Etomidate Induces Cytotoxic Effects and Gene Expression in a Murine Leukemia Macrophage Cell Line (RAW264.7)

Materials and Methods

Reagents and chemicals. Etomidate was purchased from Lipuro, B. Braun Co. Ltd. (Melsungen, Germany). Etomidate was prepared in phosphate-buffered saline (PBS), and an equal volume of PBS (0.1%) was added to the control cells as vehicle. Propidium iodide (PI) and RNase A were from Sigma-Aldrich Corp. (St. Louis, MO, USA). 4, 6-Diamidino-2-phenylindole (DAPI) was purchased from Molecular Probes/Invitrogen Life Technologies (Eugene, OR, USA). RPMI-1640 medium, fetal calf serum (FCS), penicillin and streptomycin antibiotic mixture were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The primary antibodies to Fas, cytochrome c, apoptosis-inducing factor (AIF), endonuclease G (Endo G), caspase-9, caspase-3, Bax and Bcl-2 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

RAW264.7 cell line. The RAW264.7 murine leukemia cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). The cells were maintained in RPMI-1640 medium with 2 mM L-glutamine, supplemented with 10% heat-inactivated FCS and 1% antibiotic/antimycotic and incubated at 5% CO₂ and 37°C.

Determination of cell viability and morphology. RAW264.7 cells (2×10⁵ cells/ml) maintained in 12-well plates were treated with 0, 2.5, 5, 10, 20 and 40 μg/ml etomidate for 48 h. At the end of incubation, the cells were examined and photographed by a phase-contrast microscope at ×200 or were harvested for measuring the total percentage of viable cells as previously described (8, 9). For trypan blue dye exclusion assay, cells from each treatment were collected and re-suspended in PBS, and then were mixed with 0.4% Trypan blue stain. Cells that excluded blue dye (live cells) and cells that contained blue dye (dead cells) were counted using a hemocytometer under a light microscope (10, 11). The obtained values were compared with PBS only (control) and the quantitative analysis of cell viability was carried out.

Determination of apoptotic bodies by DAPI nuclear staining. The presence of apoptotic bodies and nuclei morphology were examined by DAPI staining. RAW264.7 cells (2×10⁵ cells/well) in 12-well plates were exposed to 40 μg/ml etomidate for 24 h, then cells were fixed in 4% paraformaldehyde-PBS solution for 15 min and were stained with DAPI (300 nmol/l) for 30 min at room temperature. Cells were examined for apoptotic bodies and nuclear morphology and photographed under fluorescence microscopy. Apoptotic cells were recognized and determined based on characteristic observations including the presence of condensed, fragmented, and degraded nuclei (8, 9).

Determination of apoptosis-associated proteins by Western blot assay. RAW264.7 cells (5×10⁵ cells/ml) were placed in 6-well plates with RPMI-1640 medium with 10% FCS for 24 h. Cells were treated with or without 18 μg/ml etomidate for 48 and 72 h. Cells were individually collected from each treatment and total protein was extracted into the PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Seongnam, Gyeonggi-Do, Korea), and centrifuged at 12,000 rpm for 10 min at 4°C as previously described (12, 13). The total protein of each sample was determined by Bradford assay and all cell lysates were treated with reducing sample buffer, and boiled for 5 min at 100°C. Proteins were resolved on an SDS polyacrylamide gel via electrophoresis as described previously (14, 15). The gel was then transferred to PVDF membrane in Western transfer buffer. The membrane was stained by primary antibodies (anti-Fas, -cytochrome c, -AIF, -Endo G, -caspase-9, -caspase-3, -Bax and -Bcl-2) overnight, then washed and stained by appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, South San Francisco, CA, USA). The intensity of immunoreactive bands was determined using a densitometer (Molecular Dynamics, Sunnyvale, CA, USA) equipped with Image QuaNTsoftware (8, 9).

Determination of apoptosis-associated gene expression by microarray assay. RAW264.7 cells (5×10⁵ cells/ml) were maintained in 12-well plates in RPMI-1640 medium with 10% FCS for 24 h. Cells were treated with 0 and 18 μg/ml etomidate for 48 h. The total RNA was extracted by using Qiagen RNeasy Mini Kit (Qiagen, Inc, Valencia, CA, USA) (9). The isolated total RNA was used for cDNA synthesis and labeling and microarray hybridization, which was then followed by fluor-labeled cDNA hybridized complements on the chip (Affymetrix GeneChip Human Gene 1.0 ST array; Affymetrix, Santa Clara, CA, USA) (16). The resulting localized concentrations of fluorescent molecules were detected, quantitated (Asia BioInnovations Corporation, Taipei, Taiwan, ROC) and analysed by using Expression Console software (Affymetrix) with default RMA parameters. Genes regulated by etomidate were determined to be these showing a 2-fold change in expression (16-18)

Statistical analysis. Student's t-test was used to analyze differences between exposure to etomidate and the untreated (control) group. All data are presented as the means±S.D. of three experiments and a p-value less than 0.05 was considered significant.