Figure 1.
The structure of GSP and the characterization of A2780 and A2780/T cells.
(A) The structure diagram of catechin-3-O-2-leucocyanidin (GSP). (B) The expression of MDR1 mRNA in A2780 and A2780/T cells was analyzed by RT-qPCR. MDR1 gene was overexpressed in the resistant cells (A2780/T). **Significantly different from A2780 cells (P<0.01). (C) The protein product of MDR1 gene in A2780 and A2780/T cells was analyzed by Western blotting. P-gp protein was overexpressed in A2780/T cells.
Figure 2.
GSP increased the cytotoxicity of paclitael and adrimycin in A2780 and A2780/T cells.
(A) The cytotoxicity of GSP (3.125∼200μM) in A2780 and A2780/T cells was determined by MTT assay. Each point shows the mean ± SD of three independent experiments, performed in triplicate. Effects of GSP and paclitaxel (B and D) and adriamycin (C and E) in A2780/T (B and C) and A2780 (D and E). Cells were pretreated with or without 10 to 40 μM GSP followed by incubation with various concentrations of paclitaxel for an additional 48 h. The viability of cells was determined by MTT assay. Each point shows the mean ± SD of thee independent experiments, performed in triplicate.
Table 1.
Effect of GSP on reversing mult-drug resistance (IC50± SD μM (fold-reversal), n = 3).
Figure 3.
GSP inhibited the expression, function and transcription of MDR1 in A2780/T cells.
(A and B) The effect of GSP interacted to MDR1 mRNA in A2780/T cells was determined by RT-PCR analysis. Cells were treated with different concentrations of GSP for 48 h (A) or different time-points for the concentration of 40 μM (B). Each column shows the mean ± SD of thee independent experiments, performed in triplicate. *Significantly different from untreated cells(P<0.05), **Significantly different from untreated cells (P<0.01). (C and D) The effect of GSP interacted to MDR1 protein in A2780/T cells was determined by Western blotting analysis. Cells were treated with different concentrations for 48 h (C) or different time-points for the concentration of 40 μM (D). The GAPDH band confirms the integrity and equal loading of protein. **Significantly different from untreated cells (P<0.01). (E and F) Effect of GSP on intracellular Rho-123 accumulation in A2780 (E) and A2780/T (F) cells. Cells were treated with 0∼40 μM GSP or 40 μM verapamil (positive control) for 24 h and then exposed to 10 μM Rho-123 for 90 min. intracellular Rho-123 accumulation was then measured. Each column shows the mean ± SD of thee independent experiments, performed in triplicate. *Significantly different from untreated cells (P<0.05), **Significantly different from untreated cells (P<0.01). (G) A2780/T cells were transiently transfected with plasmids harboring MDR1 reporter gene and treated with 0∼40 μM GSP with and without 10 μM paclitaxel for 24 h. The cells were lysed, and the MDR1 activities were measured by luciferase assay. Each column shows the mean ± SD of thee independent experiments, performed in triplicate. **Significantly different from untreated cells (P<0.01); ##Significantly different from paclitaxel-treated control cells (P<0.01).
Figure 4.
GSP down-regulated the expression of MDR1 by inhibiting NF-κB and MAPK/ERK medicated YB-1 activation.
(A) A2780/T cells were incubated with 40 μM of GSP for indicated times. The lysates were subjected to Western blot analysis using anti-P-gp and the certain antibodies related to NF-κB and MAPK/ERK pathway. GSP inhibited P-gp, phospho-AKT, phosphor-ERK1/2 and phospho-iκBα levels with activated iκBα level. The nuclear and cytosolic protein was extracted from cell lysates. Western blot analysis was performed using the antibodies against nuclear p65, YB-1 and laminB1 as well as cytosolic p65, YB-1 and GAPDH. The GAPDH and phospho-protein relevant total protein band confirmed the integrity and equal loading of total protein and phospho-protein respectively. The Lamin B1 and GAPDH band confirms the integrity and equal loading of nuclear and cytosolic protein respectively. **Significantly different from untreated cells (P<0.01). (B) A2780/T cells were incubated with 0∼40 μM of GSP for 24 h. Phospho-AKT, phosphor-ERK1/2 and phospho-iκBα levels with activated iκBα level were performed by Western Blot. The nuclear and cytosolic protein was extracted from cell lysates. Western blot analysis was performed using the antibodies against nuclear p65, YB-1 and laminB1 as well as cytosolic p65, YB-1 and GAPDH. The Lamin B1 and GAPDH band confirms the integrity and equal loading of nuclear and cytosolic protein respectively. **Significantly different from untreated cells (P<0.01).
Figure 5.
GSP attenuated LPS or RANKL induced over-expression of MDR1 by inhibiting NF-κB and MAPK/ERK medicated YB-1 activation.
(A) A2780/T cells were incubated with 0∼40 μM of GSP for 6h then stimulated with or without 1 μg/ml LPS for 30min. 10μM PDTC and U1026 were used as NF-κB and MAPK/ERK inhibitor respectively. The lysates were subjected to Western blot analysis. GSP inhibited LPS-induced NF-κB, phospho-AKT, phosphor-ERK1/2 and phospho-iκBα levels with activated LPS-reduced iκBα level. The GAPDH and phospho-protein relevant total protein band confirmed the integrity and equal loading of total protein and phospho-protein respectively. *Significantly different from LPS-treated control cells (P<0.05); **Significantly different from LPS-treated control cells (P<0.01). (B) A2780/T cells were pre-incubated with 40 μM GSP for 0∼6h then simulated with or without 1 μg/ml LPS for 30 min. The nuclear and cytosolic protein were extracted from cell lysates. Western blot analysis were performed using the antibodies against nuclear p65, YB-1 and laminB1 as well as cytosolic p65, YB-1 and GAPDH. The Lamin B1 and GAPDH band confirms the integrity and equal loading of nuclear and cytosolic protein respectively. **Significantly different from untreated cells (P<0.01). (C) A2780/T cells treated with 1 μg/ml LPS or 50 ng/ml RANKL followed by pre-treatment with or without 40 μM GSP for 24 h were fixed for immunofluorescence test with staining with anti-p65 (green) and DAPI (blue). The pre-treatment with GSP before LPS or RANKL stimulation prevented the nuclear translocation of p65.