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).