromatin immunoprecipitation (ChIP) as well as luciferase reporter assay experiments using Ba/F3 cells revealed that CEBPalpha can directly bind to the PER2 promoter and induce its expression. Interestingly, treatment of Ba/F3 and several DLBCL cell lines (Ly-4, SUDHL-4, -6) with the histone deacetylase-inhibitor, SAHA, significantly induced expression of both CEBPalpha and PER2, which was associated with inhibition of cell growth and apoptosis. Our further studies explored the consequences of expressing PER2 on cell proliferation. Forced expression of PER2 in Ba/F3 cells led to substantial growth reduction, G0/G1 cell cycle arrest, and altered protein expression of apoptosis-related genes. The protein level of Bcl-X(L) was downregulated, whereas levels of Bax and PARP cleavage activity were upregulated compared with control cells transfected with empty plasmid. In summary, our results show for the first time that both CEBPalpha and its key downstream target circadian clock gene, PER2, are highly dysregulated in DLBCL. Our data strongly suggest that these genes may play a role in the pathogenesis of this disorder and should be considered for therapeutic manipulation. DisclosuresNo relevant conflicts of interest to declare.

The Use of Discontinuous Density Gradients in Stem Cell Research and Application

Discontinuous density gradients have been shown to be very efficient in sperm enrichment in assisted reproductive technologies (ART). Percoll [polyvinylpyrrolidone (PVP)-coated silica] which is a colloidal agent commonly used in such gradients is now confined to only research purposes in ART because its PVP component was considered toxic for clinical applications. The PVP component was replaced with silane instead and silane-coated colloidal silica marketed as Puresperm is currently being used for clinical sperm enhancement procedures in ART. The efficiency of these two agents has not been tested for human embryonic stem cell (hESC) and cancer cell research and clinical application. Herein we describe discontinuous density gradient protocols using PureSperm and Percoll for the separation and enrichment of hESCs and hepatocarcinoma cells from a mixed cell population. Trypan blue cell viability and CellTracker green cell counts are used to accurately quantify the extent of cell separation. We show that Puresperm provides good cell separation and enrichment with higher viable cell counts and greater number of fractions compared to Percoll. PureSperm being superior and safer compared to Percoll would be a better alternative for cell separation and enrichment in stem and cancer cell research and clinical applications.

Mesenchymal stem cells enhance the viability and proliferation of human fetal intestinal epithelial cells following hypoxic injury via paracrine mechanisms

Mesenchymal stem cells (MSCs) may be used to treat injured tissues. The ability of MSCs to treat injured fetal intestinal epithelial cells (FIEs), similar to those in infants with necrotizing enterocolitis, has not been elucidated. We hypothesized that MSCs would enhance FIE viability and proliferation after hypoxic injury via paracrine mechanisms. LLC-PK1 cells (differentiated control [DC]) and human MSCs were exposed to 1 hour of hypoxia. Cells were reoxygenated for 24 hours and cell-free conditioned media were collected. Human FIEs were exposed to 1 hour of hypoxia and plated for experiments. FIEs were reoxygenated in nonconditioned media, DC-conditioned media, or MSC-conditioned media. Supernatants were analyzed for interleukin-6 (IL-6), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) via enzyme-linked immunosorbent assay. Cell viability was assessed by trypan blue exclusion and cell counting. Proliferation was determined via 5-bromo-2'-deoxyuridine (BrdU). Expression of caspases-3 and -8 was determined via Western blot. FIEs reoxygenated in MSC-conditioned media demonstrated enhanced viability and increased proliferation after hypoxic injury. Enhanced FIE viability and proliferation were associated with increased IL-6, HGF, and VEGF, as well as decreased expression of caspase-3. MSCs may increase the viability and proliferative capacity of FIEs after hypoxic injury via the paracrine release of IL-6, HGF, and VEGF, as well as downregulation of apoptotic signaling.

Effects of histone deacetylase inhibitor MS-275 on expression of survivin and NF-kappaB in the human myeloma cell line U266 cells

To study the effects of HDACi on the expression of survivin and NF-kappaB in human myeloma cell line U266 cells. U266 cells were cultured in RPMI 1640 in the presence of MS-275, and the cell viability was evaluated by trypan blue exclusion assay and cell count. The cell morphological changes were observed with Wright-Giemsa staining. The cell cycle was analyzed by flow cytometry, and the proteins of poly (ADP-ribose) polymerase (PARP), caspase-3, survivin, p21, CDK4 and IkappaB-alpha were detected by Western blotting. MS-275 inhibited the growth of U266 cells in a dose- and time-dependent manner. The cell cycle was arrested at G(0)/G(1) phase. After exposure at 1.39 micromol/L MS-275 for 48 hours, the cell viability was decreased to 50%. The cell ratios of G(0)/G(1) phase were increased to (64.57 +/- 4.09)% for 24 h and (87.20 +/- 2.83)% for 36 h after 2 micromol/L MS-275 treatment. The visible morphological changes of U266 cells were confirmed with Wright-Giemsa staining. Cleaved-PARP, increased expression of p21, downregulation of expression of survivin, CDK4 and the phosphorylation of IkappaB-alpha was found by Western blot in MS-275 treated U266 cells. MS-275-induced apoptosis of U266 cells is mediated by downregulation of expression of survivin and inactivation of NF-kappaB survival signalling pathways.

The Effect of Atrazine on the Hypothalamic Cell Line

Atrazine is a widely used herbicide. It has been shown to exhibit endocrine disrupting properties. A few studies demonstrate that atrazine enhances estrogenic activity by stimulating aromatase or by inhibits phosphodiesterase causes elevation of cAMP in cell lines which leads to increase in estrogen production. Although evidence supports that atrazine affects the hypothalamic pituitary gonadal axis, the specific molecular mechanism of atrazine‐induced endocrine disruption is not completely understood. The goal of this study is to analyze the in vitro effects and cellular mechanisms of atrazine‐induced endocrine disruption using N38 mouse hypothalamic cell lines. Initial observation has shown that exposure of the hypothalamic cells within the EPA recommended (0.014μM) limits causes 50% cell death within 12 hours. There was about 20% cell death within 10 minutes of exposure to atrazine. Trypan Blue cell counting analysis and 3‐(4,5‐ Dimethyl‐2‐thiazolyl)‐2,5‐diphrnyltetrazolium bromide (MTT) assay revealed a similar trend. Neuronal cell death could lead to apoptotic cell death causing activation of caspases. Analyses of caspases‐3 would confer the atrazine‐induced cell death and would be done using immunocytochemical techniques. Further, aromatase (CYP19) activity would be quantitated in order to access the signaling pathway and the mechanism of action.

Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction

Adipose tissue is an abundant source of mesenchymal stem cells (MSC), which can be used for tissue-engineering purposes. The aim of our study was to determine the more suitable procedure, surgical resection or liposuction, for harvesting human adipose tissue-derived stem cells (hASC) with regard to viability, cell count and differentiation potential. After harvesting hASC, trypan blue staining and cell counting were carried out. Subsequently, hASC were cultured, analyzed by fluorescence-activated cell sorting (FACS) and differentiated under adipogenic, osteogenic and chondrogenic conditions. Histologic and functional analyzes were performed at the end of the differentiation period. No significant difference was found with regard to the cell counts of hASC from liposuction and surgically resected material (P=0.086). The percentage of viable cells was significantly higher for liposuction aspirates than for resection material (P=0.002). No significant difference was found in the adipogenic differentiation potential (P=0.179). A significantly lower number of cultures obtained from liposuction material than from resection material could be differentiated into osteocytes (P=0.049) and chondrocytes (P=0.012). Even though some lineages from lipoaspirated hASC can not be differentiated as frequently as those from surgically resected material, liposuction may be superior for some tissue-engineering purposes, particularly because of the less invasive harvesting procedure, the higher percentage of viable cells and the fact that there is no significant difference between lipoaspirated and resected hASC with regard to adipogenic differentiation potential.

Understanding the Role of TRKA Receptor in Acute Myeloid Leukemias: From Proliferation and Pro-Survival Signals to a Novel Therapeutic Approach

Background: TRKA (Tropomyosin Receptor Kinase A) is a member of the neurotrophin tyrosine kinase receptor family. Nerve Growth Factor (NGF) binds specifically to TRKA triggering multiple downstream pathways which regulate proliferation and differentiation in a number of neural, epithelial and epidermal cell types. TRKA is also expressed in hematopoietic progenitor cells and NGF has been demonstrated to promote the colony growth and differentiation of myeloid progenitor cells. Interestingly, Pralle et al. found that 44% of patients with acute myeloid leukemia (AML) express TRKA mRNA and up to 30% encode activating TRKA mutations. Furthermore, a constitutively-active TRKA variant has been shown to contribute to myeloid leukemogenesis.Purpose: To evaluate the role of NGF/TRKA signaling on AML proliferation and survival through effects on downstream signaling pathways, and evaluate the effects of TRK inhibition on human AML.Methods: TRKA protein expression was measured in a panel of human AML cell lines (U937, K562, THP1, NB4, HL60, KU812). AlamarBlue (MTT) assays were used to measure proliferation/viability. For proliferation assays, cells were stimulated with NGF (5 ng/ml) for 1–3 days with or without the addition of a TRK inhibitor AZ-23 (25nM). Cell counts, cell cycle and survival were measured using trypan blue cell counts, and propidium iodine-based flow cytometry assays. Downstream signaling events were measured following a 15 minute exposure to recombinant human NGF. Antibodies for phosphorylated and total AKT, ERK, JNK and MEKK were used. In a human AML xenograft model, mice were treated with 100 mg/kg (2.5 mg) of AZ-23 by gavage for 2 weeks without toxicity. Weekly tail vein blood collection and flow cytometry were used to evaluate leukemia tumor burden. Kaplan-Meier curves were used to assess effects on morbidity.Results: Five of six human AML cell lines showed detectable TRKA protein levels by Western. When stimulated with NGF, all five cell lines which expressed TRKA demonstrated enhanced proliferation within 24 hours with mean value of 20% (10 to 45%, p=<0.001), which was abrogated with 25nM AZ-23 (p<0.01). The HL60 cell line showed the most impressive response to NGF (fig 1). The AML cell line which did not express TRKA (U937) and a human pre-B ALL cell line (Nalm-6) did not respond to NGF stimulation, as expected. There were no significant difference between only AZ-23 and the control group (p=>0.5). Following starvation, NGF stimulation induced the phosphorylation of ERK and AKT (2–4 fold), while inhibiting the phosphorylation of MEKK and JNK. All of these signaling effects were blocked with addition of AZ-23. Importantly, human AML-engrafted mice treated with the TRKA inhibitor AZ-23 had a significant decreased AML burden at 3 weeks post-inoculation (70% vs. 18% p=0.004). Kaplan-Meier estimates showed a Hazard Ratio of 10.5 (p=0.01) fig 3.Conclusions: TRKA has been implicated in the proliferation and transformation of myeloid progenitors and may be expressed and activated in a significant subset of AML cases. Our studies provide evidence of an important role for NGF/TRKA signaling in human AML. We show that NGF induces TRK-dependent proliferation while inducing ERK and AKT phosphorylation, consistent with a proliferative and pro-survival effect. Furthermore, we show that NGF stimulation inhibits the phosphorylation of MEKK and JNK, which are associated with NGF withdrawal and promote apoptosis. Finally, we demonstrate in a human AML xenograft model that a selective TRK inhibitor AZ-23 can effectively reduce AML burden and enhance survival. Based on these results, we propose that TRK inhibition may be a therapeutic option for a subgroup of AML, and warrants further pre-clinical development. [Display omitted] [Display omitted]

Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

An analog of thyrotropin-releasing hormone (TRH) is neuroprotective against glutamate-induced toxicity in fetal rat hippocampal neurons in vitro

TRH has been found to be efficacious in treating certain neurodegenerative disorders such as epilepsy, Alzheimer's disease, neurotrauma and depression, however, its mechanism of action is poorly understood. Since glutamate (Glu) toxicity has been implicated in these disorders, we utilized primary enriched cultures of rat fetal (E 17) hippocampal neurons to test the hypothesis that an analog of TRH, 3-Methyl-Histidine TRH (3Me-H TRH), given concurrently with Glu would protect such neurons against cell damage and cell death. Cell viability was assessed via Trypan Blue exclusion cell counts, and neuronal damage was determined by assaying lactic acid dehydrogenase (LDH) released in the conditioned media. Fetal hippocampal neurons were cultured in neurobasal media for 7 days. On day 7, neurons (10 6/well) were treated with: control media, 10 μM 3Me-H TRH, 500 μM Glu or 500 μM Glu with either 10, 1, 0.1, 0.01 or 0.001 μM 3Me-H TRH. Both media and neurons were harvested 16 h after treatment. Prolonged exposure to 10 μM 3Me-H TRH was not toxic to the cells, whereas neurons exposed to 500 μM Glu resulted in maximal cell death. Notably, 10, 1 and 0.1 μM 3Me-H TRH, when co-treated with 500 μM Glu, protected fetal neurons against cell death in a concentration-dependent manner. These results provide support for an important neuroprotective effect of TRH/analogs against glutamate toxicity in primary hippocampal neuronal culture and implicate a potentially beneficial role of TRH/analogs in neurodegenerative diseases.

In vitro evaluation of the direct effect of estradiol on human osteoblasts (HOB) and human mesenchymal stem cells (h-MSCs)

Estrogen may increase the proliferation of osteoblasts depending upon their differentiation stage. Our objective was to test the hypothesis that estradiol could stimulate the proliferation of primary human osteoblast (HOB) cells or human mesenchymal stem cells (h-MSCs). To test this hypothesis, we investigated two synthetic estradiol preparations: (a) a commercially available one (in clinical use) whose effect was evaluated using MTT assay, trypan blue cell counts and total protein assays; (b) a novel synthetic preparation (not in clinical use) using Alamar Blue assays and scanning electron microscopy (SEM). Commercial estradiol increased HOB proliferation in a concentration-dependant manner between 1 nM and 8 nM reaching significance at a concentration of 8 nM (p<0.001). Small doses of 1 nM and 2 nM increased h-MSC proliferation (p<0.001) but higher doses had no effect. All novel synthetic estradiol preparations decreased HOB proliferation (p<0.001) whereas no direct effect was observed on h-MSCs. Commercial estradiol appears to induce proliferation of HOBs, although its effects on h-MSCs appears to be highly dose-dependent and requires further investigation.

Enhancement of carboplatin toxicity by Pluronic block copolymers

The objective of this study was to examine the effects of three Pluronic triblock copolymers (F127, P85, or L61) on the cytotoxicity of carboplatin to the DHB/K12/TRb rat colorectal carcinoma cell line. Studies to determine the dependence of the sensitization effect on Pluronic dose were carried out for polymer concentrations ranging from 0.0001–10% (w/w). To establish the carboplatin toxicity and its potential enhancement by Pluronic, the drug was delivered to cells in doses ranging from 0–0.5% (w/w) in the presence of Pluronic at a constant concentration. These treatment groups were compared to control groups receiving carboplatin alone. Cell cytotoxicity resulting from the treatments was determined with a mitochondrial enzyme activity assay (WST-1), while cell morphology was examined with May–Grünwald and Giemsa staining. Results indicate that the greatest enhancement of carboplatin toxicity was induced by P85, where the inhibitory concentration (IC 50) was reduced by 50% (from 0.096 mg/mL for carboplatin alone to 0.048 mg/mL in presence of P85). L61 was toxic to the cells with or without drug (viability < 3.5%), while F127 exhibited no sensitizing effect and in some cases increased the cell viability to 130% over the untreated control. The WST-1 results were confirmed by trypan blue exclusion cell counts at 0 and 24 h post treatment. Data conclusively demonstrate that Pluronic P85 is the optimal agent for increased cytotoxicity of carboplatin in this cell line and can potentially be used not only as a drug delivery scheme but also as a chemosensitizing agent in future cancer therapy.

Soluble CD44 Is Cytotoxic to Trabecular Meshwork and Retinal Ganglion Cells In Vitro

Current glaucoma research targets neuroprotective therapies for retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). The purpose of this study was to determine whether the 32-kDa ectodomain fragment of CD44-soluble CD44 (sCD44)-which is increased in the aqueous of patients with POAG, affects RGC and trabecular meshwork (TM) cell survival in vitro. sCD44 was isolated from human or fetal calf serum (FCS) by urea solubilization and immunoprecipitation. A transformed rat RGC-like cell line (RGC-5), human and bovine TM cells, and control cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS until confluent and then were incubated in medium containing 0.1% FCS and treated with various doses of purified sCD44 and 17-alpha-methyl testosterone (17-alpha-MT). The cytotoxicity of sCD44 was verified by heat-inactivation, pretreatment with a pan-caspase inhibitor, and coadministration of anti-CD44 neutralizing antibody or hyaluronic acid (HA). Cell viability was assessed by trypan blue staining, cell counting, and phase-contrast microscopy. There was a statistically significant dose- and time-dependent decrease in the number of cells and viability in the RGC-5 and TM cells treated with sCD44. Within 12 hours of sCD44 treatment, RGC-5 and TM cells displayed cell rounding, detachment, and swelling. sCD44-induced cell death was cell specific. Smooth muscle cells were resistant to sCD44, whereas human cortical neuronal-like cells were susceptible to sCD44 after 24 hours, but recovered. The cytotoxicity of sCD44 was blocked by heat-inactivation, pretreatment with a pan-caspase inhibitor, or coadministration of anti-CD44 antibody or HA. 17-alpha-MT prevented sCD44 cytotoxicity in both RGC-5 and TM cells. The results indicate that exogenous sCD44 adversely affects RGC-5 and TM cell survival in vitro by activating proapoptotic pathways.

Antitumor activity of albendazole against the human colorectal cancer cell line HT-29: in vitro and in a xenograft model of peritoneal carcinomatosis

The peritoneal surface remains an important failure site for patients with colorectal cancer. We have recently shown that albendazole (ABZ), a safe and effective anthelmintic drug, has profound antitumor activity in hepatocellular cancer. Furthermore, albendazole also possesses unique physiochemical and pharmacokinetic properties probably making it a potential drug for use in the regional treatment of peritoneal carcinomatosis (PC). The current study was therefore designed to investigate this concept under both in vitro and in vivo conditions using human colorectal cancer cells HT-29. In cell culture, studies were conducted to investigate the effect of ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO) and albendazole sulfone (ABZ-SO2) on the growth of human colorectal cell line HT-29. We also investigated the effects of ABZ on the cell cycle and the possible induction of apoptosis in these cells. Male nude mice inoculated intraperitoneally (i.p.) with HT-29 cells were treated with various schedules of ABZ given i.p. or orally for 6 weeks. Response was evaluated as the number of peritoneal tumor nodules present in animals at the end of the treatment period. In vitro, ABZ treatment of cells for 5 days led to profound inhibition of growth. (3)H-Thymidine assay and trypan blue viable cell counts confirmed the dose- and time-dependency of the ABZ effect, while recovery experiments revealed the reversible nature of this inhibition. ABZ-SO and ABZ-SO2 were also evaluated in cell culture studies and compared with the parent drug. In HT-29 cells, the IC(50) values were calculated to be 0.12 microM for ABZ and 2.35 microM for ABZ-SO. The other metabolite, ABZ-SO2, was completely inactive. Studies on the mechanism of ABZ action, revealed arrest of HT-29 cells at the G(2)/M phase of the cell cycle, while TUNEL, DNA laddering and caspase-3 activity all confirmed ABZ induced apoptosis. In nude mice with peritoneal HT-29 xenografts, ABZ profoundly inhibited peritoneal tumor growth. While alternate i.p. dosing (ABZ, 150 mg/kg) led to the highest degree of tumor growth suppression (P<0.001), schedules such as once-weekly dosing and even a single dose for the entire course of treatment (6 weeks) were also effective in reducing peritoneal tumor growth. However, no such activity was observed when ABZ was administered orally. This study shows for the first time the potent effect of regionally administered ABZ in suppressing the growth of peritoneal tumors of human colorectal origin. The effect is thought to be brought about by arresting tumor cells at the G(2)/M phase of the cycle and apoptosis. These findings provide evidence for potential value of ABZ in the treatment of regional PC arising from colorectal cell lines.

Toxic Concentrations of Exogenously Supplied Methylglyoxal in Hybridoma Cell Culture

Concentrations at which methylglyoxal, a by-product of cellular metabolism, can be toxic to hybridoma cell cultures were determined using exogenously supplied doses. Trypan blue cell counts of 6-well cultures incubated for 24 h with various methylglyoxal concentrations revealed inhibition of cell growth at 300 muM and higher, with a median inhibitory concentration of 490+/-20 muM. The primary mode of death was apoptosis, as assessed by chromatin condensation, and the effects of methylglyoxal were observed to be complete by approximately eight hours. Yet, the impact of methylglyoxal was a function of the rate of dosing; stepwise addition of MG during the first 6 h of incubation inhibited growth but caused much less cell death than a comparable bolus dose. Inhibition of cellular metabolism by MG was found to coincide with inhibition of cell growth, with a comparable median inhibitory concentration of 360+/-20 muM. The effects on viable cell density and metabolism were both linear at doses approaching zero, with lowest observable effect levels of 54 and 77 muM, respectively. These results provide quantitative estimates for concentrations of methylglyoxal that may be inhibitory to biopharmaceutical-producing cell lines.

Effects of anti-sense Smad4 gene on the biological characteristics of the fat-storing cell line CFSC

To investigate the effects of antisense Smad(4) on the biological characteristics of the fat-storing cell line CFSC. Fat-storing cells of line CFSC from rat with liver fibrosis were cultured and transfected with 50 MOI of recombinant adenoviral vector carrying antisense Smad(4) (AdvATSmad(4)) or the control empty adenovirus (Adv0), both produced by 293 packaging cells, respectively. Two, four, and six days after the transfection the cultured cells were collected to undergo trypan blue staining and cell counting. The growth curves were drawn. The presence of antisense Smad(4) was detected by RT-PCR and Western blotting. (3)H-TdR was added into the culture media to be co-cultured for 6 hours. Then the cells were collected to examine the (3)H-TdR incorporation rate. RT-PCR and immunohistochemistry were used to examine the expression of COL1A1 and type I collagen, kinds of extracellular matrix (ECM). Compared with the control CFSC and the Adv0-transfected CFSC cells, the cell growth curve, (3)H-TdR incorporation rate, proline incorporation rate, expression of Smad(4), and expression of extracellular matrix were markedly decreased in the AdvATSmad(4)-transfected CFSCs. The antisense Smad(4) gene inhibits the expression of Smad(4) mRNA and protein, proline incorporation and cell growth, thus down-regulating the production of ECM. Antisense Smad(4) gene may be used as a choice of gene therapy for liver fibrosis.

Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in human granulosa cells.

The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin. Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry. C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin. Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.