Cell lines
This study used the cell lines K562-S (IM-sensitive Bcr-Abl+ cells) and K562-R (IM-resistant Bcr-Abl+ cells), isolated from CML patients in blast phase who were sensitive or resistant to IM treatment, respectively. The cell lines were kindly provided by Dr. JPGAM. HEK-293 cells, derived from embryonic epithelial cells of human kidney, were acquired from the Rio de Janeiro Cell Bank (BCRJ: 0009) and kindly provided by Professor AML.
K562-S and K562-R cells were cultured in complete RPMI (Roswell Park Memorial Institute) 1640 medium, while HEK-293 cells were cultured in complete DMEM (Dulbecco’s Modified Eagle Medium). Both culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and the three cell lines were incubated under an atmosphere of 5% CO2 and 95% air, at 37 °C.
Isolation and purification of MjTX-I
Bothrops moojeni snake venom was donated by the Center for the Study of Venoms and Venomous Animals (CEVAP) from São Paulo State University (UNESP), Botucatu, São Paulo, Brazil, and stored at − 20 °C. The MjTX-I (B. moojeni myotoxin I) was purified from Botrops moojeni crude venom through anion-exchange chromatography on CM-Sepharose (Pharmacia) adapted from Lomonte et al. [28]. The eluted toxin homogeneity was analyzed by SDS–PAGE and reversed-phase chromatography.
Isolation of peripheral blood mononuclear cells (PBMC)
Peripheral blood was collected into vacuum tubes containing anticoagulant, from three healthy individuals aged between 30 and 40 years after their consent. The human peripheral blood mononuclear cells (PBMC) were isolated by the Ficoll-Hypaque density gradient centrifugation method, using Histopaque-1077 (Sigma Diagnostics, Inc., MO, USA). After centrifugation (500 x g for 30 min), the PBMC-rich layer was collected and suspended in 1 mL of complete RPMI. Cells were diluted in Turk’s dye and counted in the Neubauer’s chamber. Next, the cells were plated, treated with MjTX-I, and incubated under an atmosphere of 5% CO2, at 37 °C.
Cytotoxicity assay
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method described by Mosmann [29], with modifications. The tumor cell lines K562-S and K562-R and the non-tumor cells HEK-293 and PBMC (2 × 104 cells) were treated for 24 h with MjTX-I at different concentrations (3.15, 6.25, 12.5, 25, 50, 75, 100, 150, 200, 300, and 400 μg/mL). Untreated cells were used as negative control, and cells treated with 1% Triton X-100 (Bio-Rad, Hercules, CA, USA) were used as positive control. Next, 20 μL of MTT (5 mg/mL) were added to each well, and the microplate was incubated for 4 h, at 37 °C. The supernatants were discarded, and the formazan crystals were dissolved with 200 μL of dimethyl sulfoxide. After 30 min of incubation at room temperature, the absorbance was recorded at 570 nm. The percentage of cell viability was used to calculate the toxin concentration capable of killing 50% of the cells (IC50). The IC50 was calculated by using the Calcusyn 2.1 software.
Apoptosis analyses
Flow cytometric quantification of apoptosis
The toxin potential to sensitize cells to and induce apoptosis was quantified using the hypotonic fluorescent solution (HFS) method reported by Riccardi and Nicoletti [30]. K562-S and K562-R cells (1 × 105 cells) were treated for 24 h with MjTX-I at 6.25, 12.5, 50, and 100 μg/mL, as well as at the concentrations corresponding to the IC50 values for these cell lines: 257 and 191 μg/mL, respectively. Untreated cells were used as the negative control, and 25 μM Etoposide (VP-16) was used as the cell death control. Next, the cells were recovered, transferred to flow cytometry tubes, and suspended in 400 μL of HFS solution (50 μg/mL propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100). After a 15-min incubation in the dark, at 4 °C, cells were analyzed in the FACSCanto flow cytometer (BD, Sunnyvale, CA, USA), with the aid of the FACSDiva software (BD, San Diego, CA, USA). Five thousand events were acquired and the cell population was analyzed to determine the percentage of hypodiploid nuclei (apoptotic nuclei).
Western blotting detection of caspase activation and poly (ADP-ribose) polymerase (PARP) cleavage
K562-S and K562-R cells (1 × 106 cells) were treated for 24 h with MjTX-I (6.25, 12.5, 50, 100, 257, and 191 μg/mL), VP-16 25 μM (positive control), or culture media (negative control). Afterward, the cells were collected and suspended in the western blotting lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and phosphatase and protease inhibitors). Total protein concentration in the samples was determined using the BCA protein assay reagent, according to the manufacturer’s instructions (Thermo Fischer Scientific, Waltham, MA, USA). Equal amounts of protein were analyzed by 15% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), where they were separated according to their molecular weight. Next, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham, GE Healthcare Life Science, Pittsburgh, PA, USA). To detect the proteins, the membranes were first blocked for 2 h with 5% non-fat dry milk prepared in Tris-buffered saline with Tween-20 (20 mM Tris, 137 mM NaCl, 0.01% Tween-20). The PVDF membranes were incubated overnight, at 4 °C, with the following primary antibodies acquired from Cell Signaling Technology (Danvers, MA, USA): anti-caspase 3 (code 96625), anti-caspase 8 (code 9746), anti-caspase 9 (code 9502), anti-PARP (code 9541), and anti-β-tubulin (code 2146). Then, the PVDF membranes were incubated with the appropriate secondary antibodies and the expression of target proteins were detected using ECL (Amersham, GE Healthcare Life Science, Pittsburgh, PA, USA). The protein tubulin was used as an internal standard to normalize protein load among samples.
Expression of apoptosis-related genes
Total RNA extraction
Total RNA from K562-S and K562-R cells (1 × 106 cells) treated with MjTX-I at low concentrations (6.25 and 12.5 μg/mL) was extracted using the Trizol® method, following the manufacturer’s instructions (Invitrogen Life Technologies®, Carlsbad, USA). Untreated cells were used as the negative control. RNA concentration of all samples was determined by the absorbance ratio determined at 260 nm and 280 nm (A260/A280), using the NanoVue spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Complementary DNA (cDNA) synthesis and real-time polymerase chain reaction (PCR)
Total RNA (1 μg) was reverse transcribed to cDNA synthesis using the High Capacity cDNA reverse transcription® assay kit (Applied Biosystems®, Foster City, USA), according to the manufacturer’s instructions. cDNA (diluted 1:4) was used in the real time PCR assay to analyze expression of apoptosis-related genes: BAD, BAX (pro-apoptotic members from Bcl-2 family), BCL-2, BCL-XL (anti-apoptotic members from Bcl-2 family), and the c-FLIP (anti-apoptotic gene from apoptosis extrinsic pathway). Gene expression was quantified by real time PCR (three experiments assayed in duplicate) using the SYBR Green PCR Master Mix Kit (Applied Biosystems, Carlsbad, CA, USA) and the StepOnePlus™ equipment (Applied Biosystems). The results were normalized by the geometric mean of the β-actin and the B2M housekeeping genes expression and represented by 2-ΔΔCt. The sequences of the specific oligonucleotides (Invitrogen Life Technologies) used for quantification of gene expression are listed in Table 1.
Statistical data analysis
Experimental data were compared using One-way Analysis of Variance (ANOVA) followed by the Tukey’s post-hoc test, with the aid of the GraphPad Prism software, version 5.0 (GraphPad Software, San Diego, California, USA). p < 0.05 were considered as statistically significant.
Add Comment