Knockdown of PRDX2 sensitizes colon cancer cells to 5-FU by suppressing the PI3K/AKT signaling pathway

Abstract

Although, 5-Fluorouracil (5-FU) remains widely used in adjuvant therapy in patients with colon cancer, resistance to 5-FU-based chemotherapy is an important reason for treatment failure. Recent studies have reported that an enhanced reactive oxygen species (ROS) scavenging system shows drug resistance to 5-FU. Peroxiredoxin-2 (PRDX2), is an important member of the ROS scavenging system, and may be a potential target that promotes chemosensitivity to 5-FU in colon cancer. Here, we depleted PRDX2 by PRDX2-shRNA-LV transduction in two colon cancer cell lines and found that in vitro PRDX2 knockdown facilitates cell death, and apoptosis in 5-FU-treated colon cancer cells. In addition, we found that PRDX2 depletion in mice treated with 5-FU resulted in, inhibition of tumor growth, compared with mice treated with 5-FU alone. Our data also suggested that the PI3K/AKT signaling pathway links PRDX2 with 5-FU-induced apoptosis in colon cancer. Furthermore, when PRDX2 was overexpressed in colon cancer cells, we found increased p-AKT protein expression and reduced Bcl-2/Bax protein expression. PRDX2 and p-AKT protein expression were analyzed by immunohistochemistry technology in human colon carcinoma tissues. Pearson correlation coefficient is 0.873 and P<0.05. PRDX2 depletion led to reduced p-AKT expression and PI3K/AKT pathway inhibition promoted cell apoptosis in HT29 cell line. Taken together, our study suggests that decreasing the expression of PRDX2 could be a promising strategy for increasing the sensitivity of colon cancer cells to 5-FU.

Keywords: 5-FU; AKT; PRDX2; apoptosis; chemosensitivity; colon cancer.

Figure 1. PRDX2 depletion in colon cancer cells

(A) The mRNA levels of PRDX2 in shCont and shPRDX2 cells were analyzed by Q-PCR; **P<0.01. (B) The protein levels of PRDX2 in blank control (BC), shCont(NC) and shPRDX2 cells were analyzed by Western blotting; **P<0.01.

Figure 2. PRDX2 depletion promotes cell death in colon cancer cells

(A) In HT-29 cells, the shCont group and shPRDX2 group were exposed to 5-FU for 48 h and cell viability was measured by the CCK-8 assay. (B) In HCT116 cells, the shCont group and shPRDX2 group were exposed to 5-FU for 48 h and cell viability was measured by the CCK-8 assay.

Figure 3. PRDX2 depletion promotes cell apoptosis in colon cancer cells

(A) Cell apoptosis levels were evaluated by flow cytometry in HT-29 and HCT116 cells treated with 5-FU for 48 h; *P<0.05. (B) The protein levels of C-PARP and caspase-3 in HT-29 and HCT116 cells treated with 5-FU for 48 h were analyzed by Western blotting; *P<0.05, **P<0.01.

Figure 4. PRDX2 depletion reduces the resistance of colon cancer cells to 5-FU in vivo

Five female BALB/c-nu mice were placed in each group. The shCont group treated with 5-FU and shPRDX2 group with PBS injection had longer survival times than nude mice in the shCont group with PBS injection. However, nude mice in the shPRDX2 group with 5-FU treatment had the longest survival time compared with the other three groups; *P<0.05.

Figure 5. The expression of PRDX2 and p-AKT in human colon carcinoma

Representative images of PRDX2 and p-AKT expression in human colon carcinoma samples and normal adjacent tissues are shown.

Figure 6. PI3K/AKT is crucial for PRDX2-mediated 5-FU resistance in colon cancer

(A) The protein expressions of PRDX2 AKT/PI3K, Bax/Bcl-2 were analyzed by Western blotting in HT-29-shCont or HT-29-shPRDX2 cells; *P<0.05. (B) HT29 and HT116 cells were treated with LV-PRDX2 and the protein expressions of AKT, p-AKT, Bax and Bcl-2 were detected by Western blotting. (C) HT-29 cells were treated with MK-2206 2HCl for 48 h and then the protein expressions of PRDX2, AKT/PI3K, Bax/Bcl-2 were detected by Western blotting; *P<0.05. (D) Cell apoptosis levels were evaluated by flow cytometry in HT-29 cells treated with and without MK-2206 2HCl; *P<0.05.