Renal cell carcinoma (RCC) is a common urological cancer worldwide and is known to have a high risk of metastasis, which is considered responsible for more than 90% of cancer associated deaths. Honokiol is a small-molecule biphenol isolated from Magnolia spp. bark and has been shown to be a potential anticancer agent involved in multiple facets of signal transduction. In this study, we demonstrated that honokiol inhibited the invasion and colony formation of highly metastatic RCC cell line 786-0 in a dose-dependent manner. DNA-microarray data showed the significant upregulation of metastasis-suppressor gene KISS1 and its receptor, KISS1R. The upregulation was confirmed by qRT-PCR analysis. Overexpression of KISS1 and KISS1R was detected by western blotting at the translation level as well. Of note, the decreased invasive and colonized capacities were reversed by KISS1 knockdown. Taken together, the results first indicate that activation of KISS1/KISS1R signaling by honokiol suppresses multistep process of metastasis, including invasion and colony formation, in RCC cells 786-0. Honokiol may be considered as a natural agent against RCC metastasis.
Metastasis is the tendency of cancer cells to spread to distant organs in the body, which is considered responsible for more than 90% of cancer-associated deaths (1–4). It involves a multistep process including migration from the primary tumors, invasion to surrounding tissues, and proliferation leading to the colonization at distant sites (1,4). Accordingly, 25–30% of patients with renal cell carcinoma (RCC) have metastatic spread by the time they are diagnosed (5–7) and in these cases, the 5-year survival rate of patients is <10% (8,9). Moreover, 20–25% of suffers remain unresponsive to all treatments and the disease progresses rapidly (10,11). Honokiol, a small-molecule biphenolic compound isolated from Magnolia spp. Bark, has been shown to exhibit anticancer effects in different cancer types (12–17). The most widely investigated mechanism of its anticancer activities is apoptosis, which is induced in vitro and in vivo through multiple facets of signal transduction (12,14,16–25). Recently, several studies demonstrated that honokiol could also inhibit metastasis of breast, brain, gastric, lung and prostate cancer cells (13,21,26–32). However, only one study shows the metastasis suppression of RCC cells A-498 by honokiol through reversing epithelial-mesenchymal transition and blocking cancer stem cell properties (33). Definitely, there are other important targets involved in the process of RCC metastasis suppression by honokiol.
In this study, we found that honokiol inhibits the invasion and colony formation of highly metastatic RCC cells 786-0 (34) in a dose-dependent manner. DNA-microarray data showed significant upregulation of metastasis-suppressor gene KISS1 and its receptor, KISS1R. Both of the upregulation were confirmed by qRT-PCR analysis. Overexpression levels of KISS1 and KISS1R were detected by western blotting at the translation level as well. Of note, inhibition of invasion and colony formation were reversed by KISS1 knockdown. Taken together, our results indicate that honokiol suppresses the multistep process of metastasis, including invasion and colony formation, in RCC cells 786-0 via stimulation of KISS1/KISS1R signaling pathway.
Materials and methodsCell culture and reagents
Human RCC cells 786-0 were obtained from ATCC (Manassas, VA, USA). Cancer cells were maintained according to the ATCC procedures. Honokiol (98%) (HonoPure®) was provided by Econugenics Inc. (Santa Rosa, CA, USA) and dissolved in DMSO at a concentration of 80 mM then stored at −20°C. DMSO was purchased from Sigma (St. Louis, MO, USA). Anti-KISS1, anti-KISS1R and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell invasion assay
Cell invasion of 786-0 cells treated with honokiol (0–20 μM) was performed as previously described (35). Data points represent the mean ± SD of three individual filters within one representative experiment repeated at least twice.
Colony formation assay
Colony formation of the 786-0 cells incubated in the presence of honokiol (0–40 μM) was evaluated as previously described (36). Data points represent the mean ± SD in one representative experiment repeated at least twice.
DNA-microarray and quantitative RT-PCR analysis
The 786-0 cells were treated with honokiol (0, 40 μM) for 24 h and TaqMan® Array Human Tumor metastasis was performed as previously described (37). In qRT-PCR analysis, the 786-0 cells were treated with honokiol (0–40 μM) for 24 h. Isolation, quantification, reverse transcription of RNA and PCR were performed as previously described (37). Relative quantity (RQ) of gene expression was normalized to β-actin and performed using the 2−ΔΔCt method (38).
Western blot analysis
The 786-0 cells were treated with honokiol (0–40 μM) for 24 h. Whole protein extracts isolated from cells were prepared and western blot analysis with KISS1 and KISS1R antibodies were performed as previously described (39). Western blots were quantified with HP-Scanjet 550c and analyzed by UN-SCAN-IT software (Silk Scientific, Orem, UT, USA).
siRNA transfection
The 786-0 cells were transfected with human KISS1 siRNA or control siRNA-A as previously described (37). After 48 h of transfection, the cells were harvested and KISS1 knockdown was verified by western blot analysis.
Statistical analysis
All the statistical analysis was performed using SigmaPlot 11.2.0 (Systat Software Inc., San Jose, CA, USA). Data are presented as mean ± SD. Statistical comparisons were carried out using ANOVA with the significance level adjusted using the repeated t-tests with Bonferroni correction. P-value <0.05 was considered to be significant.
ResultsHonokiol inhibits invasion and colony formation of highly metastatic RCC cells
To evaluate whether honokiol (Fig. 1) suppresses invasive behavior of highly metastatic RCC cells, the 786-0 cells were treated with honokiol (0–20 μM) for 24 h and cell invasion was determined as described in Materials and methods. As shown in Fig. 2A, honokiol inhibits cell invasion through Matrigel in a dose-dependent manner. Moreover, honokiol significantly decreases the number of anchorage-independent colonies formed, which is a key step in cancer metastasis (Fig. 2B and C). In summary, honokiol significantly inhibits invasion as well as colony formation of highly meta-static RCC 786-0 cells in a dose-dependent manner.
Effect of honokiol on the expression of genes related to human tumor metastasis
In order to gain further mechanistic insight into the molecular events underlying metastasis inhibition of the 786-0 cells treated with honokiol, DNA-microarray analysis of 92 tumor metastasis-associated genes and 4 candidate endogenous control genes was performed. Table I summarizes the genes with large recurring expression differences compared with control. For example, significant upregulation was observed including the expression of metastasis suppressor gene (KISS-1, 28.56±11.17), genes encoding TIMP metalloproteinase inhibitor 4 (TIMP4, 14.25±4.04) and KISS-1 receptor (KISS-1R, 13.33±5.11). In addition, honokiol markedly suppresses expression of genes encoding chemokine (C-X-C motif) ligand 12 (CXCL12, 0.13±0.05), chemokine (C-C motif) ligand 7 (CCL7, 0.14±0.04), interleukin-18 (IL18, 0.23±0.05) and matrix metallo proteinase 7 (MMP7, 0.26±0.09).
Honokiol activates KISS1/KISS1R signaling in highly meta-static RCC cells
Since recent studies showed that activation of KISS1/KISS1R signaling by kisspeptin treatment decreases the motility and invasive capacity of conventional RCC, and overexpression of KISS1 inhibits invasion of RCC cells Caki-1 (40,41), we confirmed the significant upregulation of KISS1 and KISS1R in the 786-0 cells treated with honokiol by qRT-PCR (Fig. 3). In accordance with the change in mRNA, western blot analysis showed that honokiol stimulates expression of KISS1 and KISS1R in the 786-0 cells dose-dependently at the protein level (Fig. 4).
Silencing KISS1 reverses suppression of invasion and colony formation
To determine whether the suppression of invasion and colony formation by honokiol are associated with the activation of KISS1/KISS1R signaling in the 786-0 cells, we silenced KISS1 with siRNA as described in Materials and methods. As shown in Fig. 5, knockdown of KISS1 partially rescues the effect of honokiol on cell invasion by more than 40%. Moreover, the effect of honokiol on colony formation of the 786-0 cells is markedly reversed by KISS1 silencing (Fig. 6). These results further indicate that KISS1/KISS1R signaling is a major target of honokiol in suppressing metastasis of RCC cells.
Discussion
In the present study, we investigated the role of honokiol in the metastasis of RCC cells. Our results showed that honokiol significantly inhibited the invasion and colony formation of highly metastatic RCC cells 786-0 in a dose-dependent manner. Moreover, honokiol markedly upregulated metastasis-suppressor gene KISS1 and its receptor, KISS1R, at both transcription and translation levels. Interestingly, knockdown of KISS1 partially rescued the effect of honokiol on cell invasion and its effect on colony formation of the 786-0 cells is reversed as well, indicating that KISS1/KISS1R signaling is a major target of honokiol in suppressing metastasis of RCC cells.
Metastasis suppressors are defined as molecules whose expression results in the suppression of metastasis processes and since 1986, more than 13 metastasis suppressors have been identified (42). The KISS1 gene, initially discovered as a novel human malignant melanoma metastasis-suppressor gene (43), has been validated as an anti-metastatic gene by preclinical and clinical evidence in various types of cancer (44). The encoded KISS1 protein can be processed to a C-terminally amidated peptide termed metastin binding and activating the G-protein coupled receptor GPR54 (KISS1R) (45). Shoji et al found that metastin inhibited migration and invasion of RCC with overexpression of KISS1R (46). In addition, a recent study demonstrated that an absence of KISS1R expression was associated with rapid progression of conventional RCC in patients (40), suggesting KISS1/KISS1R signaling as a promising target in RCC.
Honokiol targets multiple signaling pathways such as nuclear factor κB (NF-κB), signal transducers and activator of transcription 3 (STAT3), mammalian target of rapamycin (mTOR) and epidermal growth factor receptor (EGFR), which have great relevance during cancer initiation and progression (47). Moreover, pharmacokinetic studies revealed that honokiol crossed the blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier (BCSFB) and had a desirable bioavailability after intravenous administration in animal models (48) thus making it a suitable agent for clinical trials.
In summary, our results indicate that activation of KISS1/KISS1R signaling by honokiol decreases the invasiveness and colonized capacity of highly metastatic RCC cells. Furthermore, we confirmed that honokiol stimulated the expression of TIMP4 dose-dependently (data not shown). It is in accordance with the finding that metastin suppresses the motility and invasive ability of RCC cells which possess KISS1R through the downregulation of MMP-2 (49). As emerging studies show that KISS1R activates a series of signaling molecules such as protein kinase C (PKC), extra-cellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and phosphatidylinositol-3-kinase (PI3K) (50), further studies are in progress to investigate the specific mechanism of honokiol, which may have the potential for use as a natural agent against RCC metastasis.
Acknowledgments
We thank Dr Zizheng Dong, Indiana University School of Medicine, for his technical assistance with the colony formation assay. This study was supported by EcoNugenics, Inc., Santa Rosa, CA, USA. One of the authors, I. Eliaz, acknowledges his interest as the formulator and owner of EcoNugenics, Inc.
ReferencesMehlenPPuisieuxAMetastasis: A question of life or deathNat Rev Cancer6449458200610.1038/nrc188616723991NguyenDXMassaguéJGenetic determinants of cancer metastasisNat Rev Genet8341352200710.1038/nrg210117440531MonteiroJFoddeRCancer stemness and metastasis: Therapeutic consequences and perspectivesEur J Cancer4611981203201010.1016/j.ejca.2010.02.03020303259DeepGAgarwalRAntimetastatic efficacy of silibinin: Molecular mechanisms and therapeutic potential against cancerCancer Metastasis Rev29447463201010.1007/s10555-010-9237-0207147883928361GuptaKMillerJDLiJZRussellMWCharbonneauCEpidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): A literature reviewCancer Treat Rev34193205200810.1016/j.ctrv.2007.12.00118313224SadlerGJAndersonMRMossMSWilsonPGMetastases from renal cell carcinoma presenting as gastrointestinal bleeding: Two case reports and a review of the literatureBMC Gastroenterol74200710.1186/1471-230X-7-4172667571800859CzarneckaAMKornakiewiczAKukwaWSzczylikCFrontiers in clinical and molecular diagnostics and staging of metastatic clear cell renal cell carcinomaFuture Oncol1010951111201410.2217/fon.13.25824941992CairnsPRenal cell carcinomaCancer Biomark94614732010PatilSManolaJElsonPNegrierSEscudierBEisenTAtkinsMBukowskiRMotzerRJImprovement in overall survival of patients with advanced renal cell carcinoma: Prognostic factor trend analysis from an international data set of clinical trialsJ Urol18820952100201210.1016/j.juro.2012.08.02623083849ButiSBersanelliMSikokisAMainesFFacchinettiFBriaEArdizzoniATortoraGMassariFChemotherapy in metastatic renal cell carcinoma today? A systematic reviewAnticancer Drugs24535554201323552469LinJDengZTanikawaCShuinTMikiTMatsudaKNakamuraYDownregulation of the tumor suppressor HSPB7, involved in the p53 pathway, in renal cell carcinoma by hypermethylationInt J Oncol44149014982014245851834027944KimDWKoSMJeonYJNohYWChoiNJChoSDMoonHSChoYSShinJCParkSMAnti-proliferative effect of honokiol in oral squamous cancer through the regulation of specificity protein 1Int J Oncol4311031110201323877711JooYNEunSYParkSWLeeJHChangKCKimHJHonokiol inhibits U87MG human glioblastoma cell invasion through endothelial cells by regulating membrane permeability and the epithelial-mesenchymal transitionInt J Oncol441871942014TianWDengYLiLHeHSunJXuDHonokiol synergizes chemotherapy drugs in multidrug resistant breast cancer cells via enhanced apoptosis and additional programmed necrotic deathInt J Oncol427217322013HahmERSakaoKSinghSVHonokiol activates reactive oxygen species-mediated cytoprotective autophagy in human prostate cancer cellsProstate7412091221201410.1002/pros.22837250432914156520LaiYJLinCIWangCLChaoJIExpression of survivin and p53 modulates honokiol-induced apoptosis in colorectal cancer cellsJ Cell Biochem11518881899201424905183ChilampalliCZhangXKaushikRSYoungAZemanDHildrethMBFahmyHDwivediCChemopreventive effects of combination of honokiol and magnolol with α-santalol on skin cancer developmentsDrug Discov Ther7109115201323917859LiangWZChouCTChangHTChengJSKuoDHKoKCChiangNNWuRFShiehPJanCRThe mechanism of honokiol-induced intracellular Ca(2+) rises and apoptosis in human glioblastoma cellsChem Biol Interact2211323201410.1016/j.cbi.2014.07.01225106108WangXBeitlerJJWangHLeeMJHuangWKoenigLNannapaneniSAminARBonnerMShinHJHonokiol enhances paclitaxel efficacy in multi-drug resistant human cancer model through the induction of apoptosisPLoS One9e86369201410.1371/journal.pone.0086369245862493934844ChangKHYanMDYaoCJLinPCLaiGMHonokiol-induced apoptosis and autophagy in glioblastoma multiforme cellsOncol Lett6143514382013241795373813738PanHCLaiDWLanKHShenCCWuSMChiuCSWangKBSheuMLHonokiol thwarts gastric tumor growth and peritoneal dissemination by inhibiting Tpl2 in an orthotopic modelCarcinogenesis3425682579201310.1093/carcin/bgt24323828905MartinSLambHKBradyCLefkoveBBonnerMYThompsonPLovatPEArbiserJLHawkinsARRedfernCPInducing apoptosis of cancer cells using small-molecule plant compounds that bind to GRP78Br J Cancer109433443201310.1038/bjc.2013.325238071683721410YaoCJLaiGMYehCTLaiMTShihPHChaoWJWhang-PengJChuangSELaiTYHonokiol eliminates human oral cancer stem-like cells accompanied with suppression of Wnt/β-catenin signaling and apoptosis inductionEvid Based Complement Alternat Med2013146136201310.1155/2013/146136ChaeJIJeonYJShimJHDownregulation of Sp1 is involved in honokiol-induced cell cycle arrest and apoptosis in human malignant pleural mesothelioma cellsOncol Rep2923182324201323525508WangYZhuXYangZZhaoXHonokiol induces caspase-independent paraptosis via reactive oxygen species production that is accompanied by apoptosis in leukemia cellsBiochem Biophys Res Commun430876882201310.1016/j.bbrc.2012.12.063AvtanskiDBNagalingamABonnerMYArbiserJLSaxenaNKSharmaDHonokiol inhibits epithelial-mesenchymal transition in breast cancer cells by targeting signal transducer and activator of transcription 3/Zeb1/E-cadherin axisMol Oncol8565580201410.1016/j.molonc.2014.01.004245080634009450SinghTKatiyarSKHonokiol inhibits non-small cell lung cancer cell migration by targeting PGE2-mediated activation of β-catenin signalingPLoS One8e60749201310.1371/journal.pone.0060749LiuSHWangKBLanKHLeeWJPanHCWuSMPengYCChenYCShenCCChengHCCalpain/SHP-1 interaction by honokiol dampening peritoneal dissemination of gastric cancer in nu/nu micePLoS One7e43711201210.1371/journal.pone.0043711229370843427156JeongJJLeeJHChangKCKimHJHonokiol exerts an anticancer effect in T98G human glioblastoma cells through the induction of apoptosis and the regulation of adhesion moleculesInt J Oncol4113581364201222895699SinghTKatiyarSKHonokiol, a phytochemical from Magnolia spp., inhibits breast cancer cell migration by targeting nitric oxide and cyclooxygenase-2Int J Oncol38769776201121225228WenJFuAFChenLJXieXJYangGLChenXCWangYSLiJChenPTangMHLiposomal honokiol inhibits VEGF-D-induced lymphangiogenesis and metastasis in xenograft tumor modelInt J Cancer12427092718200910.1002/ijc.2424419219913ShigemuraKArbiserJLSunSYZayzafoonMJohnstonePAFujisawaMGotohAWekslerBZhauHEChungLWHonokiol, a natural plant product, inhibits the bone metastatic growth of human prostate cancer cellsCancer10912791289200710.1002/cncr.2255117326044LiWWangQSuQMaDAnCMaLLiangHHonokiol suppresses renal cancer cells’ metastasis via dual-blocking epithelial-mesenchymal transition and cancer stem cell properties through modulating miR-141/ZEB2 signalingMol Cells37383388201410.14348/molcells.2014.0009248102104044309RoomiMWIvanovVKalinovskyTNiedzwieckiARathMModulation of human renal cell carcinoma 786-0 MMP-2 and MMP-9 activity by inhibitors and inducers in vitroMed Oncol23245250200610.1385/MO:23:2:24516720925LloydFPJrSlivovaVValachovicovaTSlivaDAspirin inhibits highly invasive prostate cancer cellsInt J Oncol2312771283200314532966SlivovaVValachovicovaTJiangJGanoderma lucidum inhibits invasiveness of breast cancer cellsJ Cancer Integr Med225302004ChengSEliazILinJThyagarajan-SahuASlivaDTriterpenes from Poria cocos suppress growth and invasiveness of pancreatic cancer cells through the downregulation of MMP-7Int J Oncol42186918742013235887133699575LivakKJSchmittgenTDAnalysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) methodMethods25402408200110.1006/meth.2001.1262JiangJSlivovaVHarveyKValachovicovaTSlivaDGanoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-kappaB signalingNutr Cancer49209216200410.1207/s15327914nc4902_1315489214ChenYYusenkoMVKovacsGLack of KISS1R expression is associated with rapid progression of conventional renal cell carcinomasJ Pathol2234653201110.1002/path.2764ZhangHGuoYShangCSongYWuBmiR-21 down-regulated TCF21 to inhibit KISS1 in renal cancerUrology801298302.e1201210.1016/j.urology.2012.08.013HurstDRWelchDRMetastasis suppressor genes at the interface between the environment and tumor cell growthInt Rev Cell Mol Biol2861071802011211997813575029LeeJHMieleMEHicksDJPhillipsKKTrentJMWeissmanBEWelchDRKiSS-1, a novel human malignant melanoma metastasis-suppressor geneJ Natl Cancer Inst8817311737199610.1093/jnci/88.23.17318944003BeckBHWelchDRThe KISS1 metastasis suppressor: A good night kiss for disseminated cancer cellsEur J Cancer4612831289201010.1016/j.ejca.2010.02.023203032583662025OhtakiTShintaniYHondaSMatsumotoHHoriAKanehashiKTeraoYKumanoSTakatsuYMasudaYMetastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptorNature411613617200110.1038/3507913511385580ShojiSTangXYUmemuraSItohJTakekoshiSShimaMUsuiYNagataYUchidaTOsamuraRYMetastin inhibits migration and invasion of renal cell carcinoma with overexpression of metastin receptorEur Urol55441449200910.1016/j.eururo.2008.02.048AroraSSinghSPiazzaGAContrerasCMPanyamJSinghAPHonokiol: A novel natural agent for cancer prevention and therapyCurr Mol Med1212441252201210.2174/156652412803833508228348273663139WangXDuanXYangGZhangXDengLZhengHDengCWenJWangNPengCHonokiol crosses BBB and BCSFB, and inhibits brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma modelPLoS One6e18490201110.1371/journal.pone.0018490215595103084695YoshiokaKOhnoYHoriguchiYOzuCNamikiKTachibanaMEffects of a KiSS-1 peptide, a metastasis suppressor gene, on the invasive ability of renal cell carcinoma cells through a modulation of a matrix metalloproteinase 2 expressionLife Sci83332338200810.1016/j.lfs.2008.06.01818644390CvetkovićDBabwahAVBhattacharyaMKisspeptin/KISS1R System in breast cancerJ Cancer4653661201310.7150/jca.7626
Structure of honokiol.
Effect of honokiol on the invasion and colony formation of the 786-0 cells. The 786-0 cells were treated with honokiol (A) (0–20 μM) or (B) (0–40 μM). Cell invasion through Matrigel and colony formation in agarose were determined as described in Materials and methods. Each bar represents the mean ± SD in one representative experiment repeated at least twice. Representative pictures of colony formation are shown (C). Statistical analysis by ANOVA, *P<0.05.
Honokiol stimulates mRNA expression of KISS1 and KISS1R in the 786-0 cells. The 786-0 cells were treated with honokiol (0–40 μM) for 24 h and qRT-PCR analysis on KISS1 and KISS1R were performed as described in Materials and methods. Each bar represents the mean ± SD of three independent experiments. Statistical analysis by ANOVA, *P<0.05.
Honokiol induces expression of KISS1 and KISS1R in the 786-0 cells. The 786-0 cells were treated with honokiol (0–40 μM) for 24 h and the expression of KISS1 and KISS1R were evaluated by western blot analysis as described in Materials and methods. Representative results are shown. Similar results were obtained in at least two additional experiments.
KISS1 gene silencing partially rescues the effect of honokiol on cell invasion. The 786-0 cells were transfected with scrambled siRNA (siRNA-A) or KISS1 siRNA as described in Materials and methods. (A) Western blot analysis of KISS1 was evaluated. (B) Invasion of the 786-0 cells through Matrigel was determined as described in Fig. 2A. Each bar represents the mean ± SD of three individual filters within one representative experiment repeated at least twice. Statistical analysis by ANOVA, *P<0.05.
KISS1 gene silencing reverses the effect of honokiol on colony formation. The 786-0 cells were transfected with scrambled siRNA (siRNA-A) or KISS1 siRNA as described in Materials and methods. (A) Colony formation of the 786-0 cells in agarose was determined as described in Fig. 2B. (B) Representative images of colony formation are shown. Each bar represents the mean ± SD in one representative experiment repeated at least twice. Statistical analysis by ANOVA, *P<0.05.
Effect of honokiol on the expression of human tumor metastasis genes.
Gene
Description
RQ
KISS1
KISS-1 metastasis suppressor
28.56±11.17a
TIMP4
TIMP metalloproteinase inhibitor 4
14.25±4.04a
KISS1R
KISS1 receptor
13.33±5.11a
TP53
P53 tumor suppressor
2.24±0.16
CXCL12
Chemokine (C-X-C motif) ligand 12
0.13±0.05
CCL7
Chemokine (C-C motif) ligand 7
0.14±0.04
IL18
Interleukin-18
0.23±0.05
MMP7
Matrix metalloproteinase 7
0.26±0.09
VEGFC
Vascular endothelial growth factor C
0.42±0.04
FGFR4
Fibroblast growth factor receptor 4
0.53±0.04
DNA-microarray analysis was performed on TaqMan® Array Human Tumor Metastasis as described in Materials and methods. The 786-0 cells were treated with honokiol (0 and 40 μM) for 24 h. Data are the means ± SD of three independent experiments. Analysis of the relative quantity gene expression (RQ) data was performed using the 2−ΔΔCT method. Statistical analysis by ANOVA,