RhoGDI2 up-regulates P-glycoprotein expression via Rac1 in gastric cancer cells

Abstract

Multidrug resistance (MDR) is a major clinical obstacle in treatment of gastric cancer. Previously, using 2D electrophoresis-mass spectrometry, we identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells, and also confer gastric cancer cells resistance to 5-FU. Here, we reported RhoGDI2 also induced MDR in gastric cancer cell line (MKN-45). To explore the underlining mechanism, we detected the mRNA, protein expression, activity of P-glycoprotein (P-gp) in MKN-45 stably transfected with RhoGDI2 expressing or control vector. All the mRNA, protein level, activity were increased by 130%, 230%, 35% respectively after ectopic expression of RhoGDI2. RhoGDI2 was correlated with P-gp expression in gastric cancer tissues as detected by immunohistochemistry. To further study how RhoGDI2 up-regulates P-gp expression, we tested the activity of Rac1 in MKN-45/RhoGDI2 and MKN-45/GFP. Ectopic expression of RhoGDI2 increased Rac1 activity (P < 0.05). For more important, silencing of Rac1 expression by siRNA decreased P-gp expression to undetectable level. Overall, these findings suggest that RhoGDI2 up-regulates P-gp expression via Rac1 to induce MDR.

Keywords: Gastric cancer; Multidrug resistance; P-gp; Rac1; RhoGDI2.

Figure 1

Ectopic expression of RhoGDI2 increased P-gp mRNA, protein and activity. (a) Western blotting analysis of RhoGDI2 and P-gp expression in MKN-45/RhoGDI2 and MKN-45/GFP. The immunoblot image was quantitated by densitometric analysis (right) (n = 3 independent experiments). *p < 0.05 vs MKN-45/GFP. (b) The mRNA of RhoGDI2 (left) and P-gp (right) in MKN-45/RhoGDI2 and MKN-45/GFP was detected by RT-PCR. (c) P-gp activity in MKN-45/RhoGDI2 and MKN-45/GFP was measured as described in materials and methods, MAF was plotted. Data are expressed as mean ± SD from three independent experiments; *p < 0.001 vs MKN-45/GFP.

Figure 2

Representative images of IHC staining with anti-P-gp and anti-RhoGDI2 demonstrate the co-expression of P-pg and RhoGDI2 in serial sections of gastric cancer tissue. Serial sectioning slices were stained with anti-P-gp and anti-RhoGDI2 from one case. Brown color represent positive of staining, the negative staining was contra-stained with hematoxylin and shown in blue color. Most of the cancer cells were positively stained with both anti-P-gp and anti-RhoGDI2. RhoGDI2 is expressed majorly in nuclear, whereas P-gp majorly presents in membrane. Some of the cancerous cells expressed high RhoGDI2 also were positively stained with anti-P-gp (shown in solid arrow), whereas some of the cancerous cells with lower RhoGDI2 were also weakly stained with anti-P-gp (shown in dashed arrow). The non-cancerous cells are negative for both P-gp and RhoGDI2 (marked with triangle). Magnification 400×. Scale bar = 100 μm.

Figure 3

RhoGDI2 up-regulates P-gp expression via Rac1. (a) Rac1 activity in MKN-45/RhoGDI2 and MKN-45/GFP was measured as described in materials and methods. Data are expressed as mean ± SD from three independent experiments; *p < 0.001 vs MKN-45/GFP. (b) Western blot analysis of P-gp and Rac1 expression in MKN-45/RhoGDI2, MKN-45/GFP transfected with Rac-1 or Mock siRNA (pictures are representative of three independent experiments). The immunoblot image was quantitated by densitometric analysis (c) (n = 3 independent experiments). Data was shown in folds relative to MKN-45/GFP + NC siRNA. *p < 0.05 vs MKN-45/GFP + NC siRNA, #p < 0.05 vs MKN-45/RhoGDI2 + NC siRNA.