Alamar Blue Reagent Research Articles

Abstract P2-19-02: mTOR Pathway Inhibitor Nanoparticles Beneficially Modulate Autophagy in Triple Negative Breast Cancer Cells

Abstract Background: Autophagy, a cellular degradation pathway, has shown promise as a therapeutic target for many cancers. Depending on the cell type, induction of autophagy has been reported as a cell survival mechanism, making the cells more resistant to common therapies or else a cell death mechanism, synergizing with standard therapeutic agents. We hypothesized that certain nanoparticle-drug combinations could modulate autophagy beneficially for cancer treatment, as they can be targeted to the cells of interest to decrease off-target effects in normal cells. In this study, we propose that nanoparticles containing rapamycin, would induce autophagy in a human triple negative ductal breast cancer cell line, HIM-3 (“human-in-mouse”). Materials and Methods: HIM3 cells were cultured in McCoys 5A supplemented with 10% FBS. HIM3 is a human ductal breast cancer cell line derived from a patient failing chemotherapy that can be grown in vivo in mice. Rapamycin nanoparticles were formulated with a lipid/surfactant coating and a perfluorocarbon (PFC) core. The surfactant layer composition comprised 97.7%phosphatidylcholine, 2mol % DPPE, and 0.28mol% rapamycin. After blending, the slurry was emulsified in one step at 20,000 PSI for 4 min in an ice bath (S110 Microfluidics emulsifier, Microfluidics, Newton, MA). Particle size was measured using dynamic laser light scattering (Brookhaven Instruments Corp., Holtsville, NY). Cell viability assays were performed after 6 days of treatment with Alamar Blue reagent (Invitrogen, Carlsbad, CA). Autophagy flux assays were performed in 6 hour incubations with the nanoparticles and bafilomycin (Sigma, St. Louis, MO) and western blot analysis of LC3B expression for autophagy detection. Western blot analysis was also done to assess phosphorylation of S6 ribosomal protein as evidence for mTOR inhibition. Results: In this study, cell viability decreased (LC50=5nM) in HIM3 when treated with rapamycin nanoparticles. As expected these particles inhibit phosphorylation of ribosomal protein S6, a downstream target of mTOR, by 96% after half an hour of treatment. In order to assess autophagy, an autophagy flux assay was performed over the course of six hours under blockade with bafilomycin treatment to accumulate the marker LC3B. The results from this assay demonstrates a comparable increase in autophagy by the rapamycin nanoparticles as is achieved with a one hundred fold more potent dose of free rapamycin. Discussion: We have demonstrated a novel method for delivering an autophagy modulator to a new line of triple negative breast cancer cells. By increasing direct delivery of the payload to a specific cell type and enhancing trafficking to appropriate intracellular targets, a nanoparticle carrier system appears to be more effective than the free drug in causing a decrease in cell viability through upregulation of autophagic processes. The potential for enhanced therapeutic efficacy with reduced off target effects could emerge as a benefit to this strategy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-02.

New anti-tuberculosis agents amongst known drugs

Mycobacterium tuberculosis has an on-going impact on global public health and new therapeutics to treat tuberculosis are urgently required. The emergence of drug resistant tuberculosis poses a serious threat to the control of this pathogen, and the development of drugs that are active against the resistant strains is vital. A medium-throughput assay using the Alamar Blue reagent was set-up to identify novel inhibitors of M. tuberculosis from a library of known drugs, for which there has already been extensive research investigating their suitability and safety as human therapeutics. Of the 1514 compounds screened, 53 were demonstrated to possess inhibitory properties against M. tuberculosis at a concentration of 5microM or below. Of these, 17 were novel inhibitors while 36 were known tuberculosis drugs or had been previously described as possessing anti-tuberculosis activity. Five compounds were selected as those which represent the most promising starting points for new anti-tuberculosis agents. It was demonstrated that all five were active against intracellular M. tuberculosis in a macrophage model of infection. The anti-tuberculosis agents identified in this screen represent promising new scaffolds on which future drug development efforts can be focused.

Forchlorfenuron Alters Mammalian Septin Assembly, Organization, and Dynamics

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.

Transcriptional regulation of Synoviolin is important for the proliferation of rhumatoid synovial cells

We recently cloned Synoviolin, a transmembrane protein, by immunoscreening from the human cDNA library of rheumatiod synovial cells (RSCs) using anti-RSC antibodies. Synoviolin was highly expressed in rhuematioid synovial fibroblast cells. The mice overexpressing Synoviolin developed spontaneous arthropathy. Conversely, synoviolin+/- mice were resistant to collagen-induced arthritis. These results indicate that it is important for the prevention of arthritis to reduce the amount of Synoviolin. We thus analyzed the transcriptional regulation of Synoviolin promoter and identified that an Ets binding site (EBS) was crucial for the transcription of Synoviolin in vitro and in vivo. Furthermore, to investigate the effect of downregulation of Synoviolin in RSCs, we transfected the EBS decoy oligodeoxynucleotide (ODN) or Synoviolin antisense ODN into RSCs and carried out a proliferation assay using Alamar blue reagent, which resulted in the repression of proliferation activity through the suppression of Synoviolin expression. Our results suggest that the EBS decoy ODN provides a new therapeutic approach for the treatment of arthritis.

A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity

In this study, a fluorometric method using alamarBlue has been developed for detecting cell-mediated cytotoxicity in vitro. AlamarBlue is a non-toxic metabolic indicator of viable cells that becomes fluorescent upon mitochondrial reduction. Specific lysis of targets by effector cells is quantified by comparing the total number of viable cells in wells containing effector and targets together, with wells where target and effector cells were separately seeded. Cell-mediated cytotoxic activity by alloreactive T cells and natural killer cells has been detected using a novel application of the alamarBlue technique. The assay that we have developed to detect cell-mediated cytotoxicity is extremely sensitive and specific and requires a significant lower number of effector cells than the standard 51 Cr assay. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile conditions, effector cells may be recovered at the end for further analysis or cell expansion, if desired. Direct comparison of cell-mediated cytotoxicity measured by the alamarBlue method with the standard 51 Cr release assay revealed that the former method is as specific and more sensitive than the conventional assay. Moreover, very small inter and intra-assay variations have been observed for alamarBlue cytotoxicity assays. In conclusion, this study shows that the alamarBlue assay is an extremely sensitive, economical, simple and non-toxic procedure to evaluate cell-mediated cytotoxicity that yields accurate results using a limited number of effector cells. Furthermore, since this assay is a one-step procedure, and does not involve any risk for the personnel, it may be useful to analyze automatically cell-mediated cytotoxicity in a large number of samples.

Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Saccharomyces cerevisiae infections and antifungal susceptibility studies by colorimetric and broth macrodilution methods

Saccharomyces cerevisiae was isolated in large numbers from operative specimens from two patients with perforated bowel and peritonitis and from the blood of another patient treated with extracorporeal membrane oxygenation. Susceptibility studies were performed on these three isolates and another 29 isolates that colonized or caused infection in a total of 19 patients seen over the last decade. All isolates had low minimum inhibitory concentration (MIC) values for amphotericin B (MIC 90 of ≤0.02 μg/ml) and flucytosine (MIC 90 of 0.2 μg/ ml), and a broader range of MIC values for itraconazole (MIC 90 of 0.8 μg/ml) and fluconazole (MIC 90 of 4 μg/ml). A colorimetric method using Alamar blue reagent showed good concordance with the standard broth macrodilution method for amphotericin B, flucytosine, and fluconazole, but less good concordance for itraconazole. Serious infections with S. cerevisiae probably should be treated with amphotericin B, with or without the addition of flucytosine.