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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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Quantification of PEPCK expression levels in 11 human colon cancer lines with different responses to 5-FU. (a) The expression levels of PEPCK in whole homogenates and mitochondrial fractions of different cell lines were normalized with actin and VDAC, respectively. (b) Differential responses of cell lines to 5-FU. The 5-FU IC50 was determined in 11 colon cancer cell lines 96 h after 5-FU treatment by MTT assay. (c) No correlation was observed between mPEPCK expression and the responses of cell lines to 5-FU. The 5-FU IC50 values of the various cell lines were plotted against their PEPCK expression levels in whole homogenates (upper panel) and mitochondrial fractions (lower panel) normalized to actin and VDAC, respectively.
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Figure 2: Quantification of PEPCK expression levels in 11 human colon cancer lines with different responses to 5-FU. (a) The expression levels of PEPCK in whole homogenates and mitochondrial fractions of different cell lines were normalized with actin and VDAC, respectively. (b) Differential responses of cell lines to 5-FU. The 5-FU IC50 was determined in 11 colon cancer cell lines 96 h after 5-FU treatment by MTT assay. (c) No correlation was observed between mPEPCK expression and the responses of cell lines to 5-FU. The 5-FU IC50 values of the various cell lines were plotted against their PEPCK expression levels in whole homogenates (upper panel) and mitochondrial fractions (lower panel) normalized to actin and VDAC, respectively.

Mentions: Differential PEPCK expression levels in the whole homogenates and mitochondrial fractions from 11 human colon cancer cell lines were detected by Western Blot and normalized to actin and VDAC, respectively (Figure 2a). As expected, the 5-FU IC50 (the concentration of 5-FU that results in a 50% decrease in cell survival compared with control) varied among CRC cell lines (Figure 2b). However, the PEPCK expression levels in neither the mitochondrial fractions nor the whole homogenates showed a correlation with 5-FU IC50 (Figure 2c).


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)

Quantification of PEPCK expression levels in 11 human colon cancer lines with different responses to 5-FU. (a) The expression levels of PEPCK in whole homogenates and mitochondrial fractions of different cell lines were normalized with actin and VDAC, respectively. (b) Differential responses of cell lines to 5-FU. The 5-FU IC50 was determined in 11 colon cancer cell lines 96 h after 5-FU treatment by MTT assay. (c) No correlation was observed between mPEPCK expression and the responses of cell lines to 5-FU. The 5-FU IC50 values of the various cell lines were plotted against their PEPCK expression levels in whole homogenates (upper panel) and mitochondrial fractions (lower panel) normalized to actin and VDAC, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016284&req=5

Figure 2: Quantification of PEPCK expression levels in 11 human colon cancer lines with different responses to 5-FU. (a) The expression levels of PEPCK in whole homogenates and mitochondrial fractions of different cell lines were normalized with actin and VDAC, respectively. (b) Differential responses of cell lines to 5-FU. The 5-FU IC50 was determined in 11 colon cancer cell lines 96 h after 5-FU treatment by MTT assay. (c) No correlation was observed between mPEPCK expression and the responses of cell lines to 5-FU. The 5-FU IC50 values of the various cell lines were plotted against their PEPCK expression levels in whole homogenates (upper panel) and mitochondrial fractions (lower panel) normalized to actin and VDAC, respectively.
Mentions: Differential PEPCK expression levels in the whole homogenates and mitochondrial fractions from 11 human colon cancer cell lines were detected by Western Blot and normalized to actin and VDAC, respectively (Figure 2a). As expected, the 5-FU IC50 (the concentration of 5-FU that results in a 50% decrease in cell survival compared with control) varied among CRC cell lines (Figure 2b). However, the PEPCK expression levels in neither the mitochondrial fractions nor the whole homogenates showed a correlation with 5-FU IC50 (Figure 2c).

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