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  • br Materials and methods br A panel of


    2. Materials and methods
    A panel of hematologic malignant (Nalm-6, REH, RPMI8226, KMM-1, K562, U937, KG-1, and NB4) and solid tumor (U87, SKBR3, and T4D7) cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat inactivated fetal bovine serum, 100 U/ml and 100 µg/ml streptomycin in a humidified 5% CO2 atmosphere at 37 °C under standard cell culture conditions. Stock solution of BIBR1532 (Boehringer Ingelheim, Biberach, Germany) was made in sterile DMSO. Relevant amounts of BIBR1532 were added into the culture medium to gain the desired concentrations. As negative control, equal volume of DMSO was added in control samples in which the final concentration of DMSO did not exceed more than 0.1% of total volume. r> The inhibitory effect of BIBR1532 on the metabolic activity of the cells was assessed using MTT assay. Briefly, the cells were seeded at the density of 5000/well into a 96-well culture plate and incubated with the inhibitor for 48 h. After removing the media, the cells were further incubated with MTT solution at 37 °C for 4 h. The resulting formazan solubilized with DMSO and the MG-132 was measured at 570 nm in ELISA reader.
    2.3. Trypan blue dye exclusion test of cell viability
    To evaluate cytotoxic and anti-proliferative effects of BIBR1532 on acute promyelocytic leukemia (APL) cells, NB4 cells were plated into six-well plates and were incubated in the presence of the different concentrations of the inhibitor. After 48 h of treatment, cell suspension was centrifuged and cell pellet was re-suspended in serum-free medium. Afterwards, one part of 0.4% trypan blue (Invitrogen) and one part of cell suspension were mixed and then allowed mixture to incubate 1–2 min at room temperature. The total number of unstained (viable) and stained (nonviable) cells were manually counted and determined. Finally, percentage of viable cells were calculated as follows: Viability (%) = viable cell count / total cell count × 100.
    2.4. Telomeric repeat amplification protocol (TRAP assay)
    The enzymatic activity of unique reverse transcriptase in response 
    to BIBR1532 treatment was determined by Telo TAGGG telomerase kit (Roche Diagnostics GmbH) according to the manufacturer's protocol. The PCR amplified telomerase products obtained in these assays were visualized on 8% PAGE and the ladder was detected by staining with silver nitrate (Sigma). The gel image was analyzed using Quantity One and Multi-analyst software (Bio-Rad). Percentage of inhibition was calculated by comparing telomerase activity of BIBR1532-treated cells with telomerase activity of untreated counterparts.
    2.5. Annexin-V/Propidium iodide (PI) staining assay
    To investigate the effect of BIBR1532 on induction of programmed cell death, NB4 cells were seeded into 12-well cell culture plates and were subjected to flow cytometry analysis 48 h after treatment. Annexin-V-Flous (2 µl per sample) was added, and cell suspensions were incubated for 20 min in the dark. Fluorescence was measured using flowcytometery. Annexin V-positive and PI-negative cells were considered to be in early apoptotic phase and cells having positive staining both for Annexin-V and PI were deemed to undergo late apoptosis or necrosis.
    To determine whether BIBR1532-induced apoptosis is mediated through caspase-dependent cascade, we investigated the enzymatic activity of caspase-3 using a caspase-3 assay kit (Sigma). NB4 cells were treated with BIBR1532 (60 µM) and incubated at 37 °C for 48 h. The cell pellets were lysed by centrifugation at 600 ×g for 5 min and then the lysates were centrifuged at 20,000 ×g for 10 min. In a total volume of 100 µl, 5 µg of the supernatant was incubated with 85 µl of assay buffer plus 10 µl of caspase-3 substrate in a 96-well plate at 37 °C for 2 h. Cleavage of the peptide by caspase-3 released the chromophore pNA, which was quantified spectrophotometrically at a wavelength of 405 nm.
    2.7. Flow cytometric analysis of DNA content
    For detection of the distribution of cells in different phases of cell cycle, we used propidium iodide (PI) staining. After 48 h of incubation with BIBR1532 at the concentration of 60 µM, the cells were harvested, washed with PBS and fixed with 70% ethanol at −20 °C overnight. Before staining with 50 µg/ml PI, the cells were treated with 0.5 µg/ml RNase in PBS and incubated at 37 °C. Finally, cellular DNA content was quantified from the peak analysis of flow cytometric DNA histograms.
    2.8. Assessment of cell-based NF-κB phosphorylation by ELISA
    The inhibitory effects of BIBR1532 on NF-κB phosphorylation was examined by cellular activation of signaling ELISA kit (SABioscience, Frederick) as per manufacture's protocol. NB4 cells at the density of 2 × 104 cells/well were grown in the presence of BIBR1532 in a 96-wellplate and were incubated at 37 °C for 24 h. After fixation in a 8% formaldehyde solution, the cells were subsequently stained with pri-mary and secondary antibodies followed by exposure to developing and stop solutions provided with the kit. After normalizing with the relative cell number, the ratio of phosphorylated NF-κB to total NF-κB was obtained at 450 nm.