Results

GBA exerted cytotoxicity in MT-2 cell line

We first determined the viability of MT-2 cells treated with various concentrations of GBA (5, 10, 20, 40, and 80 µM) and/or ATO (2, 4, 8, and 16µM) for 24, 48, and 72 h by alamar blue assay. As shown in Figure 1, the growth kinetics of GBA-treated MT-2 cells differs from those of solvent treated control cells and the proliferative activity of MT-2 cells decreased after exposure to GBA for 24, 48, and 72 h compared to the DMSO-treated group. GBA induced the most cytotoxicity  on MT-2 cells after 48 hours. These findings indicate that GBA is cytotoxic against MT-2 cells in a dose dependent manner with an IC50 of 80 µM at 48 h (53.3% cell viability, P < 0.0001). ATO was also cytotoxic to MT-2 cells with an IC50 of 16 µM at 72h (Data not shown).

Effects of GBA in combination with ATO against MT-2 cells

In order to study the impact of the combination of GBA and ATO, the viability of MT-2 cells treated with GBA (5, 10, and 20 µM) and ATO (2 and 4 µM) was assessed after 24, 48, and 72 h exposure. Our results show that the proliferative activity of MT-2 cells also decreased after 24, 48, and 72 h exposure to the GBA+ATO combinations compared to the control groups (Data not shown).The combination of GBA and ATO was cytotoxic against MT-2 cells in a dose dependent manner and induced the most cytotoxicity  on MT-2 cells after 48 hours (Figure 2A). As shown in figure 2B, treatment with the combination of 20 µM GBA and 4 µM ATO at 48h significantly decreased viability of MT-2 cell (67.3% viability) compared to 20 µM GBA (90.3% viability) or 4 µM ATO (98% viability) alone (p< 0.0001).

GBA in combination with ATO induced sub-G1 phase cells accumulation in MT-2 cell line

To identify whether the cytotoxicity of the GBA+ATO combination was mediated by induction of apoptotic cell death, cell cycle analysis was performed. Flow cytometric cell cycle analysis by PI staining, revealed that 20 µM GBA in combination with 4 µM ATO significantly increased the sub-G1 apoptotic population. As Figure 3 shows, the combination of 20 µM GBA and 4 µM ATO displayed a significant cell arrest in sub-G1 phase (55.56%) compared to GBA or ATO alone at equal concentrations (2.30% and 4.30%, respectively).

GBA inhibits the P-glycoprotein efflux function in MT-2 cells

The function of MDR1/P-gp or ABCB1, an ABC drug transporter, was assessed with flow cytometry using mitoxantrone. To study the P-gp function, the MT-2 cells were treated with 20 µM GBA for 48 h. Then P-gp-mediated mitoxantrone efflux was assessed. As shown in Figure 4, GBA significantly moved histogram shift to right (C) compared to untreated (A) and DMSO-treated (B) controls. From the shift in fluorescence, it is clear that GBA increased mitoxantrone accumulation in MT-2 cells compared to untreated and DMSO-treated cells (D).

GBA regulated apoptosis related genes in MT-2 cells

To study the effects of the GBA+ATO combination treatment on the expression of genes involved in regulation of cell cycle, proliferation, and apoptosis in MT-2 cells, real-time PCR was carried out. As shown in Figure 5, the expression of RelA, p53, CDK4, c-MYC, and c-FLIPS genes in GBA-treated MT-2 cells was lower than the DMSO-treated cells but only RelA (0.025 ± 0.003, p< 0.0001), CDK4 (0.09 ± 0.006, p< 0.0001), and c-MYC (0.64 ± 0.3, p< 0.001) expression reached a significant decrease in GBA-treated cells compared with the control (0.33 ± 0.005, 0.33 ± 0.02, and 3.1 ± 1.02, respectively). Whereas the gene expression of RelA, p53, CDK4, c-MYC, c-FLIPL, and c-FLIPS in GBA+ATO treated cells was dramatically lower than the DMSO-ATO treated cells, only the reduction in the expression of p53 (0.03 ± 0.002, p< 0.0001), CDK4 (0.2 ± 0.01, p< 0.0001), c-FLIPL (0.22 ± 0.1, p< 0.01), and c-FLIPS (0.12 ± 0.02, p< 0.001) genes was statistically significant (0.22 ± 0.05, 0.5 ± 0.04, 1.6 ± 0.3, and 0.9 ± 0.2). In addition, there was a significant positive correlation between the expression of c-FLIPS and p53 (p= 0.008 and r= 0.926) (Figure 6).