OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2015.3619 OL-0-0-3619 Articles Afzelin exhibits anti-cancer activity against androgen-sensitive LNCaP and androgen-independent PC-3 prostate cancer cells through the inhibition of LIM domain kinase 1 ZHUKAI-CHANG 1 2 * SUNJIAN-MEI 3 * SHENJIAN-GUO 2 JINJI-ZHONG 2 LIUFENG 2 XUXIAO-LIN 2 CHENLIN 2 LIULIN-TAO 2 LVJIA-JU 1 Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, P.R. China Department of Urology, Shanghai Jiaotong University Affiliated Sixth People's Hospital South Campus, Shanghai 201499, P.R. China Department of Neonatology, Tongji Hospital, Tongji University, Shanghai 200065, P.R. China Correspondence to: Mr. Jia-Ju Lv, Department of Urology, Shandong Provincial Hospital, Shandong University, 389 Jingwu Road, Jinan, Shandong 250021, P.R. China, E-mail: lvjiaju@hotmail.com

Contributed equally

 10 2015 18 08 2015 10 4 2359 2365 12052014 02022015 Copyright © 2015, Spandidos Publications 2015

Prostate cancer presents high occurrence worldwide. Medicinal plants are a major source of novel and potentially therapeutic molecules; therefore, the aim of the present study was to investigate the possible anti-prostate cancer activity of afzelin, a flavonol glycoside that was previously isolated from Nymphaea odorata. The effect of afzelin on the proliferation of androgen-sensitive LNCaP and androgen-independent PC-3 cells was evaluated by performing a water soluble tetrazolium salt-1 assay. In addition, the effect of afzelin on the cell cycle of the LNCaP and PC-3 prostate cancer cell lines was evaluated. Western blot analysis was performed to evaluate the effect of afzelin on the kinases responsible for the regulation of actin organization. Afzelin was identified to inhibit the proliferation of LNCaP and PC3 cells, and block the cell cycle in the G0 phase. The anticancer activity of afzelin in these cells was determined to be due to inhibition of LIM domain kinase 1 expression. Thus, the in vitro efficacy of afzelin against prostate cancer is promising; however, additional studies on different animal models are required to substantiate its anticancer potential.

 prostate cancer afzelin LNCaP cells PC-3 cells G0 phase LIM domain kinase 1 Introduction

Prostate cancer is the second most frequently diagnosed type of cancer and the sixth most common cause of cancer-associated mortality in males, worldwide (1). The typical treatment strategies of chemotherapy and radiotherapy have not provided significant survival benefits for patients with advanced prostate cancer, and the majority of available strategies are only palliative (2). Therefore, there is requirement for the prompt identification of novel molecules to treat the increasing number of prostate cancer cases, particularly cases that are resistant to current chemotherapeutic agents (3,4).

Afzelin is a flavonol glycoside found in Nymphaea odorata, which has been identified to inhibit the growth of breast cancer cells by stimulating apoptosis (5). In addition, afzelin has been demonstrated to scavenge superoxide anion radicals in RAW264.7 cells (6). The structure of this compound is shown in Fig. 1.

In eukaryotic cells, the actin cytoskeleton is essential for mediating various biological functions, including providing the structural framework of the cell, and driving cellular motility and division. In particular, dynamic reorganization of the actomyosin cytoskeleton and remodelling of the extracellular matrix drive the multistep process of tumor cell metastasis. Tumor cells are able to cross tissue boundaries into the blood and lymphatic systems and migrate to distal regions of the body. Therefore, cancer therapy has recently focused on gaining a comprehensive understanding of the biological processes that regulate actin organization (7).

Rho GTPase family proteins are key regulators of the actin cytoskeleton and, with the aid of various target proteins, maintain the tight regulation of normal cell growth and differentiation (8–11). Eukaryotic cells exhibit a predisposition to rapid and uncontrollable growth following genomic alterations or carcinogenesis. For instance, increased expression levels of LIM domain kinase 1 (LIMK1) are observed in prostate cancer. The Rho GTPase myotonic dystrophy kinase-related Cdc42-binding kinase α (MRCKα), Rho-associated coiled-coil containing protein kinase (ROCK)1 and ROCK2 are responsible for the activation of LIMK1 (9,12). Previous studies have demonstrated the ability of ROCK inhibitors to reduce the invasive ability of tumor cells in vitro and prevent the spread of tumor cells in vivo, including melanoma and fibrosarcoma cells, as well as liver, breast, lung and prostate cancer tumor cells (13–17).

Thus, inhibitors of LIMK1, MRCKα and ROCK1/2 are considered to restore normal cell proliferation and provide a key strategy for cancer treatment (18). Therefore, the aim of the present study was to evaluate the in vitro anti-prostate cancer activity of afzelin and its effect on prostate cancer-associated kinases.

 Materials and methods <sec> <title>Cell culture

Androgen-sensitive LNCaP (lymph node carcinoma of the prostate) and androgen-independent PC-3 (prostate cancer-3) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The LNCaP cells were maintained in Eagle's minimal essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, glutamine, 2% penicillin-streptomycin and 0.2% gentamicin (all obtained from Sigma-Aldrich). According to the manufacturer's instructions, the PC-3 cells were grown in Ham's F12K medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate (90%) and 10% fetal bovine serum. The two cell lines were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.

 Cell proliferation assay

LNCaP and PC-3 cells were seeded in 96-well plates at a density of 5×103 cells/well to a final volume of 100 µl. At 24 h after seeding, the medium was removed and replaced with fresh medium, containing vehicle (dimethyl sulfoxide) or increasing concentrations of afzelin (0.1, 1.0 and 10.0 µg/ml), in a final volume of 100 µl. The cultures were prepared in quadruplicate for each afzelin concentration and time point, and maintained in a CO2 incubator for three days. After 24, 48 and 72 h, 10 µl water soluble tetrazolium salt (WST)-1 labeling solution (WST-1 cell proliferation assay kit; Roche Diagnostics, Indianapolis, IN, USA) was added to the cultures and the cells were returned to the CO2 incubator for 2 h (19). The specific type of formazan product formed was detected by measuring its absorbance in a 96-well spectrophotometric plate reader (DiaSorin, Stillwater, MN, USA) at a wavelength of 420 nm, as described by the manufacturer (20).

 Cell cycle analysis

Cell cycle analysis was performed as described previously (19). Briefly, cultured LNCaP and PC-3 prostate cancer cells were exposed to various concentrations of afzelin (0.1, 1.0 and 10.0 µg/ml) for 24 h. Adherent cells were trypsinized (Sigma-Aldrich), pooled with the cells in suspension and washed three times with ice-cold phosphate-buffered saline (PBS). To determine cell viability, a fraction of the cells were stained with trypan blue (Sigma-Aldrich) and counted. The cultures were adjusted to a concentration of 1×106 cells/ml and fixed in a 2:1 ratio (v/v) of chilled methanol overnight. The fixed cells were subsequently stained with propidium iodide (Sigma-Aldrich) in the presence of RNase (Sigma-Aldrich). A minimum of 1×104 cells from each experimental group were analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA) to determine the cell cycle distribution and CellQuest cell cycle analysis software (version 5.1; BD Biosciences) to perform data analysis.

 Western blot analysis

To determine the effects of afzelin on the protein expression levels of MRCKα, LIMK1 and ROCK1, LNCaP and PC-3 prostate cancer cells were treated with varying concentrations of afzelin (0.1, 1.0 and 10.0 µg/ml). The concentration of each protein was measured spectrophotometrically using a modified Lowry assay protocol (DC Protein assay; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Next, the lysates were electrophoresed through a 7.5–12.0% denaturing polyacrylamide gel. The resolved proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA) and incubated with blocking solution (PBS containing 0.05% Tween 20 and 5% skimmed milk) to block non-specific binding. Next, the membranes were incubated with monoclonal anti-rabbit MRCKα (1:1,000; cat. no. ab96659; Abcam, Cambridge, USA), LIMK1 (1:1,000; cat. no. 3842; Cell Signaling Technology, Inc., Beverly, MA, USA), p-LIMK1 (1:1,000; cat. no. 3841; Cell Signaling Technology, Inc.), ROCK1 (1:2,000; cat. no. ab45171; Abcam) and p-ROCK1 (1:1,000; cat. no. ab203273; Abcam) primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary rabbit antibodies (1:2,000; cat. no. G33-62G-1000; SignalChem, Richmond, BC, Canada) for 1 h at room temperature. The positive protein bands were visualized using enhanced chemiluminescence solution (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

 Statistical analysis

Data are expressed as the mean ± standard deviation. Results were analyzed by one way analysis of variance and all data analysis was performed using SPSS version 21.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Afzelin decreases the growth of prostate cancer cells in vitro

The present study analyzed the effect of afzelin on the growth of two prostate cancer cell lines, PC-3 and LNCaP, by exposing the cells to increasing concentrations of afzelin (0.1, 1.0 and 10.0 µg/ml) one day after seeding. As indicated in Fig. 2A and B, the viability of the PC-3 and LNCaP cells, respectively, was monitored for 72 h using a WST-1 cell proliferation assay. The dose response curves are presented as the percentage of cell growth in the control groups on the corresponding day, and each value represents the mean of four independent experiments. The results indicate that exposure to afzelin at concentrations of 1.0 and 10.0 µg/ml markedly inhibited the growth of PC-3 and LNCaP cells at 24, 48 and 72 h. Although afzelin was a potent inhibitor of cell growth in the LNCaP and PC-3 cell lines (P<0.01), no growth inhibition was apparent in the vehicle (0.1% DMSO)-treated control cells.

 Afzelin induces apoptosis in LNCaP and PC-3 cells

Flow cytometric analysis was used to determined the cell cycle distribution of the LNCaP and PC-3 cells in the absence and presence of afzelin for 24 h. As indicated in Fig. 3A, a broad peak of cells equivalent to 27.6% of the total cell population accumulated in the G0 population of the afzelin-treated (10.0 µg/ml) PC-3 cells, compared with only 12.7% of the control cells. In addition, 43.3% of the cells accumulated in the G1 phase of the cell cycle. Thus, an increased proportion of PC-3 cells localized in the G0 region following treatment with afzelin. Notably, similar results were obtained in the LNCaP cells, where 12.0% of the control cell population localized in the G0 phase compared with 19.4% of the afzelin-treated (10.0 µg/ml) LNCaP cells (Fig. 3B). Furthermore, 50.7% of cells accumulated in G1 phase. The elevated population of G0 cells observed subsequent to afzelin treatment indicates the occurrence of extensive apoptosis or an ongoing cytotoxic response. The results indicated that afzelin exhibits a selective effect on the two cell types.

 Afzelin decreases the expression of Rho GTPase family proteins

To investigate the molecular effects of afzelin on the LNCaP and PC-3 prostate cancer cell lines, an analysis was conducted to determine the effect of afzelin treatment on the expression levels of specific molecules that are critical in cell cycle progression and apoptosis. Western blot analysis was performed to determine the effect of increasing concentrations of afzelin on the expression levels of members of the Rho GTPase family proteins, including LIMK1, MRCKα and ROCK1. The results identified that phosphorylation of these kinases progressively diminished in the LNCaP and PC-3 cells as the dose of afzelin was increased (Fig. 4A). However, densitometry evaluation indicated that this suppression was more significant in the PC-3 cells (Fig. 4B).

 Discussion

Invasion and metastasis are critical events in cancer progression, which often lead to patient mortality. Metastatic cells undergo a sequence of changes that result in loss of contact inhibition and enhanced motility, which subsequently promotes cell migration from the primary tumor site, leading to invasion and angiogenesis (21). At present, the treatments available for patients with metastatic tumors are limited. Thus, it is critical to identify and develop compounds that target the molecular events involved in cell invasion and metastasis in cancer therapy.

The contractility of the actin-myosin complex is vital for cell motility and plays an important role in tumor cell invasion and metastasis. Myosin II, a critical component of the cytoskeletal contractile machinery, is regulated by the phosphorylation of myosin II light chain proteins (MLC) at Thr18 and Ser19 (22). Furthermore, the members of the Rho GTPase family are involved with the regulation of actin cytoskeleton organization and dynamics, mediating the development of focal adhesions and stress fibers (23–25).

Rho A and C proteins promote the actomyosin contractile force generation via ROCK. ROCK1 and 2 cause the phosphorylation of several downstream target proteins, including MLC, the myosin binding subunit of MLC phosphatase, LIMK1 and LIMK2 (12,26). These target proteins catalyze various processes involved in cancer progression, such as changes in structural arrangement, alterations in cellular polarity, proliferation, migration, invasion, transformation and cytokinesis (26,27).

The phosphorylation of the LIMKs is also mediated by MRCKα and p21-activated kinase, acting downstream of the Rho/Rac/Cdc42 signaling pathway (28–31). In addition, LIMK1 and 2 have been shown to exhibit a crucial role in cancer progression, angiogenesis and metastasis (32). Previous studies have identified increased LIMK1 expression in human breast cancer cell lines; increased expression of LMK1 increased tumor invasion, whereas the inhibition of LIMK1 expression, or blockade of LIMK1 activity reduced the aggressive behavior of human MDA-MB-231 and MDA-MB-435 breast cancer cell lines (33,34). Thus inhibitors of LIMK1, ROCK1 and MRCKα may inhibit tumor invasion and metastasis effectively.

In the present study it was demonstrated that afzelin, a flavonol glycoside found in Nymphaea odorata (35), exhibits significant anti-prostate cancer activities in androgen-sensitive LNCaP and androgen-independent PC-3 prostate cancer cell lines. Afzelin has been reported to exhibit antioxidant, DNA-protective, ultra-violet radiation-absorbing and anti-inflammatory properties (36). In addition, a recent study revealed that afzelin inhibits breast cancer cell proliferation by stimulating apoptosis (5). Notbaly, afzelin attenuated asthma in a murine model of asthma (37). In the present study, afzelin inhibited cell proliferation in the two prostate cancer cell lines and blocked the cell cycle in the G0 phase. Furthermore, afzelin attenuated the expression of a number of kinases involved in the maintenance of the actin cytoskeleton.

The effect of afzelin on cell growth in different cell lines was evaluated in the current study, with afzelin identified to markedly inhibit the growth of the prostate cancer cell lines, LNCaP and PC-3. Furthermore, the effect of afzelin on cell cycle progression was evaluated in the LNCaP and PC-3 cells. The results revealed that afzelin (10.0 µg/ml) caused a markedly increased population of PC-3 and LNCaP cells to accumulate in the G0 phase when compared with the corresponding control cells. To improve current understanding of the inhibitory effect of afzelin on cell growth and cell cycle progression, the expression levels of specific kinases were evaluated in the PC-3 and LNCaP cell lines upon treatment with increasing concentrations of afzelin. Afzelin was found to attenuate the protein expression levels of LIMK1 in the LNCaP and PC-3 cells. Elevated expression of LIMK1 has been previously identified in prostate cancer cell lines; therefore, afzelin represents a novel strategy for prostate cancer treatment. Furthermore, LIMK1 is activated by MRCKα and ROCK1 (9,12); thus, the effect of afzelin on the expression of these kinases was also evaluated. The results of the present study revealed that afzelin inhibited the protein expression levels of MRCKα and ROCK1, clarifying that downregulation of LIMK1 by afzelin is due to its inhibitory effect on MRCKα and ROCK1, upstream in its signaling pathway. Previous studies have demonstrated that ROCK inhibitors appear to reduce the invasive ability of tumor cells in vitro and prevent the dissemination of tumor cells in vivo in different types of cancer, particularly in prostate cancer (10,13–17,38). Furthermore, LIMK1, MRCKα and ROCK1 are proteins of interest in the development of novel cancer therapies due to their involvement in regulating actin organization (7) and, in combination with other proteins, maintaining the tight regulation of normal cell growth and differentiation (8,9).

Increased MRCKα expression has been reported in a number of cancer types, including adenocarcinoma (39), cutaneous squamous cell carcinoma (SCC) (40), in oral, hypopharyngeal, head and neck SCC, oral cavity and tongue carcinoma (41–47). In addition, increased MRCKα expression has also been identified in in vitro studies using U937 histiocytic lymphoma, MDA-MB 231 breast cancer, A549 lung cancer and PLB-985 myelocytic leukemia cell lines (46,47). Thus, the inhibition of MRCKα may present an effective strategy to inhibit cancer. Furthermore, Lourenço et al (48) revealed that LIMK2 expression was decreased in the intestinal tumors of cancer-prone genetically modified mice, as well as in human colorectal cancer cell lines and tumors. In the present study, afzelin was found to inhibit the expression of LIMK1, ROCK1 and MRCKα, indicating that their inhibition may prevent tumor cell invasion and metastasis. These results indicate that afzelin may effectively reduce tumor progression, however, further studies are required to investigate the association between azfelin and the expression of LIMK1, ROCK1 and MRCKα as afzelin may also have upstream targets, such as RhoA, Rac1a and Cdc42 that may effect the activation and signal transduction downstream of LIMK1. ROCK1 and MRCKα.

Conventional therapies, including radiotherapy and chemotherapy, exhibit certain limitations in the treatment of hormone-refractory prostate cancer; therefore, the development of novel treatment strategies is required to enhance the number of positive treatment outcomes. The results of the present study indicate that afzelin may be an important candidate in prostate cancer therapy. However, additional studies are required to evaluate its therapeutic properties, particularly in animal models.

 Acknowledgements

This study was supported by the General Programs of Shanghai Municipal Health Bureau (grant no. 20124345), the Second Key Medical Specialties Construction Project of Shanghai (grant no. 201373) and the Science and Technology Commission of Shanghai (grant no. 20111004).

 References LoweHIWatsonCTBadalSCycloartane-3,24,25-triol inhibits MRCKα kinase and demonstrates promising anti prostate cancer activity in vitroCancer Cell Int1246201210.1186/1475-2867-12-4623151005 FerlayJSteliarova-FoucherELortet-TieulentJCancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012Eur J Cancer4913741403201310.1016/j.ejca.2012.12.02723485231 JemalABrayFCenterMMGlobal cancer statisticsCA Cancer J Clin616990201110.3322/caac.2010721296855 BertholdDRSternbergCNTannockIFManagement of advanced prostate cancer after first-line chemotherapyJ Clin Oncol2382478252200510.1200/JCO.2005.03.143516278480 DiantiniASubarnasALestariKKaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii Korth inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathwayOncol Lett310691072201222783393 MaoYWTsengHWLiangWLAnti-inflammatory and free radial scavenging activities of the constituents isolated from Machilus zuihoensisMolecules1694519466201110.3390/molecules1611945122075574 HallARho GTPases and the actin cytoskeletonScience279509514199810.1126/science.279.5350.5099438836 HeikkilaTWheatleyECrightonDCo-crystal structures of inhibitors with MRCKβ, a key regulator of tumor cell invasionPloS One6e24825201110.1371/journal.pone.002482521949762 JaffeABHallARho GTPases: biochemistry and biologyAnnu Rev Cell Dev Biol21247269200510.1146/annurev.cellbio.21.020604.15072116212495 WilkinsonSPatersonHFMarshallCJCdc42-MRCK and Rho-ROCK signaling cooperate in myosin phosphorylation and cell invasionNat Cell Biol7255261200510.1038/ncb123015723050 FritzGJustIKainaBRho GTPases are over-expressed in human tumorsInt J Cancer81682687199910.1002/(SICI)1097-0215(19990531)81:5<682::AID-IJC2>3.0.CO;2-B10328216 RientoKRidleyAJRocks: multifunctional kinases in cell behaviourNat Rev Mol Cell Biol4446456200310.1038/nrm112812778124 NakajimaMHayashiKEgiYEffect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanomaCancer Chemother Pharmacol52319324200310.1007/s00280-003-0641-912783205 NakajimaMHayashiKKatayamaKWf-536 prevents tumor metastasis by inhibiting both tumor motility and angiogenic actionsEur J Pharmacol459113120200310.1016/S0014-2999(02)02869-812524136 NakajimaMKatayamaKTamechikaIWF-536 inhibits metastatic invasion by enhancing the host cell barrier and inhibiting tumour cell motiltiyClin Exp Pharmacol Physiol30457463200310.1046/j.1440-1681.2003.03855.x12823259 GendaTSakamotoMIchidaTCell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinomaHepatology3010271036199910.1002/hep.51030042010498656 TakamuraMSakamotoMGendaTInhibition of intrahepatic metastasis of human hepatocellular carcinoma by Rho-associated protein kinase inhibitor Y-27632Hepatology33577581200110.1053/jhep.2001.2265211230737 YingHBirocSLLiWWThe Rho kinase inhibitor fasudil inhibits tumor progression in human and rat tumor modelsMol Cancer Ther521582164200610.1158/1535-7163.MCT-05-044016985048 BemisDLCapodiceJLDesaiMA concentrated aglycone isoflavone preparation (GCP) that demonstrates potent anti-prostate cancer activity in vitro and in vivoClin Cancer Res1052825292200410.1158/1078-0432.CCR-03-082815297432 NgamwongsatitPBanadaPPPanbangredWBhuniaAKWST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapiddetection of toxigenic Bacillus species using CHO cell lineJ Microbiol Meth73211215200810.1016/j.mimet.2008.03.002 ChambersAFGroomACMacDonaldICDissemination and growth of cancer cells in metastatic sitesNat Rev Cancer2563572200210.1038/nrc86512154349 OlsonMFSahaiEThe actin cytoskeleton in cancer cell motilityClin Exp Metastasis26273287200910.1007/s10585-008-9174-218498004 BanyardJAnand-ApteBSymonsMZetterBRMotility and invasion are differentially modulated by Rho family GTPasesOncogene19580591200010.1038/sj.onc.120333810698528 HallAThe cytoskeleton and cancerCancer Metastasis Rev28514200910.1007/s10555-008-9166-319153674 MorganFisherWewerUMYonedaARegulation of ROCK activity in cancerJ Histochem Cytochem61185198201310.1369/002215541247083423204112 AmanoMNakayamaMKaibuchiKRho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarityCytoskeleton (Hoboken)67545554201010.1002/cm.2047220803696 GurkarAUChuKRajLIdentification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stressNat Commun42189201310.1038/ncomms318923877263 EdwardsDCSandersLCBokochGMGillGNActivation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamicsNat Cell Biol1253259199910.1038/1296310559936 OhashiKNagataKMaekawaMIshizakiTNarumiyaSMizunoKRho associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loopJ Biol Chem27535773582200010.1074/jbc.275.5.357710652353 SumiTMatsumotoKShibuyaANakamuraTActivation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alphaJ Biol Chem2762309223096200110.1074/jbc.C10019620011340065 DanCKellyABernardOMindenACytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilinJ Biol Chem2763211532121200110.1074/jbc.M10087120011413130 VleckenDHBagowskiCPLIMK1 and LIMK2 are important for metastatic behavior and tumor cell-induced angiogenesis of pancreatic cancer cellsZebrafish6433439200910.1089/zeb.2009.060220047470 YoshiokaKFolettaVBernardOItohKA role for LIM kinase in cancer invasionProc Natl Acad Sci USA10072477252200310.1073/pnas.123234410012777619 BagheriYarmandMazumdarASahinAAKumarRLIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator systemInt J Cancer11827032710200610.1002/ijc.2165016381000 ZhangZElSohlyHNLiXCPhenolic compounds from Nymphaea odorat.J Nat Prod66548550200310.1021/np020442j12713413 ShinSWJungEKimSKimJHKimEGLeeJParkDAntagonizing effects and mechanisms of afzelin against UVB-induced cell damagePLoS One8e61971201310.1371/journal.pone.006197123626759 ZhouWNieXAfzelin attenuates asthma phenotypes by downregulation of GATA3 in a murine model of asthmaMol Med Rep127176201525738969 BenitahaSAValerónaPFAelstbLMarshallcCJLacalaJCRho GTPases in human cancer: an unresolved link to upstream and downstream transcriptional regulationBiochimicaet Biophysica Acta17051211322005 BalasenthilSChenNLottSTA migration signature and plasma biomarker panel for pancreatic adenocarcinomaCancer Prev Res (Phila)4137149201110.1158/1940-6207.CAPR-10-002521071578 LefortKMandinovaAOstanoPNotch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCK-alpha kinasesGenes Dev21562577200710.1101/gad.148470717344417 PyeonDNewtonMALambertPFFundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancersCancer Res6746054619200710.1158/0008-5472.CAN-06-361917510386 SchlingemannJHabtemichaelNIttrichCPatient-based cross-platform comparison of oligonucleotide microarray expression profilesLab Invest8510241039200510.1038/labinvest.370029316205657 GinosMAPageGPMichalowiczBSIdentification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neckCancer Res645563200410.1158/0008-5472.CAN-03-214414729608 KimSMParkYYParkESPrognostic biomarkers for esophageal adenocarcinoma identified by analysis of tumor transcriptomePLoS One5e15074201010.1371/journal.pone.001507421152079 WangSZhanMYinJTranscriptional profiling suggests that Barrett's metaplasia is an early intermediate stage in esophageal adenocarcinogenesisOncogene2533463356200610.1038/sj.onc.120935716449976 HaoYTriadafilopoulosGSahbaiePGene expression profiling reveals stromal genes expressed in common between Barrett's esophagus and adenocarcinomaGastroenterology131925933200610.1053/j.gastro.2006.04.02616952561 KimchiETPosnerMCParkJOProgression of Barrett's metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiationCancer Res6531463154200515833844 LourençoFCMunroJBrownJReduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferationGut63480493201410.1136/gutjnl-2012-30388323585469

Molecular structure of afzelin, which has a molecular formula of 5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S, 3R, 4R, 5R, 6S)-3,4,5-trihydroxy- 6-methyloxan-2-yl]oxychromen-4-one), and molecular mass of 432.38 gmol−1.

Cell proliferation assay. Inhibition of (A) androgen-independent PC-3 and (B) androgen-sensitive LNCaP prostate cancer cell line proliferation following afzelin treatment. Proliferation of the two cell lines was assessed in the presence of various concentrations of afzelin for 72 h. Absorbance was measured at a wavelength of 420 nm and expressed as a percentage of the absorbance of the control (untreated) cells. Values are expressed as the mean ± standard deviation of four independent experiments.

Cell cycle analysis of (A) PC-3 and (B) LNCaP cells treated with 10.0 µg/ml afzelin or vehicle alone (0.1% DMSO) for 24 h. Cell cycle distribution was analyzed on a minimum of 1×104 cells for each experimental condition and data analysis was performed using CellQuest cell cycle analysis software. (C) Data from the cell cycle analysis were quantified into a graph to illustrate the percentage of cells in each phase of the cell cycle. P2, P3, P4 and P5 in Fig. 3A correspond to the G0, G1, S and G2M phases of the cell cycle in Fig. 3B, respectively. *P<0.05 vs. G0.

(A) Western blot analysis of the effect of afzelin on the protein expression levels of members of the Rho GTPase family in PC-3 and LNCaP cells. Following treatment with increasing concentrations of afzelin (0.1, 1.0 and 10.0 µg/ml) for 24 h, the expression levels of LIMK1 and its downstream proteins, MRCKα and ROCK1, were evaluated in the two cell lines. Afzelin significantly attenuated the expression of all three proteins in a dose-dependent manner, with a maximum decrease observed at 10.0 µg/ml in the two cell lines. (B) PC-3 and (C) LNCaP cell blots were quantitatively analyzed using densitometry. All blots are representative of 3 independent experiments. *P<0.05 and **P<0.01, vs. control. pLIMK, phosphorylated LIM domain kinase 1; pMRCKα; phosphorylated myotonic dystrophy kinase-related Cdc42-binding kinase α; pROCK, phosphorylated Rho-associated, coiled-coil-containing protein kinase 1.