Open Access Research article

Expression of phosphoenolpyruvate carboxykinase linked to chemoradiation susceptibility of human colon cancer cells

Ji-Won Park12, Seung Cheol Kim3, Won Ki Kim1, Jun Pyu Hong1, Kyung-Hee Kim1, Hyun Yang Yeo1, Jae Yong Lee1, M Sun Kim4, Jong Heon Kim4, Se Young Yang1, Dae Yong Kim12, Jae Hwan Oh12, Jae Youl Cho5 and Byong Chul Yoo1*

Author Affiliations

1 Colorectal Cancer Branch, Research Institute, National Cancer Center, Goyang, Gyeonggi, Republic of Korea

2 Center for Colorectal Cancer, Hospital, National Cancer Center, Goyang, Gyeonggi, Republic of Korea

3 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, College of Medicine, Ewha Womans University, Seoul, Republic of Korea

4 Cancer Cell and Molecular Biology Branch, Research Institute, National Cancer Center, Goyang, Gyeonggi, Republic of Korea

5 Department of Genetic Engineering, Sungkyunkwan University, Suwon, Gyeonggi, Republic of Korea

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BMC Cancer 2014, 14:160  doi:10.1186/1471-2407-14-160

Published: 6 March 2014

Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

Our overall results demonstrate that mPEPCK is a useful predictor of a response to chemoradiotherapy in patients with rectal cancer.

Keywords:
mPEPCK; 5-FU resistance; Colon cancer; Chemoradiotherapy; Prediction