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

Park JW, Kim SC, Kim WK, Hong JP, Kim KH, Yeo HY, Lee JY, Kim MS, Kim JH, Yang SY, Kim DY, Oh JH, Cho JY, Yoo BC - BMC Cancer (2014)

Bottom Line: PEPCK was downregulated in SNU-C4R compared with its parent cell line SNU-C4.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Colorectal Cancer Branch, Research Institute, National Cancer Center, Goyang, Gyeonggi, Republic of Korea. yoo_akh@ncc.re.kr.

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.

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Effect of mPEPCK downregulation on 5-FU response in SNU-C4 cells. (a) Suppressed mPEPCK expression in SNU-C4 cells transfected with mPEPCK siRNA. The PEPCK expression levels decreased over time in the mPEPCK siRNA-transfected cells, while the nonsilenced (NS) control showed no apparent changes in the PEPCK expression levels. (b) Decreased cell proliferation rate after mPEPCK suppression. With no 5-FU or radiation treatment, artificial suppression of mPEPCK decreased the rate of cell proliferation. (c) Reduced cellular PEP level after mPEPCK suppression. (d) Increased 5-FU and radiation resistance was observed after mPEPCK suppression in SNU-C4 cells. MTT assay showed that the mPEPCK siRNA-transfected SNU-C4 cells exhibited higher survival rates than the control after combination treatment comprising 5-FU and radiation.
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Figure 3: Effect of mPEPCK downregulation on 5-FU response in SNU-C4 cells. (a) Suppressed mPEPCK expression in SNU-C4 cells transfected with mPEPCK siRNA. The PEPCK expression levels decreased over time in the mPEPCK siRNA-transfected cells, while the nonsilenced (NS) control showed no apparent changes in the PEPCK expression levels. (b) Decreased cell proliferation rate after mPEPCK suppression. With no 5-FU or radiation treatment, artificial suppression of mPEPCK decreased the rate of cell proliferation. (c) Reduced cellular PEP level after mPEPCK suppression. (d) Increased 5-FU and radiation resistance was observed after mPEPCK suppression in SNU-C4 cells. MTT assay showed that the mPEPCK siRNA-transfected SNU-C4 cells exhibited higher survival rates than the control after combination treatment comprising 5-FU and radiation.

Mentions: mPEPCK siRNA transfection was performed using SNU-C4 to investigate the role of mPEPCK in 5-FU resistance. At 96 h after siRNA transfection, significantly less PEPCK was detected in the mPEPCK-suppressed cells than in the nonsilenced (NS) control (Figure 3a). mPEPCK suppression itself reduced the proliferation rate of SNU-C4 without any 5-FU or radiation treatment (Figure 3b). Furthermore, the cellular level of PEP (an end product of PEPCK and substrate of pyruvate kinase) was also significantly decreased by the mPEPCK suppression in SNU-C4 (Figure 3c). The survival rates of the mPEPCK-suppressed SNU-4 cells after 1 μg/ml 5-FU treatment or 8-Gy radiation were higher than those of the control, but the difference did not reach statistical significance (P = 0.082 and 0.069, respectively) (Figure 3d). Suppression of mPEPCK significantly increased survival of SNU-4 cells after combined treatment comprising 5-FU and radiation (P = 0.0005) (Figure 3d).


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

Park JW, Kim SC, Kim WK, Hong JP, Kim KH, Yeo HY, Lee JY, Kim MS, Kim JH, Yang SY, Kim DY, Oh JH, Cho JY, Yoo BC - BMC Cancer (2014)

Effect of mPEPCK downregulation on 5-FU response in SNU-C4 cells. (a) Suppressed mPEPCK expression in SNU-C4 cells transfected with mPEPCK siRNA. The PEPCK expression levels decreased over time in the mPEPCK siRNA-transfected cells, while the nonsilenced (NS) control showed no apparent changes in the PEPCK expression levels. (b) Decreased cell proliferation rate after mPEPCK suppression. With no 5-FU or radiation treatment, artificial suppression of mPEPCK decreased the rate of cell proliferation. (c) Reduced cellular PEP level after mPEPCK suppression. (d) Increased 5-FU and radiation resistance was observed after mPEPCK suppression in SNU-C4 cells. MTT assay showed that the mPEPCK siRNA-transfected SNU-C4 cells exhibited higher survival rates than the control after combination treatment comprising 5-FU and radiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Effect of mPEPCK downregulation on 5-FU response in SNU-C4 cells. (a) Suppressed mPEPCK expression in SNU-C4 cells transfected with mPEPCK siRNA. The PEPCK expression levels decreased over time in the mPEPCK siRNA-transfected cells, while the nonsilenced (NS) control showed no apparent changes in the PEPCK expression levels. (b) Decreased cell proliferation rate after mPEPCK suppression. With no 5-FU or radiation treatment, artificial suppression of mPEPCK decreased the rate of cell proliferation. (c) Reduced cellular PEP level after mPEPCK suppression. (d) Increased 5-FU and radiation resistance was observed after mPEPCK suppression in SNU-C4 cells. MTT assay showed that the mPEPCK siRNA-transfected SNU-C4 cells exhibited higher survival rates than the control after combination treatment comprising 5-FU and radiation.
Mentions: mPEPCK siRNA transfection was performed using SNU-C4 to investigate the role of mPEPCK in 5-FU resistance. At 96 h after siRNA transfection, significantly less PEPCK was detected in the mPEPCK-suppressed cells than in the nonsilenced (NS) control (Figure 3a). mPEPCK suppression itself reduced the proliferation rate of SNU-C4 without any 5-FU or radiation treatment (Figure 3b). Furthermore, the cellular level of PEP (an end product of PEPCK and substrate of pyruvate kinase) was also significantly decreased by the mPEPCK suppression in SNU-C4 (Figure 3c). The survival rates of the mPEPCK-suppressed SNU-4 cells after 1 μg/ml 5-FU treatment or 8-Gy radiation were higher than those of the control, but the difference did not reach statistical significance (P = 0.082 and 0.069, respectively) (Figure 3d). Suppression of mPEPCK significantly increased survival of SNU-4 cells after combined treatment comprising 5-FU and radiation (P = 0.0005) (Figure 3d).

Bottom Line: PEPCK was downregulated in SNU-C4R compared with its parent cell line SNU-C4.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Colorectal Cancer Branch, Research Institute, National Cancer Center, Goyang, Gyeonggi, Republic of Korea. yoo_akh@ncc.re.kr.

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.

Show MeSH
Related in: MedlinePlus