BAX/BCL-2 mRNA and protein expression in human breast MCF-7 cells exposed to drug vehicles-methanol and dimethyl sulfoxide (DMSO) for 24 hrs

Kits, reagents and antibodies

Methanol and DMSO (>99% pure) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Neutral Red TOX-4 kit, Methanol, and ethylenediamine-tetraacetic acid (EDTA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit polyclonal anti-BAX antibody and mouse monoclonal anti-BCL2 were purchased from DAKO (DK-2600 Glostrup, Denmark). Secondary antibodies, Goat anti Rabbit Rhodamine conjugated and Goat anti Mouse FITC conjugated were purchased from Abcam (Cambridge, UK). The GeneJET RNA Purification kit, DNase I, RNase-free kit and O'GeneRuler Low Range DNA Ladder were purchased from Thermo Scientific (Pittsburgh PA. USA). High Capacity cDNA Reverse Transcription Kit and Power SYBR^® Green PCR Master Mix were purchased from Life technologies (California, USA). Oligos for qPCR were purchased from Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). Agarose D-1 Low EEO-GQT was purchased from Conda Laboratories (Madrid, Spain).

Cell culture and treatment

Human breast carcinoma MCF-7 cells (American Type Culture Collection Rockville, MD, USA) were cultured in DMEM medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% Fetal Bovine serum (Gibco, USA). The cells were maintained in 25cm² flasks (Corning^[®]) at 37^° C in a humidified atmosphere containing 5% CO₂. The cells were incubated with methanol and DMSO at a 0.5% (v/v) final concentration for 24 hours. Control cells were exposed to the growth medium alone. This work was approved by the ethics committee of the University of Witwatersrand Medical School.

Neutral red uptake assay

The Neutral Red TOX-4 kit was obtained from Sigma (St. Louis, MO, USA). 96-well Falcon tissue culture plates were seeded with approximately 1000 cells/well, suspended in 100 μl DMEM. The plates were incubated for 24 hours to achieve ~60–70% attachment and confluence. The medium was removed and cells were rinsed with 200 μl of phosphate buffered saline (PBS) and subsequently treated with medium containing 0.5% (v/v) methanol and DMSO for 24 hours. The negative control consisted of untreated cells, left in culture medium. Another group of cells were also exposed to 100 ug/ml efavirenz and used as a positive control. 100 ug/ml EFV was selected as a positive control because it exhibited a significant cytotoxic effect on MCF-7 cells in a separate range finder experiment that we conducted. The plate consisted of four replicates for each treatment including negative and positive controls. After 24 hours, the medium was removed and manufacturer's protocol was followed for the assay.¹⁹ Cells were rinsed 3 times in 200 μl of PBS. Neutral red medium was added to each well and plates were incubated for 2 hours for the neutral red dye to be taken up by viable cells. Thereafter the NR solution was removed and cells were rinsed with PBS. The solubilising solution (1% acetic acid in 50% ethanol) was added to each well for 10 minutes in order to extract the dye. The absorbance of the extracted dye was measured in a microplate reader (Anthos 2010 Model 17–550 Austria) at a wavelength of 540 nm. Background absorbance was read at 690 nm and subtracted from the 540 nm measurement. All experiments were repeated in three technical repeats on three different days.

Immunocytochemistry of BAX and BCL2

For immunofluorescence analysis, the ABCAM^[TM] double labelling procedure was followed. 1 × 10³ cells were plated on cover slips 1 day before the experiment. Cells were treated with 0.5% (v/v) methanol and DMSO for 24 h. Following treatment, cells were washed three times with 0.5% BSA in PBS, followed by fixation in 10% phosphate buffered formalin for 10 min. The fixed cells were rinsed in PBS and permeablized with 0.05% Triton-X 100, washed and blocked with 10% Normal goat serum in PBS for 60 mins to eliminate non-specific binding of secondary antibody. Cells were then incubated with polyclonal rabbit antihuman BAX (1:1000 DAKO) and monoclonal mouse antihuman BCL-2 (1:100 DAKO) overnight at 4^° C in a moist chamber. Following overnight incubation, coverslips were washed and incubated with secondary Antibody. The cells were washed and incubated for 2 hours with a FITC-conjugated goat anti-mouse antibody (1:500 Rockland) and Rhodamine-conjugated goat anti-rabbit antibody (1:1000 Rockland) in the dark. Slides were rinsed, nuclei counterstained in 4Ͳ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) 300 nM for 5 mins, rinsed and mounted with fluoromount (Sigma). Negative control groups were set up to ensure that the secondary antibodies were specific for their primary antibodies. The primary antibodies were substituted with 0.1% BSA/PBS and the normal protocol was carried out. In a second negative control, the cells were treated in the same way as the experimental slides, but the secondary antibodies were substituted with PBS. The positive controls used in this study were HeLa cells, which had been previously shown to express BAX and HepG2 cells, shown to express BCL-2.

Cells were visualized using a Zeiss Laser Scanning Confocal Microscope 780 under a Zeiss 100X oil immersion objective. Slides were kept dark once fluorescent antibodies had been added to prevent bleaching. To avoid the fading of fluorochromes, images were analysed within the same time period. The image acquisition settings were set to remain constant for all exposures. Using the ZEN 2010 (Carl Zeiss, Germany) image analysis software, the intensity of the fluorescence of each micrograph was analysed. This was done by using the software to initially define the regions of interest (ROIs); namely, the nucleus and cytoplasm. The mean intensity of each ROI from the treatment groups was then analysed with the statistics software JMP^® (Version 10.0 SAS Institute Inc., Cary, NC, USA). Data are reported as mean ± SEM. After verifying the normal distribution and the homogeneity of the variance using an F test (P < 0.05), a one way analysis of variance (where a significance level of P < 0.05 was set) was used to compare the results.

RNA extraction, cDNA synthesis, and real-time qPCR analysis

Cells were exposed to DMSO and methanol for 24 hours and total RNA extraction was performed using the GeneJET RNA Purification kit according to the manufacturer's instructions. RNA concentration and purity were determined by using the Nanodrop-1000 spectrophotometer. RNA integrity was checked by gel electrophoresis. According to the manufacturer's instructions, genomic DNA was removed from total RNA using the DNase I, RNase-free kit. The DNAase I treated RNA was again cleaned with the GeneJET RNA Purification kit, re quantified and stored at −80^° C until used. According to the manufacturer's instructions, cDNA was synthesized using the MultiScribe™ Reverse Transcriptase from 700 ng RNA. The reverse transcriptase reaction was carried out in a GeneAmp^® PCR System 9600 Thermal Cycler for 10 min at 25^° C, 120 min at 37^° C and then the enzyme was deactivated for 5 min at 85^° C. cDNA aliquots were then utilized in qPCR reactions for BAX and BCL-2, with TBP, RPLP0 and TFRC used as the endogenous reference genes as described previously.²⁰ Briefly, PCR reactions were amplified for 40 cycles prior, then the AmpliTaq Gold^® DNA polymerase was activated for 10 min at 95^° C. Each cycle consisted of a denaturing step for 15 sec at 95^° C, and annealing/extension step for 1 min at 60^° C. PCR amplification was performed in a final volume of 20 μL using the Power SYBR^® Green PCR Master Mix with the ABI 7500 real-time PCR machine. Primer sequences and PCR product sizes are indicated in Table 1. To confirm the absence of nonspecific amplification, PCR products were separated on 3% agarose gels, stained with ethidium bromide and images acquired with the BioRad Gel Doc^® XR (Model 170–8170 Segrate, Milan. Italy). Melt curves were generated for each PCR product. The relative mRNA expression levels of target genes in each sample were calculated using the qbase^PLUS software (Biogazelle, Zulte, Belgium). The expression stability of the reference genes was evaluated using qbase^PLUS version 2.3.²¹ PCR base line Cq values were imported into the qbase^PLUS software. The relative quantity of each target/sample combination was scaled to the average Cq of corresponding target (scale set to untreated control in the qbase^PLUS software). The relative expression of specific genes in the experiment were normalised as a ratio to the amount of the two most stably expressed reference genes according to the -^[ΔΔC] T method of Livak and Schmittgen.²²

Statistical analysis

Statistical analysis was performed using JMP^® (Version 10.0 SAS Institute Inc., Cary, NC). Data are reported as mean ± SEM. After verifying the normal distribution and the homogeneity of the variance using an F test (P < 0.05), a one way analysis of variance (where a significance level of P < 0.05 was set) was used to compare the results. All means were then compared using the Tukey-Kramer HSD.

Neutral red assay

Methanol and DMSO, individually at 0.5% (v/v) concentration did not have any significant (P < 0.0001) cytotoxic effect on MCF-7 cells following a 24 hour incubation as demonstrated by the Neutral Red assay [Figure 1]. Methanol and DMSO at 0.5% (v/v) concentration did not exhibit any significantly different cytotoxic or viability effect from each other (P < 0.0001). 100 ug/ml efavirenz, which was used as a positive control, however exhibited a significant cytotoxic effect on MCF-7 cells [Figure 1].

Immunofluorescence of BAX and BCL2 proteins in 0.5% DMSO and 0.5% methanol exposed MCF-7 cells

Immunofluorescence of the cells was assessed. The untreated group [Figure 2a] shows the expression and co localisation of BAX and BCL2 proteins. Figure 2b illustrates the effect of Methanol on expression of BAX and BCL-2 in MCF-7 cell cultures. While BCL-2 expression is not altered, BAX protein expression evidently is decreased in the 0.5% methanol exposed MCF-7 cell cultures [Figure 2b] compared to the untreated cells seen in Figure 2a. Figure 2c shows the effect of DMSO on expression of BAX and BCL-2 in MCF-7 cells. BAX and BCL-2 were not altered in the 0.5% DMSO exposed MCF-7 cells compared to the untreated [Figure 2a]. These observations were confirmed by statistically analysing the mean fluorescence intensities of BAX and BCL-2 in the nuclei and cytoplasm between the untreated and the treated groups, (Graphical representation shown in Figure 3). There are no differences in fluorescence intensities of BAX and BCL-2 between MCF-7 cells exposed to growth medium alone and 0.5% DMSO. 0.5% methanol however significantly reduced the expression of BAX but not BCL-2 in MCF-7 cells [Figure 3].

BAX and BCL-2 mRNA expression in 0.5%DMSO and 0.5%methanol exposed MCF-7 cells

In the MCF-7 cells exposed to 0.5% DMSO, the differences in fold changes were not significantly (P < 0.05) different from the untreated controls for BAX and BCL-2 [Figure 4]. The cells exposed to 0.5% methanol demonstrated significant differences (P < 0.05) in fold changes when compared to the untreated controls for BAX but not for BCL-2 [Figure 4]. In the 0.5% methanol exposed cells, BAX was significantly down-regulated. This result agrees with the immunofluorescence results.