Cancer Res Treat.  2015 Jul;47(3):501-508. 10.4143/crt.2014.054.

p21-Activated Kinase 4 (PAK4) as a Predictive Marker of Gemcitabine Sensitivity in Pancreatic Cancer Cell Lines

• Moon SU
• Kim JW
• Sung JH
• Kang MH
• Kim SH
• Chang H
• Lee JO
• Kim YJ
• Lee KW
• Kim JH
• Bang SM
• Lee JS

• KMID: 2148501
• DOI: http://doi.org/10.4143/crt.2014.054

Abstract

PURPOSE
p21-activated kinases (PAKs) are involved in cytoskeletal reorganization, gene transcription, cell proliferation and survival, and oncogenic transformation. Therefore, we hypothesized that PAK expression levels could predict the sensitivity of pancreatic cancer cells to gemcitabine treatment, and PAKs could be therapeutic targets.
MATERIALS AND METHODS
Cell viability inhibition by gemcitabine was evaluated in human pancreatic cancer cell lines (Capan-1, Capan-2, MIA PaCa-2, PANC-1, Aspc-1, SNU-213, and SNU-410). Protein expression and mRNA of molecules was detected by immunoblot analysis and reverse transcription polymerase chain reaction. To define the function of PAK4, PAK4 was controlled using PAK4 siRNA.
RESULTS
Capan-2, PANC-1, and SNU-410 cells were resistant to gemcitabine treatment. Immunoblot analysis of signaling molecules reported to indicate gemcitabine sensitivity showed higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1), a well-known predictive marker for gemcitabine activity, in the resistant cell lines. Knockdown of PAK4 using siRNA induced the upregulation of hENT1. In resistant cell lines (Capan-2, PANC-1, and SNU-410), knockdown of PAK4 by siRNA resulted in restoration of sensitivity to gemcitabine.
CONCLUSION
PAK4 could be a predictive marker of gemcitabine sensitivity and a potential therapeutic target to increase gemcitabine sensitivity in pancreatic cancer.

Keyword

PAK4; hENT1; Gemcitabine; Pancreatic neoplasms

MeSH Terms

Cell Line*
Cell Proliferation
Cell Survival
Equilibrative Nucleoside Transporter 1
Humans
p21-Activated Kinases
Pancreatic Neoplasms*
Phosphotransferases*
Polymerase Chain Reaction
Reverse Transcription
RNA, Messenger
RNA, Small Interfering
Up-Regulation
Equilibrative Nucleoside Transporter 1
Phosphotransferases
RNA, Messenger
RNA, Small Interfering
p21-Activated Kinases

Figure

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  Fig. 1. The cytotoxic effect of gemcitabine on human pancreatic cancer cell lines. The cell viability of three pancreatic cancer cell lines (Capan-2, PANC-1, and SNU-410) reflected increased resistance to gemcitabine, whereas the other cancer cell lines (Capan-1, MIA PaCa-2, Aspc-1, and SNU-213) showed relatively good sensitivity to gemcitabine.

• 

  Fig. 2. Protein expression of p21-activated kinases (PAKs) and signaling molecules related to gemcitabine sensitivity. An inverse relationship was observed between PAK4 and human equilibrative nucleoside transporter 1 (hENT1) protein expression. (A) In gemcitabine-resistant cell lines (Capan-2, PANC-1, and SNU-410), higher PAK4 and lower hENT1 protein expression are shown. (B) There is no correlation between molecules of the epidermal growth factor receptor (EGFR) pathway (EGFR, p-EGFR, AKT, p-AKT, ERK, and p-ERK) and sensitivity to gemcitabine. The quantitative graph of densitometry (C) shows a significant correlation between PAK4 and hENT1 expression in pancreatic cancer cell lines.

• 

  Fig. 3. The effect of p21-activated kinase 4 (PAK4) knockdown on human equilibrative nucleoside transporter 1 (hENT1) expression in pancreatic cancer cell lines with higher PAK4 expression. PAK4 knockdown induced higher hENT1 protein expression in pancreatic cancer cell lines (Capan-2, PANC-1, and SNU-410). mRNA levels of hENT1 were also upregulated when PAK4 was knocked down in the pancreatic cancer cell lines.

• 

  Fig. 4. The effect of p21-activated kinase 4 (PAK4) expression on sensitivity to gemcitabine. Treatment with siRNA reduced PAK4 expression in Capan-2 (A), PANC-1 (B), SNU-410 (C), and MIA PaCa-2 cell lines (D). Pancreatic cell lines with PAK4 knockdown had decreased cell viability when compared to those with higher PAK4 expression (Capan-2, PANC-1, and SNU-410). When gemcitabine was added in combination with PAK4 knockdown, cell viability was significantly reduced compared to that of cell lines with high-level PAK4 expression treated with gemcitabine alone. MIA PaCa-2 cells, which have lower PAK4 expression, were used as a negative control. Although PAK4 siRNA alone did not induce a significant reduction in cell viability, the combination of PAK4 knockdown with gemcitabine showed a modest effect. Results are expressed as mean±SE (*p < 0.001). GEM, gemcitabine.

Reference

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