Reversal of multidrug resistance of hepatocellular carcinoma cells by metformin through inhibiting NF-κB gene transcription

Figure 1 The levels of P-glycoprotein, mdr1 and nuclear factor-κB expression in different cell lines. A and C: The levels of P-gp and NF-κB expression in HepG2 or HepG2/ADM cells were determined by Western blot. The number indicates the ratio of HepG2/ADM cells to HepG2 cells (n = 3, mean ± SD); B and D: The gray intensity images of Figure 1A and Figure 1C, respectively; E: The levels of mdr1 and NF-κB mRNA expression were determined by qRT-PCR. ^bP < 0.01 (n = 3, mean ± SD), compared with hepG2 or LO2 cell line. P-gp: P-glycoprotein; NF-κB: Nuclear factor-κB; ADM: Adriamycin.

Figure 2 Metformin enhances adriamycin-induced apoptosis of HepG2/adriamycin cells. Cell early apoptosis was measured by Annexin-V-PE/7-AAD double staining assay in triplicate. A: The blank group (untreated); B: The control group (only treated with adriamycin); C: The experiment group (treated with metformin plus adriamycin). AAD: AnnexinV apoptosis detection.

Figure 3 Metformin down-regulates P-glycoprotein expression via the nuclear factor-κB signaling pathway. A: After HepG2/ADM cells were treated with different doses of metformin for 24 h, the levels of P-gp and p-p65 expression analyzed by Western blot were decreased in a dose-dependent manner, and the cytoplasma p65 increased in a dose-dependent manner; B: The gray intensity images of Figure 3A. ^aP < 0.05, ^bP < 0.01 vs the blank group (n = 3, mean ± SD); C: After HepG2/ADM cells were treated with 1 mmol/L metformin for different time periods, the levels of P-gp and p-p65 expression analyzed by Western blot were decreased in a time-dependent manner, and the cytoplasma p65 increased in a time-dependent manner; D: The gray intensity images of Figure 3C. ^aP < 0.05, ^bP < 0.01 vs the blank group (n = 3, mean ± SD). P-gp: P-glycoprotein; NF-κB: Nuclear factor-κB; ADM: Adriamycin.

Figure 4 Alteration of P-glycoprotein expression in cells treated with metformin plus nuclear factor-κB-small interference RNA. A: Alteration of P-gp expression in different groups of cells. A significant decrease of P-gp expression analyzed by Western blot was found in HepG2/ADM cells treated with the metformin plus NF-κB-siRNA; B: The gray intensity images of Figure 4A. ^bP < 0.01 vs the blank group (n = 3, mean ± SD). P-gp: P-glycoprotein; NF-κB: Nuclear factor-κB; ADM: Adriamycin; siRNA: Small interference RNA.