Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells
© Cho et al.; licensee BioMed Central Ltd. 2014
Received: 4 April 2014
Accepted: 25 July 2014
Published: 30 July 2014
The essential oil of Pinus koraiensis (EOPK) is biologically active compound obtained from the leaves of P. koraiensis. The goal of this study was to investigate the anti-cancer mechanism of EOPK in HCT116 colorectal cancer cells.
HCT116 cell proliferation was assessed by conducting crystal violet and BrdU assays. To assess the effects of EOPK on cell migration, we performed a wound-healing assay. Further, the contribution of PAK1 to EOPK-induced AKT and extracellular signal-regulated kinase (ERK) suppression was assessed by siRNA-mediated PAK1 knockdown. Changes to the expression and phosphorylation of PAK1 and its effectors were determined by western blotting, and changes to the actin cytoskeleton were determined by performing an immunofluorescence assay.
EOPK significantly decreased HCT116 cell proliferation and migration, and induced G1 arrest without affecting normal cells. Additionally, EOPK suppressed the expression of PAK1, and decreased ERK and AKT phosphorylation in HCT116 cells. Finally, EOPK suppressed β-catenin, cyclin D1, and CDK4/6 expression.
Our studies indicate that EOPK significantly reduced proliferation and migration of colorectal cancer cells. Furthermore, EOPK suppressed PAK1 expression in a dose-dependent manner, and this suppression of PAK1 led to inhibition of ERK, AKT, and β-catenin activities. Our findings suggest that EOPK exerts its anticancer activity via the inhibition of PAK1 expression, suggesting it may be a potent chemotherapeutic agent for colorectal cancer.
KeywordsEOPK PAK1 Colorectal cancer Proliferation Migration
Colorectal cancer is one of the most common forms of cancer in the Western world . The pathogenesis of colorectal cancer involves several processes, including enhanced cell survival, differentiation, and proliferation . Importantly, K-Ras and B-Raf mutations have been identified in 40% and 10% of colorectal cancers, respectively [3, 4]. Mutations in K-Ras and B-Raf activate the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways. In colorectal cancer, overexpression of PI3K/AKT and MAPK/ERK promotes cell survival, proliferation, and migration [5, 6].
The p21 activated kinase (PAK) family of proteins has been identified as a novel target for cancer therapies . PAKs are downstream effectors of the Rho family GTPases, which regulate cell motility and survival. There are six isoforms of PAK that are classified into two families, Group I (PAK1-3) and Group II (PAK4-6). Group I PAKs differ from Group II PAKs in both structure and mechanisms of activation . PAK1 is an important effector of Rac and Cdc42 that regulates cell transformation and tumor proliferation . Activated PAK1 enhances cell survival and migration via the AKT pathway, and stimulates transformation through the Ras/Raf/ERK/MAPK pathway. PAK1 knockdown was shown to inhibit the activation of β-catenin in colorectal cancer, thereby inhibiting Wnt/β-catenin signaling and proliferation . In addition, PAK1 overexpression occurs in a variety of human cancers, including breast, ovarian, and colorectal cancer. Thus, PAK1 may play an important role in cell growth, adhesion, migration, and survival in colorectal cancer, through the activation of AKT, ERK and β-catenin [11–14].
Pinus koraiensis, generally called the Korean nut pine, is an evergreen tree found in Korea, China, Japan, and eastern Russia. Unlike the common Pinaceae plants, P. koraiensis shows leaves (needles) in fascicles (bundles) of five. Essential oil derived from P. koraiensis (EOPK) contains a number of components, including D-limonene, β-pinene, 4-carene, camphene, β-phellandrene, and fencyl . Much is known about the role of EOPK in obesity. For example, our group previously reported that EOPK has an >anti-hyperlipidemic effect through the up-regulation of the low-density lipoprotein receptor and the inhibition of acyl-coenzyme A . Further, a recent report on the effects of EOPK indicated that the oil has anti-obesity and hypolipidemic activity in vitro and in vivo, using 3 T3-L1 cells and high fat diet-fed rats . Additional studies have shown that EOPK is a potential anti-diabetic agents and that the nut of P. koraiensis has antioxidant activity in vivo[17, 18]. Others studies have demonstrated that the Korean pine nut is an appetite suppressant, and reduced weight gain in mice fed a high fat diet [19, 20]. Nevertheless, it remains unclear whether EOPK may have an anti-cancer effect. In the present study, we examined the antitumor mechanism of EOPK in vitro in HCT116 colorectal cancer cells.
Preparation of essential oil from P. koraiensis leaves
Leaves were harvested from a Korean pine tree that was more than 30 years old at the end of October in Gangwondo Honchengun where is famous for producing excellent Korean pine nuts in Korea. The leaves were purchased as a dried condition from agricultural corporation, Beaksongyounglim (Ganwondo, Korea) and identified by the department of Oriental medicine Biotechnology at the Kyung Hee University. A voucher specimen (no.KMH-0081025) was maintained at the herbarium of the department of plant physiology at the Kyung Hee University. EOPK was prepared using a hydrodistillation method . Dried and pulverized P. koraiensis leaves were immersed in distilled water and steam distilled using an apparatus with a condenser (Hanil Labtech, Seoul, Korea) for 3 to 4 h at 90°C. The volatile compounds were contained in the water-soluble fraction, and were allowed to settle for 20 min. The essential oil layer was separated and purified by microfiltration.
Colon26L5, a murine colorectal cancer cell line; NIH-3 T3, a fibroblast cell line; HCT116, a human colorectal cancer cell line; and HCT15, HT29, and SW620, three human colorectal adenocarcinoma cell lines, were purchased from American Type Culture Collection (ATCC) (Rockville, MD), and maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 μM l-glutamine, and penicillin/streptomycin (WelGene, Deagu, South Korea) in a humidified atmosphere of 5% CO2 at 37°C.
Cytotoxicity of EOPK was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, St Louis, MO) assay. The cell were seeded at density of 2 × 104 cells per well in a 96 well plate, cultured for 24 h, and then treated with various concentrations of EOPK (0, 25, 50, 100 μg/ml). After 24 h incubation, Per 50 μl of MTT solution (1 mg/ml) was add to each well and incubated for 2 h at 37°C in dark. The viable cell number was correlated with the production of formazan, which was dissolved with dimethyl sulfoxide (DMSO) and optical density (O.D.) was measured by microplate reader (Molecular Devices Co., Sunnyvale, CA) at 570 nm. Cell viability was calculated by the following equation. Cell viability(%) = [O.D.(EOPK)-O.D.(blank)]/[O.D(control)-O.D.(blank)] × 100.
Western blot analysis
Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors cocktail). Protein samples were quantified using the Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA), separated by electrophoresis on an 8 to 10% SDS-PAGE gel, and transferred onto a Hybond ECL transfer membrane (Amersham Pharmacia, Piscataway, NJ). The membranes were blocked in 3% nonfat skim milk and probed with primary antibodies for PAK1 (Abcam, Cambridge, UK), PI3K (Millipore, Billerica, MA, USA), phospho-ERK, ERK, β-catenin (Cell Signaling, Beverly, MA), phospho-AKT, AKT, PTEN (Santa Cruz Biotechnologies, Santa Cruz, CA), or β-actin (Sigma Aldrich Co., St. Louis, MO). Membranes were exposed to horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit secondary antibodies. Protein expression was examined by using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ).
The PAK1 small interfering RNA (siRNA) I and II were purchased from Cell Signaling. A control siRNA were purchased from Santa Cruz Biotechnology. To transfect the siRNA, HCT116 cells were plated at a density of 1 × 105 cells per well in a six-well plate. Cells were transfected using 100 nM of PAK1 siRNA with siRNA transfection reagent for 48 h. After treatment, cells were stimulated for Western blot or immunofluorescence assay.
Wound healing assay
The ability of cells to migrate was assayed by wound healing assay. The HCT116 cells (1 × 106 cells/ml) were seeded in a 6-well plate and incubated at 37°C. When confluent, the cells were scratched with a 200-μL pipette tip, followed by washing with PBS. The cells were then treated with EOPK in complete medium for 24 h. After incubation, the cells were fixed and stained with Diff-Quick. Randomly chosen fields were photographed under a fluorescence microscope (AXIO observer A1, ZEISS, Germany). The number of cells that migrated into the scratched area was calculated.
Cell growth assay
The cell growth assay was performed to measure the anti-proliferative effect of EOPK. HCT116 cells (1 × 105 cells/ml) were seeded in a 6-well plate and incubated at 37°C for 24 h, followed by treatment with various concentrations of EOPK (0, 25, 50, 100 μg/ml). Cells were incubated for 5 d, with daily addition of fresh media and EOPK. To assess proliferation, a crystal violet assay was performed. The medium was removed carefully by mild suction, and 2 ml of 1% glutaraldehyde solution (JUNSEl, Tokyo, Japan) in PBS was added to each well for 15 min at 37°C. After washing with PBS, 2 ml of 0.05% crystal violet (Sigma Aldrich, St Louis, MO) was added for 30 min. Following incubation, the crystal violet solution was removed, and the cells were washed gently with DI water. The plates were then dried room temperature overnight. The following day, a 70% ethanol solution was added (2 ml/well) to each well of the 6-well plate. To release the crystal violet, the plates were placed on a rotary shaker for 2 h at room temperature. The optical density (OD) was measured using a microplate reader (Molecular Devices Co., Sunnyvale, CA) at 570 nm, with a reference filter at 405 nm.
Cell proliferation in HCT116 cells following EOPK treatment was evaluated using a cell proliferation ELISA kit (Roche, Swiss) according to the manufacturer’s instruction. Briefly, after a 24- or 48-h treatment with EOPK, the cells were treated with bromodeoxyuridine (BrdU, 10 μL/well), and incubated for 4 h at 37°C. Following incubation, the BrdU solution was removed, and 200 μl of FixDenat was added to each well. After incubation for 30 min at room temperature, the FixDenat solution was removed and 100 μl of anti-BrdU-POD was added to each well. After washing with PBS three times, 100 μl of substrate solution was added to each well, and the optical density was measured at 450 nm using a microplate reader (Molecular Devices Co., Sunnyvale, CA, USA). All samples were prepared in triplicate, and the assay was repeated at least three times.
Cell cycle assay
HCT116 cells were treated with EOPK (100 μg/ml) for 24 h. The cells were washed and fixed in 70% ethanol overnight at -20°C. The following day, the cells were treated with RNase A (10 mg/ml) for 1 h at 37°C. The cells were stained by adding 1 ml of propidium iodide (PI) (50 μg/ml). After filtering with nylon mesh (40 μm), the DNA contents of stained cells were analyzed using Cellquest Software (BD Biosciences, San Jose, CA) with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Cells were plated onto poly-L-lysine coated coverslips and fixed with 4% (v/v) methanol-free formaldehyde solution (pH 7.4) at 4°C for 25 min. The cells were permeabilized with 0.2% Triton-X 100 in PBS for 5 min, and blocked in blocking buffer (5% bovine serum albumin, 0.5% Tween-20 in PBS) for 2 h. The cells were incubated with 200 μl of 100 nM rhodamine phalloidin (Cytoskeleton, USA) at room temperature in the dark for 30 min. The DNA was counterstained for 30 s with 300 μl of 100 nM DAPI in PBS. The slides were mounted with mounting medium (Vector Laboratories, Inc., Burlingame, CA) and visualized using the DV Elite™ Imaging System (Deltavision, Applied Precision, Inc., USA).
All data were shown as mean ± SE. Statistically significant differences were evaluated using Student’s t-test and a Tukey-Kramer multiple-comparison post-test.
PAK1 mediates the MEK/ERK, PI3K/AKT, and Wnt/β-catenin signaling pathways in colorectal cancer cells
PAK1 is a central node for various oncogenic signaling pathways, including the Ras/Raf/MAPK/ERK and the PI3K/AKT signaling pathways [8, 22, 23]. To assess the contribution of PAK1 to oncogenic signaling pathways in colon cancer cells, we knocked down PAK1 expression with siRNA. siRNA-mediated PAK1 knockdown attenuated both β-catenin expression and the phosphorylation of AKT and ERK (Figure 1B).
EOPK inhibits the expression of PAK1
To investigate the effect of EOPK on PAK1, the cells were treated with EOPK (100 μg/ml) and Western blotting was performed. EOPK significantly suppressed the protein expression of PAK1 in colon cancer cell lines, as shown in Figure 1C. Further, we assessed the cytotoxicity of EOPK in NIH-3 T3 cells as a normal cell control. EOPK had no significant cytotoxic effect in NIH-3 T3 cells (Figure 1D).
EOPK induces G1-arrest and inhibits cell proliferation and migration
EOPK attenuates β-catenin expression and AKT, and ERK phosphorylation via PAK1
PAK1 mediates EOPK-induced G1 arrest, suppression of cell proliferation and migration, and altered actin cytoskeletal arrangement
Our findings show that EOPK efficaciously inhibits the proliferation and migration of colon cancer cells in vitro. Fluorouracil (5-FU) is a standard therapeutic agent used in the treatment of colorectal cancer since the 1950s. However, not all of patients respond to 5-FU . Thus, recent work identifying the antitumor effects of natural products indicates they may be a promising alternative therapeutic for the treatment of colorectal cancer. Many extracts from vegetables, fruits, and plants, including epigallocatechin gallate (EGCG) and Morus alba leaf extract, can inhibit cell proliferation and survival, and induce apoptosis in colorectal cancer cell [29, 30]. Here, we show that EOPK significantly decreases proliferation and migration in colorectal cancer cells, without affecting normal cells. Recent reports have indicated that EOPK has anti-obesity and hypolipidemic effects [15, 16]. Moreover, essential oil from P. koraiensis seeds, leaves, nuts, and cones have been shown to have biological efficacy [17, 18, 20, 31, 32]. However, the effect of EOPK on tumor proliferation and survival has not been examined to date.
PAK1 was recently shown to be an effective therapeutic target for cancer treatment . Interestingly, we found that EOPK treatment significantly reduced PAK1 expression in several colon cancer cells. It is well known that PAK1 mediates cell proliferation, migration, invasion, and survival through the ERK and AKT signaling pathways . Thus, to confirm the involvement of PAK1 in EOPK-mediated inhibition of cell proliferation and migration, we analyzed the effects of EOPK on AKT and ERK phosphorylation in HCT116 cells. Knockdown of PAK1 with siRNA enhanced the inhibitory effect of EOPK on AKT and ERK phosphorylation in HCT116 cells. Further, PAK1 knockdown enhanced EOPK-mediated inhibition of PAK1 activity. Combined, these data suggest that EOPK suppresses cell growth and migration via the inhibition of PAK1, AKT, and ERK signaling in colon cancer cells. In addition, PAK1 is known to be involved in macrophage spreading and migration, and previous work indicates that PAK1 acts via ERK1/2 to regulate lamellipodia formation . We showed that EOPK significantly suppressed actin cytoskeletal rearrangement in HCT116 colorectal cancer cells. Furthermore, EOPK significantly decreased proliferation and migration, induced G1-arrest, and inhibited the expression of phospho-ERK, phospho-AKT, β-catenin, cyclin D1 and CDK4/6 in colorectal cancer cells.
In summary, our results demonstrate that EOPK reduces proliferation of HCT116 cells through G1 arrest, without affecting normal cells. EOPK inhibits cell migration and alters cytoskeletal structure, through the reduction of basal spread and cell elongation and increased cell rounding. EOPK-induced G1 arrest, inhibited cell proliferation and migration, and suppressed actin cytoskeletal rearrangement via the inhibition of PAK1. This inhibition of PAK1 resulted in decreases in ERK and AKT phosphorylation and β-catenin expression. Combined, our findings provide evidence that EOPK may be a novel therapeutic for the treatment of colorectal cancer. There is great potential for the development of novel chemotherapeutics from the leaves of P. koraiensis.
Essential oils of Pinus koraiensis leaves
p21-activated kinase 1.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2007–0054931).
- Sala-Vila A, Folkes J, Calder PC: The effect of three lipid emulsions differing in fatty acid composition on growth, apoptosis and cell cycle arrest in the HT-29 colorectal cancer cell line. Clin Nutr. 2010, 29 (4): 519-524. 10.1016/j.clnu.2009.11.004.View ArticlePubMedGoogle Scholar
- Calvert PM, Frucht H: The genetics of colorectal cancer. Ann Intern Med. 2002, 137 (7): 603-612.View ArticlePubMedGoogle Scholar
- Yuen ST, Davies H, Chan TL, Ho JW, Bignell GR, Cox C, Stephens P, Edkins S, Tsui WW, Chan AS, Futreal PA, Stratton MR, Wooster R, Leung SY: Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Res. 2002, 62 (22): 6451-6455.PubMedGoogle Scholar
- Baldus SE, Schaefer KL, Engers R, Hartleb D, Stoecklein NH, Gabbert HE: Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res. 2010, 16 (3): 790-799. 10.1158/1078-0432.CCR-09-2446.View ArticlePubMedGoogle Scholar
- Fransen K, Klintenas M, Osterstrom A, Dimberg J, Monstein HJ, Soderkvist P: Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas. Carcinogenesis. 2004, 25 (4): 527-533.View ArticlePubMedGoogle Scholar
- Huynh N, Liu KH, Baldwin GS, He H: P21-activated kinase 1 stimulates colon cancer cell growth and migration/invasion via ERK- and AKT-dependent pathways. Biochim Biophys Acta. 2010, 1803 (9): 1106-1113. 10.1016/j.bbamcr.2010.05.007.View ArticlePubMedGoogle Scholar
- Arias-Romero LE, Chernoff J: A tale of two Paks. Biol Cell. 2008, 100 (2): 97-108. 10.1042/BC20070109.View ArticlePubMedGoogle Scholar
- Bokoch GM: Biology of the p21-activated kinases. Annu Rev Biochem. 2003, 72: 743-781. 10.1146/annurev.biochem.72.121801.161742.View ArticlePubMedGoogle Scholar
- Eswaran J, Soundararajan M, Kumar R, Knapp S: UnPAKing the class differences among p21-activated kinases. Trends Biochem Sci. 2008, 33 (8): 394-403. 10.1016/j.tibs.2008.06.002.View ArticlePubMedGoogle Scholar
- Zhu G, Wang Y, Huang B, Liang J, Ding Y, Xu A, Wu W: A Rac1/PAK1 cascade controls beta-catenin activation in colon cancer cells. Oncogene. 2012, 31 (8): 1001-1012. 10.1038/onc.2011.294.View ArticlePubMedGoogle Scholar
- Carter JH, Douglass LE, Deddens JA, Colligan BM, Bhatt TR, Pemberton JO, Konicek S, Hom J, Marshall M, Graff JR: Pak-1 expression increases with progression of colorectal carcinomas to metastasis. Clin Cancer Res. 2004, 10 (10): 3448-3456. 10.1158/1078-0432.CCR-03-0210.View ArticlePubMedGoogle Scholar
- Coniglio SJ, Zavarella S, Symons MH: Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms. Mol Cell Biol. 2008, 28 (12): 4162-4172. 10.1128/MCB.01532-07.View ArticlePubMedPubMed CentralGoogle Scholar
- Schurmann A, Mooney AF, Sanders LC, Sells MA, Wang HG, Reed JC, Bokoch GM: p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol Cell Biol. 2000, 20 (2): 453-461. 10.1128/MCB.20.2.453-461.2000.View ArticlePubMedPubMed CentralGoogle Scholar
- Whale A, Hashim FN, Fram S, Jones GE, Wells CM: Signalling to cancer cell invasion through PAK family kinases. Front Biosci (Landmark Ed). 2011, 16: 849-864. 10.2741/3724.View ArticleGoogle Scholar
- Kim JH, Lee HJ, Jeong SJ, Lee MH, Kim SH: Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase. Phytother Res. 26 (9): 1314-1319.
- Ko HS, Lee HJ, Sohn EJ, Yun M, Lee MH, Kim SH: Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling. Evid Based Complement Alternat Med. 2013, 2013: 947037-PubMedPubMed CentralGoogle Scholar
- Joo HE, Lee HJ, Sohn EJ, Lee MH, Ko HS, Jeong SJ, Kim SH: Anti-Diabetic Potential of the Essential Oil of Pinus koraiensis Leaves toward Streptozotocin-Treated Mice and HIT-T15 Pancreatic beta Cells. Biosci Biotechnol Biochem. 2013, 77 (10): 1997-2001. 10.1271/bbb.130254.View ArticlePubMedGoogle Scholar
- Chen X, Zhang Y, Wang Z, Zu Y: In vivo antioxidant activity of Pinus koraiensis nut oil obtained by optimised supercritical carbon dioxide extraction. Nat Prod Res. 25 (19): 1807-1816.
- Park S, Lim Y, Shin S, Han SN: Impact of Korean pine nut oil on weight gain and immune responses in high-fat diet-induced obese mice. Nutr Res Pract. 2013, 7 (5): 352-358. 10.4162/nrp.2013.7.5.352.View ArticlePubMedPubMed CentralGoogle Scholar
- Pasman WJ, Heimerikx J, Rubingh CM, van den Berg R, O'Shea M, Gambelli L, Hendriks HF, Einerhand AW, Scott C, Keizer HG, Mennen LI: The effect of Korean pine nut oil on in vitro CCK release, on appetite sensations and on gut hormones in post-menopausal overweight women. Lipids Health Dis. 2008, 7: 10-10.1186/1476-511X-7-10.View ArticlePubMedPubMed CentralGoogle Scholar
- Hong EJ, Na KJ, Choi IG, Choi KC, Jeung EB: Antibacterial and antifungal effects of essential oils from coniferous trees. Biol Pharm Bull. 2004, 27 (6): 863-866.View ArticlePubMedGoogle Scholar
- Higuchi M, Onishi K, Kikuchi C, Gotoh Y: Scaffolding function of PAK in the PDK1-Akt pathway. Nat Cell Biol. 2008, 10 (11): 1356-1364.View ArticlePubMedGoogle Scholar
- Mao K, Kobayashi S, Jaffer ZM, Huang Y, Volden P, Chernoff J, Liang Q: Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes. J Mol Cell Cardiol. 2008, 44 (2): 429-434. 10.1016/j.yjmcc.2007.10.016.View ArticlePubMedGoogle Scholar
- Kichina JV, Goc A, Al-Husein B, Somanath PR, Kandel ES: PAK1 as a therapeutic target. Expert Opin Ther Targets. 2010, 14 (7): 703-725. 10.1517/14728222.2010.492779.View ArticlePubMedPubMed CentralGoogle Scholar
- Ong CC, Jubb AM, Zhou W, Haverty PM, Harris AL, Belvin M, Friedman LS, Koeppen H, Hoeflich KP: p21-activated kinase 1: PAK'ed with potential. Oncotarget. 2011, 2 (6): 491-496.View ArticlePubMedPubMed CentralGoogle Scholar
- Edwards DC, Sanders LC, Bokoch GM, Gill GN: Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol. 1999, 1 (5): 253-259.View ArticlePubMedGoogle Scholar
- Suzuki-Inoue K, Yatomi Y, Asazuma N, Kainoh M, Tanaka T, Satoh K, Ozaki Y: Rac, a small guanosine triphosphate-binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin alpha(2)beta(1). Blood. 2001, 98 (13): 3708-3716. 10.1182/blood.V98.13.3708.View ArticlePubMedGoogle Scholar
- Royce ME, Medgyesy D, Zukowski TH, Dwivedy S, Hoff PM, Pazdur R: Colorectal cancer: chemotherapy treatment overview. Oncology (Williston Park). 2000, 14 (12 Suppl 14): 40-46.Google Scholar
- Chen XQ, Wu JB, Yang XQ, Song YG, Li MS: Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression. Nan Fang Yi Ke Da Xue Xue Bao. 2009, 29 (8): 1568-1570.PubMedGoogle Scholar
- Deepa M, Sureshkumar T, Satheeshkumar PK, Priya S: Antioxidant rich Morus alba leaf extract induces apoptosis in human colon and breast cancer cells by the downregulation of nitric oxide produced by inducible nitric oxide synthase. Nutr Cancer. 2013, 65 (2): 305-310. 10.1080/01635581.2013.748924.View ArticlePubMedGoogle Scholar
- Sugano M, Ikeda I, Wakamatsu K, Oka T: Influence of Korean pine (Pinus koraiensis)-seed oil containing cis-5, cis-9, cis-12-octadecatrienoic acid on polyunsaturated fatty acid metabolism, eicosanoid production and blood pressure of rats. Br J Nutr. 1994, 72 (5): 775-783. 10.1079/BJN19940079.View ArticlePubMedGoogle Scholar
- Speijers GJ, Dederen LH, Keizer H: A sub-chronic (13 weeks) oral toxicity study in rats and an in vitro genotoxicity study with Korean pine nut oil (PinnoThin TG). Regul Toxicol Pharmacol. 2009, 55 (2): 158-165. 10.1016/j.yrtph.2009.06.010.View ArticlePubMedGoogle Scholar
- Abdel-Magid AF: PAK1: A Therapeutic Target for Cancer Treatment. ACS Med Chem Lett. 2013, 4 (5): 431-432. 10.1021/ml400101g.View ArticlePubMedPubMed CentralGoogle Scholar
- Smith SD, Jaffer ZM, Chernoff J, Ridley AJ: PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics. J Cell Sci. 2008, 121 (Pt 22): 3729-3736.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/275/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.