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Expression of phosphoenolpyruvate carboxykinase linked to chemoradiation susceptibility of human colon cancer cells
- Ji-Won Park†1, 2,
- Seung Cheol Kim†3,
- Won Ki Kim†1,
- Jun Pyu Hong1,
- Kyung-Hee Kim1,
- Hyun Yang Yeo1,
- Jae Yong Lee1,
- M Sun Kim4,
- Jong Heon Kim4,
- Se Young Yang1,
- Dae Yong Kim1, 2,
- Jae Hwan Oh1, 2,
- Jae Youl Cho5 and
- Byong Chul Yoo1Email author
© Park et al.; licensee BioMed Central Ltd. 2014
Received: 31 May 2013
Accepted: 28 February 2014
Published: 6 March 2014
Resistance to 5-fluorouracil (5-FU) in patients with colorectal cancer prevents effective treatment and leads to unnecessary and burdensome chemotherapy. Therefore, prediction of 5-FU resistance is imperative.
To identify the proteins linked to 5-FU resistance, two-dimensional gel electrophoresis-based proteomics was performed using the human colon cancer cell line SNU-C4R with induced 5-FU resistance. Proteins showing altered expression in SNU-C4R were identified by matrix-associated laser desorption/ionization–time-of-flight analysis, and their roles in susceptibility to 5-FU or radiation were evaluated in various cell lines by transfection of specific siRNA or creation of overexpression constructs. Changes in cellular signaling and expression of mitochondrial apoptotic factors were investigated by Western Blot analysis. A mitochondrial membrane potential probe (JC-1 dye) and a flow cytometry system were employed to determine the mitochondrial membrane potential. Finally, protein levels were determined by Western Blot analysis in tissues from 122 patients with rectal cancer to clarify whether each identified protein is a useful predictor of a chemoradiation response.
We identified mitochondrial phosphoenolpyruvate carboxykinase (mPEPCK) as a candidate predictor of 5-FU resistance. PEPCK was downregulated in SNU-C4R compared with its parent cell line SNU-C4. Overexpression of mPEPCK did not significantly alter the susceptibility to either 5-FU or radiation. Suppression of mPEPCK led to a decrease in both the cellular level of phosphoenolpyruvate and the susceptibility to 5-FU and radiation. Furthermore, the cellular levels of phosphoenolpyruvate (an end product of PEPCK and a substrate of pyruvate kinase), phosphorylated AKT, and phosphorylated 4EBP1 were decreased significantly secondary to the mPEPCK suppression in SNU-C4. However, mPEPCK siRNA transfection induced changes in neither the mitochondrial membrane potential nor the expression levels of mitochondrial apoptotic factors such as Bax, Bcl-2, and Bad. Downregulation of total PEPCK was observed in tissues from patients with rectal cancer who displayed poor responses to preoperative 5-FU-based radiation therapy.
Our overall results demonstrate that mPEPCK is a useful predictor of a response to chemoradiotherapy in patients with rectal cancer.
The use of 5-fluorouracil (5-FU) as a chemotherapeutic agent for colorectal cancer (CRC) is pervasive in the field of medicine because of its ability to act as an S-phase-specific antimetabolite and to suppress cell proliferation [1, 2]. In chemoradiotherapy (CRT), radiation is often performed in conjunction with 5-FU . However, resistance to radiation has been reported in some patients with CRC, and their poor response to either 5-FU or radiation treatment makes preoperative CRT ineffective and burdensome. Activated NF-κB along with other NF-κB–regulated gene products, such as Bcl-xL, cyclin D1, matrix metalloproteinase 9, vascular endothelial growth factor, or COX-2, might contribute to radiation resistance by promoting prosurvival signaling . Ashele et al. found that 5-FU resistance may be caused by impaired polyglutamylation of the thymidylate synthase cofactor CH2FH4 and decreased incorporation of 5-FU into RNA .
Phosphoenolpyruvate carboxykinase (PEPCK) is known to exist in both the cytosol and mitochondria in the mouse, human, and chicken. This enzyme catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form phosphoenolpyruvate (PEP) and GDP [6, 7]. Cytosolic PEPCK has been investigated extensively and is considered to be a key enzyme in gluconeogenesis and glyceroneogenesis [8, 9]. In contrast, the mitochondrial isoform of PEPCK (mPEPCK) has a metabolic role that is complementary to but distinct from that of cytosolic PEPCK in the regulation of gluconeogenesis .
In this study, we found that mPEPCK is downregulated in the human colon cancer cell line SNU-C4R with induced 5-FU resistance. We herein present the results of this study and discuss how the total amount of PEPCK may affect the CRT response.
Human colon cancer cell lines and establishment of 5-FU–resistant cell lines
The human colon cancer cell lines SNU-C4, SNU-C5, SNU-81, SNU-407, DLD-1, SW620, LoVo, HCT-116, NCI-H747, NCI-H508, and CaCo2 were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell line SNU-C4R, which is resistant to the anticancer agent 5-FU (Choongwae Pharma Corporation, Gyeonggi, Korea), was derived from SNU-C4 as described previously .
Tissue from patients with CRC
Characteristics of patients with rectal cancer
Patients number (%)
Age (years), median (range)
Tumor distance from anal verge (cm), median (range)
Pretreatment CEA (ng/mL), median (range)
Tumor size (cm), median (range)
A colorimetric assay using the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was used to assess suppression of cell survival by 5-FU (Dong-A Pharmaceutical Co., Ltd., Seoul, Korea) and irradiation. Cells grown in a 96-well plate were incubated with 5-FU and/or irradiated in a self-contained X-ray system (X-RAD 320; Precision X-Ray, Inc., North Branford, CT, USA) at 290 kV and 10 mA. The dose rate was 8 Gy/min. Single-cell suspensions were prepared, and the cell density was measured. MTT assays were performed as described previously . All experiments were performed three times, and the mean and standard deviation of the half-maximal inhibitory concentration (IC50, μg/ml) were calculated.
Two-dimensional gel electrophoresis-based comparative proteomics
Two-dimensional gel electrophoresis (2-DE) analysis was performed as described previously . A 0.15-mg protein sample was applied to 13-cm immobilized nonlinear gradient strips (pH 3–10), focused at 8000 V within 3 h, and separated in 10% polyacrylamide gels (Serva, Heidelberg, Germany; Bio-Rad, Hercules, CA). The 2-DE gels were stained with Colloidal Coomassie Blue (Invitrogen, Carlsbad, CA) for 24 h and then destained with deionized water. Proteins showing abnormal expression were subjected to matrix-associated laser desorption/ionization–mass spectroscopy (MALDI-MS) analysis for identification. MALDI-MS analysis of 2-DE protein spots was performed as previously described . Mass spectra were first calibrated in the closed external mode using the 4700 Proteomics Analyzer Calibration Mixture (AB SCIEX, Foster City, CA) and analyzed with GPS Explorer software, version 3.5 (AB SCIEX). The acquired MS/MS spectra were searched against the Swiss-Prot and NCBI databases by an in-house version of MASCOT.
Whole-protein extraction and subcellular fractionation
Cells or tissues were homogenized in four volumes of cell lysis buffer (Pro-Prep; iNtRON Biotechnology, Gyeonggi, Republic of Korea) using a Sample Grinding Kit (GE Healthcare, Piscataway, NJ). The total homogenate was incubated on ice for 20 min and centrifuged at 600 × g for 5 min. The supernatant was used as a whole protein extract. The cytosolic fraction was obtained by centrifugation of the whole protein extract at 11,000 × g for 10 min.
For isolation of an enriched, functional mitochondrial fraction from cells, the Mitochondria Isolation Kit (Catalog No. MITISO1; Sigma, Saint Louis, MO) was used as recommended by the manufacturer. Briefly, cells were suspended with 10 volumes of the extraction buffer (20 mM MOPS, pH 7.5, containing 110 mM KCl, 1 mM EGTA, and 0.25 mg/ml trypsin) and incubated on ice for 3 min. The cells were then centrifuged for a few seconds. The supernatant was removed by aspiration, and eight volumes of the extraction buffer were added. After incubation on ice for 20 min, the albumin solution was added to a final concentration of 10 mg/ml to quench the proteolytic reaction. The solution was then centrifuged for a few seconds. The supernatant was removed by aspiration, and the pellet was washed with 8 volumes of the extraction buffer. This step was repeated. The pellet was then homogenized and centrifuged at 600 × g for 5 min. The supernatant liquid was transferred to a new tube and centrifuged at 11,000 × g for 10 min. The pellet was suspended in the storage buffer (10 mM HEPES, pH 7.4, containing 250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 5 mM sodium succinate, 2 mM K2HPO4, and 1 mM DTT [~40 ml per 100 mg of tissue]) and used as a mitochondrial fraction.
Western blot analysis
Western Blot analysis was performed as described previously . Supernatants of cell homogenates containing equivalent amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated for 2 h at room temperature with primary anti-PEPCK antibody that attaches to both cytosolic and mitochondrial PEPCK) (Catalog No. sc-32879; Santa Cruz Biotechnology, Inc., Dallas, TX), VDAC (Abcam, Cambridge, UK), actin (Capital Bioscience, Rockville, MD), Bax (Abcam), Bcl-2 (Abcam), Bad (Abcam), AKT (Cell Signaling Technology, Inc., Danvers, MA), 4EBP1 (Cell Signaling Technology, Inc.), mTOR (Cell Signaling Technology, Inc.), p-AKT (Ser 473; Cell Signaling Technology, Inc.), p-mTOR (Ser 2448; Cell Signaling Technology, Inc.), or p-4EBP1 (Thr 70; Cell Signaling Technology, Inc.). The membranes were washed, incubated with diluted HRP-conjugated secondary antibody (SouthernBiotech, Birmingham, AL), and exposed to film (Blue XB-1; Kodak, Rochester, NY).
Measurement of PEP
The level of intracellular PEP was measured using a PEP assay kit (BioVision Inc., Milpitas, CA) as recommended by the manufacturer.
Transfection of mPEPCK siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) and negative control siRNA (Qiagen, Chatsworth, CA) was performed with the HiPerFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
mPEPCK expression construct
To generate pEGFPc1-mPEPCK, a human fetal liver cDNA library (Clontech, Mountain View, CA) was PCR-amplified with the following oligomers specific to human mPEPCK (Macrogen, Seoul, Korea): sense, 5′-GGAATTCCATGGCCGCATTGTACCGCC-3′ and antisense, 5′-CGGGATCCTCAGGTCACATTTTGTGCACACGTC-3′. The amplified DNA was digested with EcoRI-BamHI and then inserted into pEGFPc1 (Clontech). The construct was verified by DNA sequencing (Cosmo Genetech, Seoul, Korea).
Detection of mitochondrial membrane potential
Cells were treated with 5-FU and radiation as described above, then dislodged by trypsin. After centrifugation, the pelleted cells were washed with PBS and suspended in 5 μg/ml of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 dye; Molecular Probes, Eugene, OR) for 15 min at 37°C in an incubation chamber. The cells were washed again with PBS and then resuspended in PBS. The stained cells were analyzed for their relative apoptosis content using a flow cytometry system (FACSCalibur; BD Biosciences, Franklin Lakes, NJ).
Between-group differences were calculated using Student’s t-test, and within-group correlations were calculated using Spearman’s rank-coefficient. Statistical significance was set at P < 0.05.
Downregulated mPEPCK in human colon cancer cell line SNU-C4R with induced 5-FU resistance and its correlation with 5-FU response
Decreased cellular level of PEP and reduced susceptibility to 5-FU/radiation induced by mPEPCK suppression in SNU-C4
Effect of mPEPCK suppression on mitochondrial membrane potential and signal-transducing molecules
Examination of the phosphorylation level of signal-transducing molecules after mPEPCK siRNA transfection revealed lower levels of phosphorylated AKT (p-AKT) and phosphorylated 4EBP1 (p-4EBP1) in mPEPCK siRNA-transfected cells than in the NS control or normal, nontransfected SNU-C4 line (Figure 5c).
Poor response to CRT in patients with rectal cancer is associated with low PEPCK expression
In this study, we investigated the possible role of mPEPCK as a predictor of 5-FU and radiation susceptibility in patients with CRC. Using different CRC cell lines and tumor tissues of patients with CRC who were treated with 5-FU-based CRT, we showed that mPEPCK was downregulated in 5-FU–resistant cells and tumor tissues of patients who were resistant to 5-FU–based CRT. Several mechanisms of reduced PEPCK expression have been proposed. Antioxidants can suppress PEPCK expression through the transcription factor activator protein 1, or PEPCK can be degraded by proteasome pathways in response to high glucose levels [15, 16]. Although the cause of PEPCK downregulation in nonresponders to 5-FU–based CRT has not yet been elucidated, low PEPCK expression seems to be related to a poor response.
Downregulation of mPEPCK in the human colon cancer cell line SNU-C4R with induced 5-FU resistance was confirmed by 2-DE and MALDI-MS (Figure 1a and b). mPEPCK expression and responses to 5-FU differed among the 11 human colon cancer cell lines (Figure 2). However, there was no significant correlation between mPEPCK expression and 5-FU susceptibility in any of these 11 colon cancer cell lines (Figure 2c). This finding suggests that downregulation of mPEPCK may be linked to induced 5-FU resistance, not intrinsic 5-FU susceptibility, in colon cancer cells. mPEPCK was suppressed using mPEPCK siRNA to clarify the role of mPEPCK in the cellular response to 5-FU, and successful suppression was confirmed by the observation of decreasing PEPCK expression levels over time after siRNA transfection (Figure 3a). Compared with the NS control, the mPEPCK-suppressed SNU-C4 cells demonstrated slower proliferation (Figure 3b), but a higher survival rate, after combined treatment comprising 5-FU and radiation (P = 0.0005) (Figure 3d). These findings suggest the involvement of mPEPCK in response to both 5-FU and radiation. Slow growth may be caused by downregulated energy metabolism. Because PEP is the final product of PEPCK as well as the substrate of pyruvate kinase, which is an enzyme involved in a rate-limiting step of glycolysis, a low cellular level of PEP followed by mPEPCK suppression may lead to slow proliferation of colon cancer cells (Figure 3b and c). Our previous reports showed that low energy metabolism and slow proliferation can decrease the susceptibility of cancer cells to anticancer drugs [17, 18]. Although upregulated expression of mPEPCK showed the potential to slightly increase the 5-FU susceptibility in both the SNU-C4 and LoVo cell lines, the effects were not statistically significant (Figure 4). Because overexpressed mPEPCK was not found in the mitochondrial fraction (data not shown), further investigations are necessary to validate the effect of subcellular localization of this enzyme on CRT susceptibility.
Although the metabolic characteristics of mPECK remain largely unknown, recent reports suggest that mPEPCK plays various roles in gluconeogenesis and anaplerotic reactions. For example, mPEPCK plays a role in mitochondrial GTP synthesis with insulin release through anaplerotic PEP cycling  and cooperates with cytosolic PEPCK to adjust gluconeogenic/TCA flux in response to changes in substrate or energy availability . At present, we cannot explain the molecular mechanism linking mPEPCK expression and susceptibility of CRC cells to 5-FU or radiation. However, expression of both cytosolic PEPCK and mPEPCK may indicate changes in gluconeogenic flux because PEPCK expression can be exquisitely coordinated in parallel with glucose requirements . Our previous reports have shown that the slow energy metabolic process caused by downregulation of key enzymes, such as mitochondrial ATP synthase and pyruvate kinase M2, lead to differential responses of cancer cells to anticancer drugs [17, 18]. Thus, mPEPCK downregulation could also lead to slow energy metabolism in CRC cells, and it may subsequently reduce the susceptibility of CRC cells to 5-FU or radiation.
5-FU is known to act as an S-phase-specific antimetabolite that suppresses cell proliferation [1, 2]. When it enters cells, fluorouracil is converted to 5-fluoro-2′-deoxyuridylate, which can competitively inhibit thymidylate synthase and block the formation of deoxythymidylate, which is required for DNA synthesis . However, the flow cytometry results showed that after 1 μg/ml 5-FU treatment or 8-Gy radiation, the mPEPCK siRNA-transfected cells did not exhibit a significantly different mitochondrial membrane potential or level of apoptosis compared with the NS control (Figure 5a). This finding suggests that downregulation of mPEPCK may affect the energy metabolism of cancer cells, leading to slow proliferation, but that mPEPCK itself cannot augment apoptotic cell death by either 5-FU or radiation treatment. Bad forms a heterodimer with Bcl-2, which is known to induce cells to persist in a G-0 state and to promote cell survival by inhibiting the caspase cascade activation induced by cytochrome c [19, 20]. Therefore, in effect, this heterodimer formation counters the antiapoptotic effects of Bcl-2, allowing Bax/Bak-triggered apoptosis . Previous studies have also found that Bax interacts with the permeability transition pore to lower the mitochondrial membrane potential and release cytochrome c, inducing apoptosis . We therefore examined the expression levels of mitochondrial molecules such as Bax, Bcl-2, and Bad in our study. However, in line with the flow cytometry results, the mitochondrial expression levels of Bax, Bcl-2, and Bad were not altered in the mPEPCK siRNA-transfected cells (Figure 5b). To identify a link between mPEPCK and cellular events, we finally investigated the expression of the main cellular signaling pathway molecules, such as AKT, 4EBP1, and mTOR, which are involved in important pathways downstream of Ras (Figure 5c). Lower amounts of p-4EBP1 and p-AKT were detected in the mPEPCK-silenced cells than in the nontransfected SNU-C4 or the NS control (Figure 5c). 4EBP1 plays an important role in the control of protein synthesis, survival, and cell growth [23, 24]. When phosphorylated, 4EBP1 has a reduced binding affinity for EIF4E, which leads to the release of EIF4E and the initiation of cap-dependent translation . p-AKT promotes growth factor-mediated cell growth and phosphorylates Bad, hindering apoptosis . Decreased expression levels of p-AKT and p-4EBP1 in the mPEPCK-silenced cells implies that mPEPCK downregulation may not only decrease normal cellular metabolism (Figure 3c), but may be linked to broad cellular events such as abnormal protein synthesis and slow cell proliferation and growth.
To clarify whether our in vitro results can be applied clinically, PEPCK expression levels were determined in tissues from patients with rectal cancer who had undergone preoperative CRT. Because of the small amount of tissues available, the mitochondrial fraction could not be isolated from the tissues. However, lower expression levels of total PEPCK (cytosolic and mitochondrial isoforms) in patients’ tumors seemed to be correlated with poorer responses to CRT (Figure 5b). Predicting the tumor response to preoperative CRT can help to tailor treatment plans for locally advanced rectal cancer. PEPCK can be used as a candidate biomarker for predicting the rectal tumor response to CRT. Further studies using standardized measurements and validation will be needed to fully elucidate the potential clinical application of PEPCK as a predictor.
In conclusion, further studies are needed to elucidate the critical role of PEPCK in 5-FU–based CRT responses. Additionally, a larger sample size is required to confirm the use of PEPCK as a prognostic factor for CRC. However, the present results demonstrate that the slow energy metabolic process caused by downregulation of PEPCK may lead to different responses of CRC cells to 5-FU, radiation, or a combination of the two treatments.
This study was supported by a grant (HI12C0050) from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea; the Ewha Global Top 5 Grant 2011 of Ewha Womans University; and the Bio-Signal Analysis Technology Innovation Program (NRF-2008-2005479) of the Ministry of Education, Science and Technology, Korea.
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