Materials and Methods
Isolation of GBA
GBA (C24H30O5: 2-cyclohexene-1-butanoic acid) was extracted from the
herbal roots of Ferula Szowitsiana over a previously described
method (Iranshahi et al., 2007). This study did not
require ethical approval.
Cell culture
The MT-2 cell line (a HTLV-1 transformed human T cell line) was cultured
in RPMI 1640 (KBC, Iran) medium supplemented with 10% fetal bovine
serum (Gibco, USA), 1% penicillin/streptomycin (Capricorn, Germany),
and 0.5% L- Glutamine (Serena, Germany) at 37°C with 5%
CO2 and a humidity of 96%.
Cytotoxicity assay
The cytotoxicity of GBA and ATO was determined by alamar blue assay. The
MT-2 cells were seeded at the density of 50 × 103 per
well in a 96-well cell culture plate, and then treated with various
concentrations of GBA (5, 10, 20, 40, and 80 µM) and/or ATO
(Sigma–Aldrich, St Louis, MO, USA) at the concentrations of 2, 4, 8,
and 16 µM for 24, 48, and 72 h. The effect of GBA and/or ATO on the
viability of MT-2 cells was then assessed by alamar blue assay (Sigma,
Germany). Alamar blue solution (0.15 mg/ml) was incubated until a pink
fluorescent resorufin was constituted. The optical density (O. D) was
measured using a microplate reader (Epoch, BioTek, USA) at 600 nm and
cell viability was calculated. When the IC50 values of
GBA and ATO were determined, the MT-2 cells were treated with GBA and
ATO, alone or in combination, at concentrations lower than
IC50 with weak cytotoxic effects, and then the cell
viability was assessed with alamar blue assay.
Cell cycle analysis
To study the cell cycle distribution in GBA+ATO treated cells, the MT-2
cells were seeded at the density of 40 × 104 per well
in a 6-well cell culture plate and treated with 20 µM GBA and 4 µM ATO
alone or in combination at equal concentrations for 48 h. Then, the
treated cells were washed and resuspended in 480 µl of PI solution (100
µg/mL PI, 0.1% Triton X-100, and 0.1% sodium citrate in PBS) (Sigma,
Germany) and incubated for 30 min at 37°C in dark. Then the flow
cytometric analysis was performed with the FACS Calibur flow cytometer
(BD Biosciences, USA) using FL2 filter. Data obtained from flow
cytometric analysis of cell cycle was then analyzed in winMDI data
analysis software version 2.8.
Efflux assay
To study the activity of P-gp in the presence of GBA, the MT-2 cells
were seeded at the density of 40 × 104 per well in a
6-well cell culture plate and treated with 20 µM GBA for 48 h. After
centrifugation, the cell pellets were resuspended in mitoxantrone (10
µM) and incubated for 30 min at 37°C in dark (accumulation phase). After
washing twice with ice cold PBS, the cell pellets were resuspended in
culture medium and incubated for 60 min at 37°C in dark (the efflux
phase). Finally, the activity of the ATP-binding cassette (ABC)
transporter ABCB1 or MDR1/P-glycoprotein (P-gp) was analyzed by FACS
Calibur flow cytometer (BD Biosciences, USA) using FL3 filter. Data
obtained from flow cytometric analysis of P-gp function was then
analyzed in winMDI data analysis software version 2.8.
cDNA synthesis and real-time
PCR
The MT-2 cells were treated with 20 µM GBA and 4 µM ATO alone or in
combination at equal concentrations for 48 h. Total RNA was extracted
from the treated cells using Tripure isolation reagent (Roche, Germany)
according to the manufacturer’s instruction. Complementary DNA (cDNA)
was then synthesized using reverse transcriptase and random primers
according to manufacturer’s instruction (Thermo Fisher Scientific, USA).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used to
control the cDNA synthesis efficacy.
Primers and probes were designed by Allele ID, version 5 after obtaining
the desired sequences for genes (RelA, p53, CDK4, c-MYC, c-FLIPL, and
c-FLIPS) from the National Center for Biotechnology Information (NCBI)
GenBank. Real-time PCR was performed on Rotor gene 6000 cycler (Qiagen,
Germany), using TaqMan and SYBR Green reagents (Takara, Japan) according
to the manufacturer’s instruction. The gene expression of RelA, p53, and
CDK4 was evaluated with Taq Man real-time PCR assay and SYBR Green
real-time PCR method was performed for c-MYC, c-FLIPL, and c-FLIPS. β2
microglobulin (β2M) was considered as a cellular reference gene in both
methods. Table 1 represents specific primers and probes used for
detection of desired genes.
Statistical analysis
Statistical analysis was performed using Statistical Package for Social
Sciences (SPSS) software (version 16.0, SPSS, Inc, Chicago, IL,
USA).Variables with normal distribution were compared between groups
using one-way ANOVA. Bonferroni post-hoc analysis was used for
between-group comparisons. Additionally, the relationship between genes
was assessed using Spearman’s correlation analysis. P values
<0.05 were considered to be statistically significant. All
data were expressed as means ± standard deviation (SD).