Biomolecules & Therapeutics 2018; 26(3): 313-321  https://doi.org/10.4062/biomolther.2017.033
Cellular Prion Protein Enhances Drug Resistance of Colorectal Cancer Cells via Regulation of a Survival Signal Pathway
Jun Hee Lee1, Chul Won Yun2, and Sang Hun Lee2,3,*
1Department of Pharmacology and Toxicology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, USA, 2Medical Science Research Institute, Soonchunhyang University Seoul Hospital, Seoul 04401, Republic of Korea, 3Department of Medical Bioscience, Soonchunhyang University, Asan 31151, Republic of Korea
E-mail: ykckss1114@nate.com, jhlee0407@sch.ac.kr, Tel: +82-2-709-9029, Fax: +82-2-792-5812
Received: February 20, 2017; Revised: April 27, 2017; Accepted: June 14, 2017; Published online: August 21, 2017.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Anti-cancer drug resistance is a major problem in colorectal cancer (CRC) research. Although several studies have revealed the mechanism of cancer drug resistance, molecular targets for chemotherapeutic combinations remain elusive. To address this issue, we focused on the expression of cellular prion protein (PrPC) in 5-FU-resistant CRC cells. In 5-FU-resistant CRC cells, PrPC expression is significantly increased, compared with that in normal CRC cells. In the presence of 5-FU, PrPC increased CRC cell survival and proliferation by maintaining the activation of the PI3K-Akt signaling pathway and the expression of cell cycle-associated proteins, including cyclin E, CDK2, cyclin D1, and CDK4. In addition, PrPC inhibited the activation of the stress-associated proteins p38, JNK, and p53. Moreover, after treatment of 5-FU-resistant CRC cells with 5-FU, silencing of PrPC triggered apoptosis via the activation of caspase-3. These results indicate that PrPC plays a key role in CRC drug resistance. The novel strategy of combining chemotherapy with PrPC targeting may yield efficacious treatments of colorectal cancer.

Keywords: Colorectal cancer, Prion protein, Drug resistance, 5-fluorouracil, Anticancer
INTRODUCTION

Colorectal cancer (CRC) is the third most frequently diagnosed cancer among males and females, the second leading cause of cancer related death among males, and third leading cause among females (Siegel et al., 2017). Although surgical techniques, chemotherapies, and molecular therapies have improved, clinical outcomes have not kept pace. This lag is mostly because of changes in lifestyle, local recurrence, distal metastasis, and resistance to chemotherapeutics and molecularly targeted therapies (Holohan et al., 2013; Oliphant et al., 2013). Among these hurdles, drug resistance limits therapeutic efficacy, because chemotherapy is one of the principal modes of cancer therapy (Holohan et al., 2013). In particular, chemotherapy failure caused by anti-cancer drug resistance induces most cancer related deaths due to decreases in drug delivery and changes in metabolic enzymes (Hammond et al., 2016). Therefore, understanding the mechanisms underlying drug resistance plays a pivotal role in developing reversal strategies and effective cancer therapeutic combinations.

5-fluorouracil (5-FU) is widely used in the treatment for several cancers, particularly for CRC. It is an antimetabolite drug that inhibits thymidylate synthase and is incorporated into RNA, single DNA, and double DNA helix, leading to cancer cell death (Longley et al., 2003). Several clinical studies have shown that 5-FU-based chemotherapies and chemoradiotherapies increased the survival of patients with several cancers (Pignon et al., 2000; Adelstein et al., 2006; Tsukuda et al., 2010). However, despite the benefits of 5-FU to cancer therapy, acquired resistance to 5-FU is a major clinical problem. Accumulated evidence shows that abnormally high activities of thymidylate synthase, deoxyuridine triphosphatase, Bcl-2, Bcl-XL, and Mcl-1 lead to 5-FU resistance (Zhang et al., 2008). However, the detailed molecular mechanisms for 5-FU chemoresistance in CRC require further investigation.

Cellular prion protein (PrPC) is highly expressed in a variety of cells, such as lymphocytes, and tissues, including muscle, heart, skin, and nervous tissues (Liang and Kong, 2012). Although studies on PrPC have initially focused on the nervous system, recent evidence indicates that PrPC regulates not only self-renewal of stem/progenitor cells and stem cell fate but also proliferation and resistance to apoptosis in cancer cells (Martin-Lanneree et al., 2014). In CRC, PrPC promotes cancer cell survival by increasing uptake of glucose (Li et al., 2011). PrPC augmented CRC metastasis through the Fyn-SP1-SATB1 pathway (Wang et al., 2012). Our previous study reveals that silencing PrPC inhibits colon cancer cell growth (Yun et al., 2016). However, there is little evidence of a relationship between PrPC and anti-cancer chemoresistance in CRC cells. In this study, we investigated the effect of PrPC on proliferation and survival in 5-FU resistant CRC cells. Moreover, we explored the underlying mechanism of resistance to 5-FU in CRC cells through regulation of PrPC expression.

MATERIALS AND METHODS

Preparation of 5-fluorouracil and oxaliplatin

5-Fluorouracil and oxaliplatin were obtained from Sigma (St. Louis, MO, USA). 5-Fluorouracil and oxaliplatin powder were dissolved in dimethyl sulfoxide (DMSO), and aliquots were stored at 4°C until use.

Cell culture

The human colon cancer cell line (SNU-C5/WT), 5-FU resistant cell line (SNU-C5/5FUR) and oxaliplatin-resistant cell line (SNU-C5/Oxal-R) were obtained from the Chosun University Research Center for Resistant Cells (Gwangju, Korea). The cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, l-glutamine, and antibiotics (Biological Industries, Beit Haemek, Israel) at 37°C with 5% CO2 in a humidified incubator.

Western blot analysis

Total cell protein was extracted by utilizing RIPA lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA). Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies against PrPC, phospho-phosphatidylinositol-3-kinase (PI3K), total PI3K, phospho-AKT, total AKT, cyclin dependent kinase (CDK) 2, CDK4, cyclin D1, cyclin E, phosphor-p38, total p38, phosphor-c-JUN N-terminal kinase (JNK), total JNK, cleaved caspase-3, cleaved poly [ADP-ribose] polymerase 1 (PARP1), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-p53, total p53 (Cell Signaling Technology, Danvers, MA, USA), phospho-ataxia-telangiectasia mutated (ATM), and total ATM (Thermo Fisher Scientific). After incubation of membranes with peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Santa Cruz biotechnology), bands were detected by utilizing enhanced chemiluminescence reagents (Amersham Biosciences, Uppsala, Sweden).

Flow cytometry

SNU-C5/WT and SNU-C5/5FUR cells were subjected to flow cytometry analysis using anti-PrPC antibody (Santa Cruz Biotechnology). Flow cytometry was performed using a Cyflow Cube 8 (Partec, Münster, Germany). Data analysis was performed using standard FSC Express (De Novo software; Los Angeles, USA).

Immunocytochemistry

SNU-C5/WT and SNU-C5/5FUR cells on the cover glass slide were fixed in 4% paraformaldehyde solution for 10 min. After washing three times in PBS, cells were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against PrPC (Santa Cruz Biotechnology) at 4°C for 24 h. After washing three times in PBS, cells were incubated with secondary antibodies conjugated with Alexa-488 (Thermo Fisher Scientific) for 1 h. Immunostained slides were imaged by fluorescent microscope (ZEISS, Oberkochen, Germany).

Silencing of PrPC expression by small interfering RNA

SNU-C5/5FUR and SNU-C5/Oxal-R cells were seeded in 60 mm plates and grown up to 75% confluence. Cells were transfected with siRNA in serum-free Opti-MEM (Thermo Fisher Scientific), using Lipofectamine 2000 (Thermo Fisher Scientific) by following the manufacturer’s protocols. At 48 h after transfection, the level of PrPC was analyzed by western blot. The siRNA used to target PRNP and a scrambled sequence were synthesized by Dharmacon (Lafayette, CO, USA).

Kinase assays

The cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific). Cdk4 kinase assays were performed using a CKD4 Kinase Assay Kit (Cusabio, Baltimore, USA). Briefly, 10 mM ATP was added to 1.25 ml of 6 μM substrate peptide. The mixture was diluted with dH2O to 2.5 ml to yield a 2× ATP/substrate cocktail. Then 1 ml of 10× kinase buffer was added to 1.5 ml of dH2O to yield a 2.5 ml 4× reaction buffer and the enzyme was diluted in reaction buffer to give the reaction cocktail. The reaction cocktail was added to 12.5 μl/well of prediluted compound of interest (usually approximately 10 μM) and incubated for 5 min at room temperature. ATP/substrate cocktail was added to 25 μl/well preincubated reaction cocktail/compound. The final assay conditions for a 50-μl reaction were therefore 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM b-glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT, 200 μM ATP, 1.5 μM peptide, and 50 ng CDK4 kinase. The reaction was incubated at room temperature for 30 min, then the stop buffer was added. From each reaction, 25 μl was transferred to a 96-well streptavidin-coated plate and incubated at room temperature for 1 h. Plates were washed three times with PBS. Phospho-Rb (Ser780) antibody was added at 100 μl/well and incubated at room temperature for 2 h. Plates were washed three times with PBS. HRP-avidin was added to each well, and incubated at 37°C for 1 h. Plates were washed five times with PBS. TMB substrate and stop solution were added. The absorbance was read at 450 nm with a microtiter plate reader (Tecan Group AG, Mänedorf, Switzerland).

Cell cycle analysis

SNU-C5/5FUR cells were harvested and fixed with 70% ethanol at −20°C for 2 h. After washing twice with cold PBS, cells were subsequently incubated with RNase and propidium iodide (PI; Sigma). The cell cycle histograms were assessed using a Cyflow Cube 8 (Partec). Results were analyzed using the FCS Express software package (De Novo Software).

Propidium iodide/Annexin V flow cytometric analysis

Apoptosis of SNU-C5/5FUR and SNU-C5/Oxal-R cells was evaluated with a Cyflow Cube 8 (Partec) following staining of the cells with Annexin V-FITC and PI (Sigma). Data analysis was performed using the FCS Express software package (De Novo Software).

Statistical analyses

Data are expressed as means ± SEM. Statistical significance was assessed using the Student’s t test, where p<0.05 was considered significant.

RESULTS

Expression of PrPC in drug resistant CRC cells

Several studies have shown that the expression of PrPC is increased in gastric, breast, and CRC cells (Yang et al., 2014). In addition, PrPC is associated with multidrug resistance in gastric and breast cancer cells (Liang et al., 2009; Cheng et al., 2014). To explore whether PrPC is upregulated in drug resistant CRC cells, we analyzed the expression of PrPC in wild type (SNU-C5/WT) and 5-FU resistant colorectal cancer cell lines (SNU-C5/5FUR) by western blot assay (Fig. 1A). The expression level of PrPC was significantly increased in SNU-C5/5FUR, compared with that in SNU-C5/WT (Fig. 1B). In addition, flow cytometry analysis showed that the ratio of PrPC positive cells was significantly increased in SNU-C5/5FUR (Fig. 1C, 1D). Immunofluorescent staining also revealed an increased level of PrPC in SNU-C5/5FUR (Fig. 1E). These results indicate that 5-FU-induced drug resistant CRC cells express high levels of PrPC.

PrPC regulates CRC cell survival via the PI3K-Akt axis

The PI3K-Akt signaling pathway is a pivotal regulator and central node in cell survival signaling downstream of growth factor, cytokines, and several cellular stimuli (Luo et al., 2003; Manning and Cantley, 2007). To investigate whether PrPC regulates the PI3K-Akt signaling pathway, we evaluated the phosphorylation of PI3K and Akt in SNU-C5/WT and SNU-C5/5FUR (Fig. 2A). The phosphorylation of PI3K and Akt were significantly enhanced in SNU-C5/5FUR, compared with that in SNU-C5/WT (Fig. 2B). To investigate whether the expression of PrPC is associated with regulation of the PI3K-Akt signaling pathway in 5-FU-resistant CRC cells, we confirmed activation of PI3K-Akt in the presence or absence of 5-FU (140 μM). Treatment of SNU-C5/WT with 5-FU increased the level of PrPC (Fig. 2C, 2D). In addition, the level of PrPC was significantly increased in SNU-C5/5FUR in the presence and absence of 5-FU, compared with in SNU-C5/WT (Fig. 2C, 2D). The expression of PrPC was suppressed using PRNP siRNA (si-PRNP) in SNU-C5/5FUR (Fig. 2E, 2F). After treatment of SNU-C5/5FUR with 5-FU, the suppression of PrPC induced a decrease in phosphorylation of PI3K and Akt (Fig. 2G, 2H). These data suggest that PrPC regulates the PI3K-Akt signaling pathway in 5-FU resistant CRC cells.

PrPC is involved in cancer cell proliferation through the regulation of cell cycle-associated proteins

To confirm the effect of PrPC on cell proliferation in 5-FU resistant CRC cells, the expression of cell cycle-associated proteins, such as cyclin E, CDK2, cyclin D1, and CDK4, was analyzed by western blot assay after treatment of SNU-C5/5FUR with 5-FU (Fig. 3A). SNU-C5/5FUR transfected with PRNP siRNA had a significant decrease in the levels of cyclin E, CDK2, cyclin D1, and CDK4 after treatment with 5-FU (Fig. 3B). In addition, SNU-C5/5FUR transfected with PRNP siRNA showed a significant decrease in CDK4 kinase activity after treatment with 5-FU (Fig. 3C). Furthermore, flow cytometry analysis for PI staining showed that PrPC knockdown SNU-C5/5FUR exhibited a significant decrease in the ratio of S phase (Fig. 3D, 3E) after treatment with 5-FU. These findings suggest that PrPC regulates proliferation of 5-FU resistant CRC cells via regulation of cell cycle-associated proteins in the presence of this anti-cancer drug.

PrPC regulates anti-cancer drug-induced stress-associated signaling

The main mechanism of 5-FU is the inhibition of DNA synthesis and mRNA translation (Alvarez et al., 2012). To explore whether PrPC regulates stress-associated proteins in the presence of this drug, we assessed the phosphorylation of stress-associated proteins, including p38, JNK, p53, and ATM, after treatment of SNU-C5/5FUR with 5-FU. SNU-C5/5FUR transfected with PRNP siRNA exhibited a significant increase in the phosphorylation of p38, JNK, and p53 (Fig. 4A–4F) after treatment with 5-FU. However, although knockdown of PrPC induced the phosphorylation of ATM in the PBS-treated group, there was no significant increase in the phosphorylation of ATM after treatment with 5-FU, implying that PrPC is not involved in ATM signaling in the presence of 5-FU (Fig. 4G, 4H). These results indicate that PrPC inhibits the activation of stress-associated signaling against an anti-cancer drug through suppression of p38, JNK, and p53 phosphorylation.

PrPC inhibits apoptosis of anti-cancer drug resistant CRC cells via suppression of caspase-3 activation and PARP1 cleavage

To investigate whether PrPC is involved in apoptosis of 5-FU resistant CRC cells, activation of caspase-3 and cleavage of PARP1 were assessed using a western blot after treatment of SNU-C5/5FUR with 5-FU (Fig. 5A). The levels of cleaved caspase-3 (C-Caspase-3) and PARP1 (C-PARP1) were significantly increased in treatment with 5-FU, compared with those in treatment with PBS (Fig. 5B, 5C). SNU-C5/5FUR transfected with PRNP siRNA had a significant increase in the levels of C-Caspase-3 and C-PARP1 after treatment with 5-FU (Fig. 5B, 5C). In addition, flow cytometry for PI/Annexin V staining showed that silencing of PrPC significantly increased in the percentage of early and late apoptotic cells after treatment with 5-FU (Fig. 5D, 5E). These data indicate that PrPC inhibits apoptosis of CRC cells in anti-cancer drug conditions via suppression of caspase-3 activation and PARP1 cleavage. In addition, SNU-C5/5FUR transfected with PRNP siRNA showed a significant decrease in the kinase activity of CDK4 after treatment with 5-FU (Fig. 3C).

siRRNP enhanced apoptosis in oxaliplatin-resistant CRC cells

We also confirmed apoptosis inhibition by the level of PrPC expression in other drug-resistant CRC cells. We analyzed the expression of PrPC in wild-type (SNU-C5/WT) and oxaliplatin-resistant colorectal cancer cell lines (SNU-C5/Oxal-R) by western blot assay (Fig. 6A, 6B). SNU-C5/Oxal-R transfected with PRNP siRNA showed a significant decrease in CDK4 kinase activity after treatment with oxaliplatin (Fig. 6C). In addition, flow cytometry for PI/Annexin V staining showed that silencing of PrPC significantly increased the percentage of early and late apoptotic cells after treatment with oxaliplatin (Fig. 6D).

DISCUSSION

In this study, we demonstrated that the level of PrPC is increased in 5-FU resistant CRC cells, implying that PrPC increased cell survival in anti-cancer drug conditions by regulating survival and apoptosis signaling pathways. After 5-FU treatment of SNU-C5/5FUR, a 5-FU resistant colorectal cancer cell line, silencing of PrPC yielded the opposite effects, including inhibition of proliferation and augmentation of apoptosis. Previous studies have shown that PrPC contributes to protection from oxidative stress, regulation of copper metabolism, cell cycle control, and cell adhesion (Brown et al., 1997; McLennan et al., 2004; Liang et al., 2007; Malaga-Trillo et al., 2009). In addition, PrPC promotes pro-proliferative and anti-apoptotic effects in stem cells, such as hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and human embryonic stem cells (Martin-Lanneree et al., 2014). In cancer cell biology, PrPC accelerates cell proliferation, invasion, metastasis, and resistance to cytotoxic drugs (Liang et al., 2007; Mehrpour and Codogno, 2010; Wang et al., 2012; Du et al., 2013). Our results revealed for the first time that 5-FU resistant CRC cells express high levels of PrPC. In gastric cancer cells, PrPC expression enhanced drug resistance via an increase in Bcl-2 expression (Wang et al., 2011). Moreover, treatment of HCT116 CRC cells with PrP-specific antibodies inhibited cancer cell growth and promoted the effects of anti-cancer drugs such as 5-FU, cisplatin, and doxorubicin (McEwan et al., 2009). We also confirmed that the level of PrPC expression was significantly increased in oxaliplatin-resistant SNU-C5 cells, compared with wild-type cells (Fig. 6A, 6B). Silencing of PrPC decreased the kinase activity of CDK4 (Fig. 6C) and induced cell death in oxaliplatin-resistant CRC cells after treatment with oxaliplatin (Fig. 6D). These findings suggest that PrPC could be a key protein for resistance to anti-cancer drugs in CRC cells.

The PI3K signaling pathway is involved in various cellular processes, including cell survival, metabolism, inflammation, motility, and cancer progression (Vanhaesebroeck et al., 2010). In particular, the PI3K-Akt signal axis regulates survival and proliferation in cancer cells (Yuan and Cantley, 2008). In human lung cancer cells, Akt amplification plays a pivotal role in acquiring cisplatinum resistance (Liu et al., 2007). Our results indicated that the phosphorylation of PI3K and Akt are significantly increased in 5-FU resistant CRC cells, and inhibition of PrPC expression reduced the activation of PI3K and Akt in the presence of 5-FU. Activation of Akt also promotes cell proliferation through multiple downstream targets for cell cycle regulation (Manning and Cantley, 2007). We also revealed that downregulation of PrPC inhibited proliferation via suppression of cell cycle-associated proteins, such as cyclin E, CDK2, cyclin D1, and CDK4 after 5-FU treatment of 5-FU resistant cells. PrPC accelerated the G1/S transition via the PI3K-Aktcyclin D1 axis (Liang et al., 2007). These findings suggest that PrPC plays pivotal roles in cell survival and proliferation in drug resistant CRC cells via regulation of the PI3K-Akt signaling pathway.

Activation of PI3K-Akt pathway by PrPC expression promotes anti-apoptotic effect (Vassallo et al., 2005). Conversely, downregulation of PrPC promotes caspase-3 activation by reducing Akt activation (Weise et al., 2006). In hypoxic conditions, increased PrPC inhibits TRAIL-mediated apoptosis (Park et al., 2015). Our previous study revealed that silencing of PrPC increased apoptosis of CRC cells. To explore the relationship between PrPC and anti-cancer drug resistance, we showed that treatment with 5-FU induced the activation of stress-associated proteins, such as p38, JNK, and p53, and suppression of PrPC triggered additional activation of these proteins. This additional activation implies that PrPC inhibits the activation of stress-associated proteins in drug resistant CRC cells. Furthermore, PrPC suppressed the activation of caspase-3 after 5-FU treatment of drug-resistant CRC cells. These results suggest that the level of PrPC plays an important role in the development of chemoresistance by CRC cells.

Our findings reveal for the first time that 5-FU resistant colorectal cancer cells express high levels of PrPC, and that PrPC facilitates anti-cancer drug resistance by regulating survival, proliferation, and apoptosis signaling pathways (Fig. 7). Further studies are required to understand how the expression of PrPC is induced during the acquisition of anti-cancer drug resistance. This study may help clarify the physiological function of PrPC in drug resistant colorectal cancer cells. The strategy of targeting PrPC may enable novel molecular target combination chemotherapies.

ACKNOWLEDGEMENTS

This work was supported by the Soonchunhyang University Research Fund, a National Research Foundation grant funded by the Korean government (NRF-2016R1D-1A3B01007727), and a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI14C2253). The funders had no role in the study design, data collection or analysis, the decision to publish, or preparation of the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

Figures
Fig. 1. The levels of cellular prion proteins (PrPC) in SNU-C5/WT and SNU-C5/5FUR cells. (A) Expression of PrPC in SNU-C5/WT and SNU-C5/5FUR cells. (B) The expression level of PrPC was determined by densitometry, relative to β-actin expression. (C) The expression of PrPC in SNU-C5/WT and SNU-C5/5FUR cells was analyzed by flow cytometry. (D) Standard quantification of the percentage of PrPC positive cells. (E) Immunocytochemistry for PrPC (green) in SNU-C5/WT and SNU-C5/5FUR cells. Nuclei were stained by DAPI (blue). Scale bar=100 μm. (B, D) Data are expressed as the mean ± SEM of three independent experiments. **p<0.01 vs. SNU-C5/WT.
Fig. 2. The effect of PrPC on cell survival-associated signaling pathways in colorectal cancer cells. (A) The level of phosphor-PI3K (p-PI3K) and phospho-AKT (p-AKT) in SNU-C5/WT and SNU-C5/5FUR cells. (B) The expression level of p-PI3K and p-AKT was determined by densitometry, relative to total PI3K and AKT expression, respectively. Measurements are reported as means ± SEM. **p<0.01 vs. SNU-C5/WT. (C) The level of PrPC in SNU-C5/WT cells and SNU-C5/5FUR cells after treatment with 5-FU (140 μM). (D) The expression level of PrPC was determined by densitometry, relative to β-actin expression. Measurements are reported as means ± SEM. **p<0.01 vs. SNU-C5/WT treated with PBS, $$p<0.01 vs. SNU-C5/5FUR treated with PBS, and ##p<0.01 vs. SNU-C5/WT treated with 5-FU. (E) The expression of PrPC after transfection of SNU-C5/5FUR cells with PRNP siRNA (si-PRNP). (F) The expression level of PrPC was determined by densitometry, relative to β-actin expression. Measurements are reported as means ± SEM. **p<0.01 vs. SNU-C5/5FUR transfected with scrambled siRNA (siscr). (G) The level of phosphor-PI3K (p-PI3K) and phospho-AKT (p-AKT) in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (H) The expression level of p-PI3K and p-AKT was determined by densitometry, relative to total PI3K and AKT expression, respectively. Measurements are reported as means ± SEM. *p<0.05, **p<0.01 vs. si-scr treated with PBS, $$p<0.01 vs. si-PRNP treated with PBS, and ##p<0.01 vs. si-scr treated with 5-FU.
Fig. 3. The effect of PrPC on proliferation in colorectal cancer cells. (A) The level of cyclin E, CDK2, cyclin D1, and CDK4 in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (B) The expression level of cyclin E, CDK2, cyclin D1, and CDK4 was determined by densitometry, relative to β-actin expression. (C) The kinase activity of CDK4 in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (D) The percentage of G1, S, and G2/M phase in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU was determined by flow cytometry analysis of PI-stained cells. (E) The quantitation of the percentage of cells in S phase. (B, C, E) Data are expressed as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01 vs. scrambled siRNA-transfected SNUC5/5FUR (si-scr) treated with PBS, $p<0.05, $$p<0.01 vs. si-PRNP treated with PBS, and ##p<0.01 vs. si-scr treated with 5-FU.
Fig. 4. The effect of PrPC on cell stress-associated protein in colorectal cancer cells. (A) The phosphorylation of p38 in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (B) The level of p-p38 was determined by densitometry, relative to total p38 expression. (C) The phosphorylation of JNK in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (D) The level of p-JNK was determined by densitometry, relative to total JNK expression. (E) The phosphorylation of p53 in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (F) The level of p-p53 was determined by densitometry, relative to total p53 expression. (G) The phosphorylation of ATM in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (H) The level of p-ATM was determined by densitometry, relative to total ATM expression. (B, D, F, H) Data are expressed as the mean ± SEM of three independent experiments. **p<0.01 vs. scrambled siRNA-transfected SNU-C5/5FUR (si-scr) treated with PBS, $$p<0.01 vs. si-PRNP treated with PBS, and ##p<0.01 vs. si-scr treated with 5-FU.
Fig. 5. The effect of PrPC on apoptosis in colorectal cancer cells through inhibition of caspase-3 activation and PARP1 cleavage. (A) The level of cleaved caspase-3 (C-Caspase3) and cleaved PARP1 (C-PARP1) in SNU-C5/5FUR cells transfected with si-scr or si-PRNP after treatment with PBS or 5-FU (140 μM) for 48 h. (B) The level of C-Caspase3 was determined by densitometry, relative to β-actin expression. (C) The level of C-PARP1 was determined by densitometry, relative to β-actin expression. (D) Apoptosis was measured using flow cytometry analysis for PI/Annexin V staining. (E) The quantitation of the percentage of apoptotic cells. (B, C, E) Data are expressed as the mean ± SEM of three independent experiments. **p<0.01 vs. scrambled siRNA-transfected SNU-C5/5FUR (siscr) treated with PBS, $p<0.05, $$p<0.01 vs. si-PRNP treated with PBS, and ##p<0.01 vs. si-scr treated with 5-FU.
Fig. 6. The effect of PrPC on proliferation and survival in oxaliplatin-resistant colorectal cancer cells. (A) Expression of PrPC in SNU-C5/WT and oxaliplatin-resistant SNU-C5 (SNU-C5/Oxal-R) cells. (B) The expression level of PrPC was determined by densitometry, relative to β-actin expression. (C) The kinase activity of CDK4 in SNU-C5/Oxal-R cells transfected with si-scr or si-PRNP after treatment with PBS or oxaliplatin (10 μM) for 48 h. (D) Apoptosis was measured using flow cytometry analysis for PI/Annexin V staining. (B, C) Data are expressed as the mean ± SEM of three independent experiments. **p<0.01 vs. si-scr treated with PBS, $$p<0.01 vs. si-PRNP treated with PBS, and ##p<0.01 vs. si-scr treated with oxaliplatin.
Fig. 7. Schematic illustrating the protective mechanism of PrPC on 5-FU in drug resistant cancer cells. In 5-FU resistant cancer cells, the expression level of PrPC is increased. In the presence of 5-FU, PrPC promotes cell survival, proliferation, and apoptosis via activation of the PI3K-Akt signaling pathway, suppression of cell cycle-associated protein inhibition, and blockage of stress- and apoptosis-mediated protein activation.
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