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

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.


Introduction
Colorectal cancer is the fifth most common cancer in China and one of the leading causes of cancer-related deaths worldwide [1]. Although, chemotherapy is very effective after surgery, resistance to drugs contributes to therapy failure [2]. Therefore, understanding the molecular mechanisms involved in drug resistance and identifying better targets to promote sensitivity to chemotherapeutics is of great importance.
5-Fluorouracil (5-FU) is one of the most widely used anticancer agents, which inhibits thymidylate synthetase and incorporates into both RNA and DNA. 5-FU induces tumor cell apoptosis by increasing the intracellular concentration of reactive oxygen species (ROS); however, an enhanced ROS scavenging system also shows drug resistance to 5-FU [3,4,5]. Peroxiredoxins (PRDX), belong to a family of antioxidant enzymes that protect cells from oxidative stress. PRDX2 is overexpressed in colon cancer and depletion of PRDX2 expression inhibits colon cancer cell growth [6,7]. These findings indicate that PRDX2 could be a potential target to address the problem of 5-FU resistance in colon cancer.
In our study, we used PRDX2-shRNA-LV to deplete PRDX2 expression in colon cancer cells. We showed that knocking down PRDX2 in vitro facilitates cell death and apoptosis in colon cancer cells treated with 5-FU. In addition, PRDX2 depletion in vivo in combination with 5-FU treatment, markedly inhibited tumor growth compared with treatment with 5-FU alone. Furthermore, we found that the PI3K/AKT signaling pathway plays a role in 5-FU-induced apoptosis in colon cancer. Therefore, decreasing the expression of PRDX2 could be a promising strategy for increasing the sensitivity of colon cancer cells to 5-FU.

Cells and reagents
HT-29 and HCT116 human colon cancer cell lines were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in an RPMI 1640 medium (Gibco, U.S.A.) containing 10% FBS (PAN, Germany) and antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin), in a humidified incubator maintained at 5% CO 2 at 37 • C.

Transfection and stable cell line construction
Lentiviral constructs expressing PRDX2 shRNA (PRDX2-shRNA-LV) were purchased from Genechem (Shanghai, China). The PRDX2 shRNA vector sequence was as follows: forward: 5 -TCC TCT TTA TCA TCG ATG GCA ACT CGA GTT GCC ATC GAT GAT AAA GAG GTT TTT TC-3 ; reverse: 3 -TCG AGA AAA AAC CTC TTT ATC ATC GAT GGC AAC TCG AGT TGC CAT CGA TGA TAA AGA GGA-5 . The positive experimental group (sh-PRDX2) consisted of PRDX2-shRNA-LV transduced into cells at a multiplicity of infection (MOI) of 60 using polybrene (10 μg/ml) and enhanced infection solution (Genechem, China). The negative control group (shCont) consisted of NC-shRNA-LV transduced into cells. After 72 h, cells that were transduced with the lentivirus containing shPRDX2 or shCont were selected with medium containing 5 μg/ml of puromycin. qRT-PCR and Western blotting were used to detect the inhibition rates of lentivirus-mediated shRNA targetting PRDX2.

RNA extraction and qRT-PCR analysis
Cellular RNA was extracted from cells using the RNAiso plus reagent (Takara). RNA (1 μg) was reverse transcribed into cDNA with the PrimeScript TM RT Reagent Kit and gDNA Eraser (Takara). qRT-PCR was performed in an ABI Q6 qRT-PCR system (Applied Biosystems Inc., U.S.A.), according to the manufacturer's instructions. The primers for PRDX2 were 5 -GCTGGGCTGTGAAGTGCTGG-3 (forward) and 5 -ACGCCGTAATCCTCAGACAAGC-3 (reverse), and B2M was used as an internal control. The primers for B2M were 5 -CTCTTTCTGGCCTGGAGGCTAT-3 (forward) and 5 -AGTCAACTTCAATGTCGGATGGAT-3 (reverse). All the reactions were performed in triplicates. The relative quantitation of gene expression was analyzed according to the C t method.

In vitro cytotoxicity assay
Cells were seeded in 96-well plates at 1 ×

Cell apoptosis assay
Colon cancer cells were seeded in a culture flask at a density of 1 × 10 6 cells and treated with 5-FU for 48 h. Then, the cells were stained with PE-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) and apoptosis was detected using the Annexin V-PE Apoptosis Detection Kit (Beyotime, China). After staining and incubating, according to the manufacturer's protocol, apoptosis was measured by flow cytometry (BD Biosciences, San Jose, CA, U.S.A.).

Protein extraction and Western blot assay
Cells were lysed in RIPA lysis buffer (Beyotime, China) supplemented with PMSF (Beyotime, China). Cell lysates were cleared by centrifugation at 12000 rpm at 4 • C for 20 min and collected. Protein concentration was determined by the BCA protein assay (Beyotime, China). Proteins were separated by SDS/PAGE (10% gel) and transferred on to PVDF membranes (Immobilon-P, Millipore, Germany). Membranes were blotted with the appropriate primary antibodies overnight at 4 • C and then secondary antibodies for 1 h at room temperature. The antigen-antibody complexes were detected by ECL substrate (Advansta, California, U.S.A.).

Immunohistochemical staining
All human colon carcinoma tissues were collected from colon cancer patients during surgery at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). The study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). Informed consent was obtained for experiments with human subjects. Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin and processed as 5-μm thick sections. Sections were deparaffinized in xylene and rehydrated in graded ethanol. Antigen retrieval was performed by boiling in sodium citrate buffer (0.01 mmol/l). Endogenous peroxidases were inactivated with 3% H 2 O 2 , followed by incubation with goat serum for 20 min at 37 • C, and the primary antibody overnight at 4 • C. Finally, sections were incubated with the secondary antibody for 20 min at 37 • C in a humidified chamber. Peroxidases bound to the antibody complex were visualized by treatment with 3,3 -diaminobenzidine chromogenic substrate solution. Immunolabeled sections were dehydrated in a series of graded ethanol and defatted in xylenes. The sections were then examined with an Olympus BX51 microscope (Olympus, Japan) under bright field illumination, and images were acquired with an Olympus DP70 camera (Olympus, Japan).

Experiments with 5-FU treatment in nude mice
All studies involving animals were approved by the Ethics Committee of Chongqing Medical University. To establish a colon cancer mouse model, HCT116-shCont and HCT116-shPRDX2 cells (5 × 10 6 ) in 0.2 ml PBS were injected intraperitoneally into the flanks of 4-week-old female BALB/c-nu mice (Animal Center of Chongqing Medical University, China). Tumors were allowed to grow for 5 days and then the animals were divided into four groups: shCont + PBS, shPRDX2 + PBS, shCont + 5-FU and shPRDX2 + 5-FU (each group: n=5). Five mice per group were injected intraperitoneally with either PBS or 5-FU (50 mg/kg per day, every 3 days, respectively). The survival of nude mice was measured on a regular basis.

Lentivirus-mediated shRNA inhibition of PRDX2 in colon cancer cells
To investigate the role of PRDX2 in colon cancer cells, we transfected PRDX2-shRNA-LV and NC-shRNA-LV into the HT-29 and HCT116 cell lines. The mRNA and protein expressions of PRDX2 were significantly decreased in the sh-PRDX2 group compared with the shCont group ( Figure 1A,B). These results demonstrate that the lentivirus-mediated shRNA targetted PRDX2 effectively and knocked down PRDX2 expression in colon cancer cells.

PRDX2 depletion promotes cell death in colon cancer cells
To determine the role of PRDX2 in colon cancer cells treated with 5-FU, we used the CCK-8 assay to detect the survival rate of colon cancer cells that were stably transfected with NC-shRNA-LV and PRDX2-shRNA-LV and treated with 5-FU at different concentrations for 48 h. We observed a lower survival rate in the shPRDX2 group, compared with the shCont group (Figure 2A,B). These results indicated that knocking down the expression of PRDX2 increased the chemosensitivity of colon cancer cells to 5-FU, in a dose-dependent manner.

PRDX2 depletion promotes cell apoptosis in colon cancer cells
We analyzed the cell apoptosis by flow cytometry and found that knocking down PRDX2 expression in FU-treated colon cancer cells markedly increased apoptosis and protein levels of cleaved PARP and caspase-3 ( Figure 3A,B). Collectively, our results suggest that PRDX2 depletion, combined with 5-FU, promotes cell apoptosis in colon cancer cells.

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.

PRDX2 depletion reduces colon cancer cell resistance to 5-FU in vivo
To ascertain the effect of PRDX2 knockdown in colon tumor growth, we examined the in vivo efficacy of 5-FU in mice bearing abdominal tumors that originated from HCT116-shPRDX2 or HCT116-shCont cells. Using Kaplan-Meier analysis with the log-rank test, we found that the mice in the shCont group treated with 5-FU and the untreated shPRDX2 group had longer survival times than the mice in the untreated shCont group. However, the mice in the shPRDX2 group treated with 5-FU exhibited the longest survival time of the four groups ( Figure 4). These in vivo results suggest that PRDX2 contributes to anticancer drug resistance and that decreasing the expression of PRDX2 in combination with 5-FU treatment reduces tumor development in colon cancer.

Expression of PRDX2 and p-AKT in human colon carcinoma
To investigate whether PI3K/AKT contributes to PRDX2-induced drug resistance in colon cancer, we used immunohistochemistry to detect PRDX2 and p-AKT protein levels in tumor tissues and their corresponding normal colorectal mucosal tissues from 50 colon cancer patients. We found that PRDX2 and p-AKT were strongly expressed in the cytoplasm of colon cancer cells, and were weakly expressed in normal colon mucosal tissues ( Figure 5). The proportion  of PRDX2-positive cells and p-AKT-positive cells were 86% (43/50) and 88% (44/50), respectively. On further analysis, we found that PRDX2 and p-AKT showed a significant positive correlation (r =0.738, P<0.05) through Pearson correlation ( Table 1). These results demonstrate that PI3K/AKT contributes to PRDX2-mediated 5-FU resistance.

PI3K/AKT is crucial for PRDX2-mediated 5-FU resistance in colon cancer
The protein expressions of AKT, p-AKT and p-PI3K were analyzed by Western blotting in HT-29-shCont or HT-29-shPRDX2 cells. We found that depleting PRDX2 resulted in reduced p-AKT and p-PI3K expressions ( Figure  6A). Furthermore, as shown in Figure 6B Figure 6C). These results were validated by measuring apoptosis by using flow cytometry after cells were treated 5-FU in the presence or absence of MK-2206 HCl . We observed higher C-PARP and Bax protein expression and a higher rate of apoptosis in colon cancer cells treated with MK-2206 2HCl ( Figure 6C,D). Together, these data suggest that 5-FU targets PRDX2 and down-regulates its expression through the AKT/PI3K pathway.

Discussion
In general, higher levels of ROS contribute to tumor progression and development in most tumors compared with normal tissues [8]. Moreover, oxidative stress is associated with the efficacy of cancer treatments, including chemosensitivity and apoptosis [9]. However, extremely high ROS concentrations are deadly to tumor cells [10]. 5-FU inhibits thymidylate synthetase and incorporates into both RNA and DNA, inducing intracellular increases in O 2− levels in colon cancer [5,11]. This results in a new redox balance with a higher ROS level and an enhanced ROS scavenging system, that is resistant to 5-FU [12]. For these tumors, combining ROS generating drugs and restraining the cellular antioxidant capacity is effective. For example, combining arsenic trioxide and ascorbic acid, induces GSH depletion, and is effective in treating relapsed or refractory multiple myeloma [13]. In fact, PRDX3 knockdown combined with 5-FU increased cell death and tumor suppression in colon cancer [14]. PRDX2, is an important member of the ROS scavenging system, and exerts an anti-apoptotic effect on colon cancer cells. Lu et al. [15] showed that PRDX2 is up-regulated and protects cells from oxidative stress in colorectal cancer. In our study, we found that knockdown of PRDX2 expression promotes sensitivity to 5-FU in colon cancer cells. These results suggest that depleting PRDX2 combined with 5-FU treatment could be a promising strategy for anticancer drug resistance. Hwang et al. [4] found that 5-FU treatment induced ROS generation in human lung carcinoma cells, leading to a higher level of antioxidant enzymes and resistance to 5-FU. PRDX proteins protect MCF-7 breast cancer cells from doxorubicin-induced toxicity [16]. PRDX6 overexpression attenuated cisplatin-induced apoptosis in human ovarian cancer cells [17]. Kalinina et al. [18] found that cisplatin resistance in cancer cells was accompanied with a significantly increased expression of PRDX2 gene. In pancreatic cancer, a proteomic study using gemcitabine-sensitive KLM1 and -resistant KLM1-R cells showed that PRDX2 was significantly up-regulated in KLM1-R cells [19]. The expression of PRDX2 was higher in osteosarcoma patients that were poor responders to induction chemotherapy. In addition, PRDX2 depletion contributed to increased sensitivity to chemotherapeutic drugs in osteosarcoma cells