Figure 1. Time-based dose response analysis of MT-2 cells to GBA after 24, 48, and 72 h. The MT-2 cell line was treated with various concentrations of GBA (5, 10, 20, 40, and 80 µM) for 24, 48, and 72 h. The cell viability was assessed with alamar blue assay. Viability assessment was carried for at least three times and results are presented as mean ± SD. MT-2 cells treated with 0.5% DMSO were considered as relevant control. Statistical significance from control;*P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001.
Figure 2. (A) Viability of MT-2 cells after treatment with various concentrations of GBA and ATO at 48 h. In each combination, cells treated with 0.5% DMSO-ATO (with similar dose) were considered as relevant control. (B)Viability of MT-2 cells significantly decreased after treatment with 20 µM GBA in combination with 4 µM ATO after 48 h compared to treatment with each agent alone at equal concentrations. Viability assessment was carried for at least three times and results are presented as mean ± SD. Statistical significance from control;*P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001.
Figure 3. Effects of GBA/ATO on apoptotic cell death in MT-2 cell line. The MT-2 cells were treated with 20 µM GBA and 4 µM ATO alone or in combination for 48 h and stained with PI. Then the sub-G1 population was analyzed by flow cytometry. PI staining reveals distribution of cells in four major phases of cell cycle including, sub-G1, G1, S, and G2/M. (A) Untreated, (B) 0.5% DMSO-treated, (C) 20 µM GBA-treated, (D) 4 µM ATO-treated, (E) 4 µM 0.5% DMSO-ATO-treated, and (F) 20 µM GBA-4 µM ATO-treated cells.
Figure 4. Flow cytometric analysis of Mitoxantrone uptake and efflux by MT-2 cells. P-gp function was assayed by flow cytometry in MT-2 cells after exposure to 10 µ Mmitoxantrone. As shown by the shift in fluorescence, GBA decreased mitoxantrone efflux in MT-2 cells (C) compared to Untreated (A) and DMSO-treated (B) cells.
Figure 5. The mRNA expression levels of (A) RelA, (B) p53, (C) CDK4, (D) c-MYC, (E) c-FLIPL, and (F) c-FLIPS in MT-2 cells treated with 20 µM GBA and 4 µM ATO alone and in combination. Statistical significance from control, *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001.
Figure 6. Pearson’s correlation analysis of the expression of p53 and c-FLIPS in 6 groups of MT-2 cells treated with 20 µM GBA and/or 4 µM ATO alone and in combination.