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).