1521-0111/88/2/326–334$25.00 http://dx.doi.org/10.1124/mol.115.097758MOLECULAR PHARMACOLOGY Mol Pharmacol 88:326–334, August 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Neuritogenic Activity of Tetradecyl 2,3-Dihydroxybenzoate IsMediated through the Insulin-Like Growth Factor 1 Receptor/Phosphatidylinositol 3 Kinase/Mitogen-Activated Protein KinaseSignaling Pathway s

Ruiqi Tang, Lijuan Gao, Makoto Kawatani, Jianzhong Chen, Xueli Cao, Hiroyuki Osada,Lan Xiang, and Jianhua QiCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China (R.T., L.G., J.C., X.C., L.X.,J.Q.); and Chemical Biology Core Facility, RIKEN, Advanced Science Institute, Saitama, Japan (M.K., H.O.)

Received January 7, 2015; accepted May 26, 2015

ABSTRACTTetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead com-pound derived from neuritogenic gentisides. In the presentstudy, we investigated the mechanism by which ABG-001induces neurite outgrowth in a rat adrenal pheochromocytomacell line (PC12). Inhibitors of insulin-like growth factor 1 (IGF-1)receptor, phosphatidylinositol 3-kinase (PI3K), and extracellularsignal-regulated kinase (ERK) 1/2 significantly decreasedABG-001–induced neurite outgrowth. Western blot analysis re-vealed that ABG-001 significantly induced phosphorylation ofIGF-1 receptor, protein kinase B (Akt), ERK, and cAMP responsive

element-binding protein (CREB). These effects were markedlyreduced by addition of the corresponding inhibitors. We also foundthat ABG-001–induced neurite outgrowth was reduced by pro-tein kinase C inhibitor as well as small-interfering RNA againstthe IGF-1 receptor. Furthermore, like ABG-001, IGF-1 also inducedneurite outgrowth of PC12 cells, and low-dose nerve growth factoraugmented the observed effects of ABG-001 on neurite outgrowth.These results suggest that ABG-001 targets the IGF-1 receptorand activates PI3K, mitogen-activated protein kinase, and theirdownstream signaling cascades to induce neurite outgrowth.

IntroductionNeurotrophic factors such as nerve growth factors (NGFs)

participate in neuronal development and in the survivaland functional maintenance of neurons (McAllister 2001).Several studies have indicated that reduced neurotrophic

support is a contributing factor in the pathogenesis ofAlzheimer’s disease, Parkinson’s disease, and amyotrophiclateral sclerosis (Dawbarn and Allen, 2003). Therefore,neurotrophic factors are strong-candidate therapeuticagents for chronic neurodegenerative diseases. Unfortu-nately, NGF cannot penetrate the blood-brain barrier becauseof its size, and this limitation hinders its applications inclinical therapy. As such, screening forNGF-mimicking chemicalcompounds with low molecular weights and the ability topass through the blood-brain barrier is an active area ofresearch.Natural compounds such as termitomycesphins A–D

(Qi et al., 2000), linckosides A–E (Qi et al., 2002, 2004),panax ginseng (Yamazaki et al., 2001), and nardosinone

This work was supported by the Natural Science Foundation of ZhejiangProvince, People’s Republic of China [Grant Y2110105], the International Scienceand Technology Cooperation Program of China [No. 2014DFG32690], the NationalNatural Science Foundation of China [Grants 30873152 and 81072536], the Projectfor Science and Technology of Yunnan Province, China [Grant 2012AE002]. Thiswork was inspired by the international and interdisciplinary environments of theJSPS Asian CORE Program, “Asian Chemical Biology Initiative.”

dx.doi.org/10.1124/mol.115.097758.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: ABG-001, tetradecyl 2,3-dihydroxybenzoate; AG1024, 2-(3-bromo-5-(tert-butyl)-4-hydroxybenylidene)malononitrile; Akt, proteinkinase B; AZ628, 3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl]benzamide; DMEM, Dulbecco’s modifiedEagle’s medium; DMSO, dimethylsulfoxide; ERK, extracellular signal-regulated kinase; FAM, 5-carboxy-fluorescein; GF109203X, bisindolylmaleimide I;Gö6983, 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; HNMPA(AM)3, hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester; JNK, c-Jun N-terminalkinase; K252a, (9S-(9a,10b,12a))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3ʹ,2ʹ,1ʹ-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one; LY294002, 2-morpholin-4-yl-8-phenylchromen-4-one; MAPK, mitogen-activated protein kinase; MEK,mitogen-activated protein-extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NGF, nerve growthfactor; PD98059, 2-(2-amino-3-methoxyphenyl)chromen-4-one; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C;PLC, phospholipase C; Ro318220, 3-(1-(3-(amidinothio) propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide; S3131, sulindac sulfide; SAPK,stress-activated protein kinase; SB203580, 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine; siRNA, small-interfering RNA;SP600125, 1,9-pyrazoloanthrone; T9576, picropodophyllotoxin; TrkA, tyrosine kinase A; U0126, (2Z,3Z )-2,3-bis[amino-(2-aminophenyl)sulfanylme-thylidene]butanedinitrile; U73122, 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione; U73343, 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione.

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(Li et al., 2003) as well as synthetic compounds such as 4,5-bis-epi-neovibsanin A (Chen et al., 2010), xaliproden (Sabbagh,2009), and synthetic verbena chalcone derivatives (Clementet al., 2009) have recently been shown to have NGF-mimickingor NGF-enhancing activity related to neurite outgrowth in a ratadrenal pheochromocytoma cell line (PC12). Previous studieshave identified diverse molecular signaling pathways partici-pating in the neuron-protective activities of small naturalcompounds (Macready et al., 2009; Weng et al., 2011). Thesepathways include selective actions of a number of protein andlipid kinase signaling cascades, the most notable of whichinvolve the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) and mitogen-activated protein kinase (MAPK)pathways that regulate prosurvival transcription factors andgene expression.Gentiana rigescens Franch (G. rigescens) is a traditional

Chinesemedicine that is believed to remove heat and dampnessfrom the human body. It is used to treat hypertension,cholecystitis, acute infective hepatitis, and cystitis. Pharma-codynamic studies have indicated that G. rigescens has anti-inflammation, antipathogen, antioxidant, antitumor, andproimmunity functions (Tang et al., 1977). In our previousstudy, we described the isolation of gentisides A2K, which arenew neuritogenic compounds, from G. rigescens (Gao et al.,2010a,b). These gentisides showed good neuritogenic activi-ties in PC12 cells. To study their structure-activity relation-ships and to facilitate the discovery of the lead compounds ingentisides, we synthesized more than a hundred derivativesof gentisides and tested their neuritogenic activities in PC12cells. We found that ABG-001 (tetradecyl 2,3-dihydroxybenzoate;Fig. 1A) exhibited strong NGF-mimicking effects via theextracellular signal-regulated kinase (ERK) signaling pathway(Luo et al., 2011). However, the molecular target of gentisidesand the mechanism behind induction of neurite outgrowth inPC12 cells remain unknown.In the present study, we used inhibitors of the NGF

signaling pathway and small-interfering RNAs (siRNAs) incombination with Western blot assays to investigate themechanism behind improvements in neurite outgrowth in-duced by ABG-001. We report that ABG-001 may target theinsulin-like growth factor 1 (IGF-1) receptor and activate thePI3K, protein kinase C (PKC)/MAPK, and the associateddownstream signaling cascades to induce neuritogenicactivity.

Materials and MethodsChemicals and Reagents. ABG-001 was synthesized and puri-

fied using high-pressure liquid chromatography in our laboratory(Luo et al., 2011). Dimethylsulfoxide (DMSO); NGF; the PI3Kinhibitors LY294002 (2-morpholin-4-yl-8-phenylchromen-4-one) andwortmannin; the mitogen-activated protein-extracellular signal-regulated kinase (MEK)/ERK inhibitors U0126 [(2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile] andPD98059 [2-(2-amino-3-methoxyphenyl)chromen-4-one]; the IGF-1receptor kinase inhibitor T9576 (picropodophyllotoxin); the PKCinhibitors GF109203X (bisindolylmaleimide I), Gö6983 [3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione]); and the protein kinase A (PKA) inhibitor H-89([N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide])were purchased from Sigma-Aldrich (St. Louis, MO). Ras inhibitors(sulindac sulfide and farnesylthiosalicylic acid) were purchasedfrom Sigma-Aldrich and Cayman Chemical (Ann Arbor, MI). The

phospholipase C (PLC) inhibitorsU73122 (1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione) and U73343(1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), the PKC inhibitors Ro318220 [3-(1-(3-(amidino-thio) propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide], theJunN-terminal kinase (JNK) inhibitor SP600125 (1,9-pyrazoloanthrone),the p38 MAPK inhibitor SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine), the tyrosine kinaseA (TrkA) inhibitor K252a [(9S-(9a,10b,12a))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3ʹ,2ʹ,1ʹ-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one], the insulin re-ceptor inhibitorHNMPA(AM)3 [hydroxy-2-naphthalenylmethylphosphonicacid tris acetoxymethyl ester], the IGF-1 inhibitor AG1024 [2-(3-bromo-5-(tert-butyl)-4-hydroxybenylidene)malononitrile], and the Raf inhibitorAZ628 [3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl]benzamide] were purchased from Santa Cruz Bio-technology (Dallas, TX). IGF-1 was purchased from Sino BiologicCompany (Beijing, People’s Republic of China).

Cell Culture. The rat adrenal pheochromocytoma cell line PC12was purchased from the Type Culture Collection of the ChineseAcademy of Sciences (Shanghai, People’s Republic of China). PC12cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)(Thermo Scientific, Waltham, MA) containing 4 mM glutamine, 1.5 g/lsodium bicarbonate, 4.5 g/l glucose, 10 mMHEPES, and 1 mM sodiumpyruvate and supplemented with 10% horse serum and 5% fetal bovineserum (Invitrogen, Grand Island, NY) in a 5% CO2 incubator at 37°C.Before treatment, cells were subcultured and allowed to attachovernight.

Neurite Outgrowth Assay. Briefly, 2 � 104 PC12 cells wereseeded in the wells of a 24-well microplate and cultured undera humidified atmosphere of 5% CO2 at 37°C. The medium wasreplaced with 1 ml of serum-free DMEM containing the test sample orDMSO (0.5%) after 24 hours. NGF was used as a positive control. Thenumber of neurite-bearing cells was measured by counting cells inthree arbitrary areas on the 24-well plate containing at least 100single cells (not aggregated). A cell was identified as positive forneurite outgrowth if the outgrowths were at least twice the celldiameter (Jeon et al., 2010a,b). Cells were visualized using phasecontrast (200-fold magnification) with an Olympus microscope (ModelCK-2; Olympus China, Beijing, People’s Republic of China).

About 100 cells were counted from a random site, and each ex-periment was repeated three times. If the neurite outgrowth of 100cells observed in an experiment were twice the cell diameter, then theoutgrowth was considered to be 100%. The results are expressed asmean 6 S.E.M. For experiments involving inhibitors, we first per-formed a dose-dependent investigation. The optimum concentrationof inhibitors was then used to conduct the subsequent experiments.

Analysis of Cell Viability by MTT Assay. Cell viability wasdetermined based on mitochondria-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) topurple formazan. Briefly, the cells were incubated with ABG-001 atconcentrations of 0, 0.3, 0.6, or 1 mM for 48 hours. The medium wascarefully removed by aspiration, and 0.5 ml of fresh mediumcontaining MTT (0.2 mg/ml) was added to each well, and the plateswere incubated at 37°C for 2 hours. The medium was then completelyremoved, and 0.2 ml of DMSO was added to each well to solubilize theformazan crystals. The resultant formazan was detected at 570 nmusing a plate reader. All experiments were repeated at least threetimes.

Real-Time Polymerase Chain Reaction Analysis. Cells treatedwith siRNA against the IGF-1 receptor and ABG-001, respectively,were collected. The total RNA was extracted using TRIzol reagent(Beijing Cowin Biotech; Beijing, People’s Republic of China), and theRNA content was determined using a spectrophotometer. Transcrip-tion was performed using 2.5 mg of total RNA, Oligo(dT)20 primers,and reverse transcriptase (Beijing Cowin Biotech). Transcript levelswere quantified by real-time polymerase chain reaction (AB SCIEX,

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Framingham, MA) and SYBR Premix EX Taq (Takara, Otsu, Japan).The polymerase chain reaction primers for rat IGF-1 receptor and 18SRNA were as follows: for IGF-1 receptor, sense: 59-ATG GCT TCGTTA TCC ACG AC-39, and anti-sense: 59-CGA ATC GAT GGT TTTCGT TT-39; for 18S RNA, sense: 59-TAA CCCGTTGAACCCCAT T-39,and anti-sense: 59-CCA TCC AAT CGG TAG TAG CG-39. The cDNAwas then amplified using Takara SYBR Premix Ex Taq under thefollowing conditions: 95°C for 2 minutes, followed by 40 cycles for15 seconds at 95°C, 15 seconds at 54.2°C, and 20 seconds at 68°C. Allresults were normalized to 18S RNA levels, and the relative mRNAtranscript levels were calculated using the DDCt formula. All sampleswere run in triplicate, and the average values were calculated.

Western Blot Analysis. Approximately 1 � 106 PC12 cells wereseeded in a 60-mm culture dish containing 5 ml DMEMand incubatedfor 24 hours. To investigate the dose-dependent effect of ABG-001,various ABG-001 concentrations were added to the cultures atconcentrations of 0, 0.3, 0.6, or 1.0 mM, after which they wereincubated for 30 minutes or 16 hours. To investigate the time-dependent effects of ABG-001, ABG-001 was added at a finalconcentration of 1 mM, after which the cultures were incubated forspecific time periods.

To investigate the effects of siRNA IGF-1 receptor on neuriteoutgrowth induced by ABG-001, ABG-001 was added after celltransfection of the negative control siRNA against IGF-1 receptorfor 6 hours.

To prepare protein lysates, cells were collected in lysis buffer (1%Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mMEDTA, and1% phosphatase inhibitor) and then sonicated using an ultrasonicator(Ningbo Scientz Biotechnology, Ningbo, People’s Republic of China).The supernatant containing the proteins was collected by centrifuga-tion at 12 � 103 rpm for 15 minutes. The protein concentration wasmeasured using the Bio-Rad Protein Assay Dye Reagent Concentrate(Bio-Rad Laboratories, Hercules, CA) at 595 nm. Protein lysates(15 mg) were separated by SDS-PAGE and then transferred ontopolyvinylidene difluoride membranes. The membrane was incubatedwith primary antibodies followed by horseradish peroxidase–conjugated secondary antibodies. Antigens were visualized using

chemiluminescent substrates (Amersham Biosciences/GE Health-care, Little Chalfont, United Kingdom).

The primary antibodies used for immunoblotting are asfollows: anti–44/42 MAPK antibody, anti–phospho-p44/42 MAPK(Thr202/Tyr204) polyclonal antibody, anti–IGF-1 receptor b anti-body, anti–phospho-IGF-1 receptor b (Tyr1135/1136)/insulin re-ceptor b (Tyr1150/1151), anti–phospho-CREB (Ser133), anti-CREBand anti-Akt antibodies (Cell Signaling Technology, Beverly, MA),anti–phospho-Akt (Ser473) (Abcam, Hong Kong, People’s Republicof China), and GAPDH antibody (Beijing Cowin Biotech). Thesecondary antibodies used in this study are as follows: horseradishperoxidase–linked anti-rabbit and anti-mouse IgGs (Beijing CowinBiotech).

Cell Transfection. PC12 cells were transfected with 5-carboxy-fluorescein (FAM)–labeled siRNA to investigate transfection effi-ciency. A concentration of 120 nM, at which the transfection efficiencyreached 90%, was used to perform the experiment. The followingprimer sequences were used to generate siRNAs targeting the ratIGF-1 receptor and the negative control (Shanghai GenePharmaCompany, Shanghai, People’s Republic of China): for IGF-1 receptor-530, sense: 59-GCG GUG UCC AAU AAC UAC ATT-39, anti-sense:59-UGU AGU UAU UGG ACA CCG CTT-39; for negative control,sense: 59-UUCGAACGUGUCACGUTT-39, anti-sense: 59-ACGUGACAC GUU CGG AGA ATT-39.

Transfection of PC12 cells with siRNA was performed according tothe manufacturer’s protocol (Invitrogen). Briefly, the day beforetransfection, 2 � 104 cells were seeded in each well of 24-well platesand allowed to reach 70–90% confluence in growth medium withoutantibiotics. Then siRNA against IGF-1 receptor or the negativecontrol siRNA was used at a concentration of 120 nM with Lipofect-amine 2000 (Invitrogen) as the transfection agent. After 6 hours oftransfection, the medium in the plates was replaced with freshmedium containing 1 mM ABG-001 and incubated for an additional24 hours. Cell morphologic features were observed and recorded usinga microscope fitted with a camera. The lengths of neurites weremeasured using ImageJ software (National Institutes of Health,Bethesda, MD).

Fig. 1. (A) Chemical structure of ABG-001.(B) Neurite outgrowth in PC12 cells ofABG-001 at different concentrations. (C) Per-centage of viable PC12 cells after treatmentwith ABG-001 for 48 hours. (D) Effects ofTrkA inhibitor on the neurogenic effects ofABG-001 in PC12 cells. Percentage of neuriteoutgrowth as monitored using a phase-contrastmicroscope 48 hours after ABG-001 treatment.Neurites were identified as cells bearing neu-rites that were at least twice the cell diameter.(Control: DMSO, 0.5%; positive control: NGF,40 ng/ml). *Significantly different from thecontrol group at the same time point atP, 0.05.***Significantly different from the controlgroup at the same time point at P , 0.001.###Significantly different from the NGF groupat the same time point at P , 0.001.

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Statistical Analysis. All experiments were independentlyperformed three times, and each experiment was conducted withtriplicate samples. Data are presented as mean 6 S.E.M. Thestatistically significant differences between groups were deter-mined by analysis of variance, followed by two-tailed multiplet tests with Student-Newman-Keuls through SPSS, biostatisticssoftware (IBM, Chicago, IL). P , 0.05 was considered statisticallysignificant.

ResultsABG-001 Induces Neurite Outgrowth in PC12

Cells. Figure 1B shows the neuritogenic activity of ABG-001in PC12 cells. ABG-001 induced neurite outgrowth in PC12cells in a dose-dependent manner. The percentages of neurite-bearing cells treated with 0.3, 0.6, and 1.0 mM ABG-001 for48 hours were 57.7 6 5.4%, 70.1 6 3.6%, and 83.9 6 5.8%,respectively, and were significantly higher than that of thecontrol group (P , 0.001). This result suggests that ABG-001has significant effects on neurite outgrowth in PC12 cells andis consistent with the findings of a previous study (Luo et al.,2011).Effects of ABG-001 on PC12 Cell Viability. The effect

of ABG-001 on PC12 cell viability was determined by MTTassay. Figure 1C illustrates that PC12 cells treated withABG-001 concentrations of 0.3, 0.6, and 1 mM for 48 hoursshow viabilities of 82.6 6 4.7%, 78.5 6 11.3%, and 75.5 67.1%, respectively. These results indicate that ABG-001 isweakly cytotoxic toward PC12 cells.

Effects of ABG-001 on the TrkA Signaling Pathway.We investigated the function of the TrkA receptor in neuriteoutgrowth induced by ABG-001 using the TrkA receptorinhibitor K252a. Application of this inhibitor did not affect theneuritogenic activity of ABG-001 in PC12 cells (Fig. 1D;Supplementary Fig. 1A).Effects of ABG-001 on the MAPK/ERK/CREB Signal-

ing Pathway. We investigated the function of ERK1/2activation in ABG-001–induced neurite outgrowth in PC12cells. U0126 significantly attenuated the percentage ofneurite-bearing cells from an initial value of 71.8 6 5.5% to32.46 2% after the cells had been treated with 1 mMABG-001(Fig. 2, A and B; Supplemental Fig. 2B). The average ofneurite length of PC12 cells induced by NGF and ABG-001was significantly reduced by U0126 (Fig. 2C).Furthermore, we determined the effects of ABG-001 on

ERK phosphorylation at the protein level. ERK1/2 phosphoryla-tion was enhanced by ABG-001 at concentrations of 0, 0.6,1.0, and 1.2 mM and reduced by U0126 (Fig. 2D). ERK1/2phosphorylation induced by ABG-001 began 8 hours aftertreatment, and then gradually decreased after 48 hours(Fig. 2E).CREB phosphorylation induced by ABG-001 was initiated

8 hours after treatment and was maintained for 48 hours (Fig.2F). Parallel blots were run and probed with antibodies todetect total ERK1/2, CREB, and GAPDH levels. The resultsdemonstrated that all proteins had been loaded at equivalentlevels. These findings indicate that ERK/CREB signaling is

Fig. 2. (A) Photomicrographs of PC12 cells after treatment with ABG-001 andU0126 for 48 hours: (a) Control (0.5%DMSO), (b) NGF (40 ng/ml), (c) 1 mMABG-001, (d) 40 ng/ml NGF + 10 mMU0126, and (e) 1mMABG-001 + 10 mMU0126. (B) Effect of U0126 on the neurite outgrowth in PC12 cells induced byABG-001 at 1 mM. (C) The average of neurite length for control group was 0.175 6 0.025 (mm); NGF at 40 ng/ml, 1.303 6 0.117 (***); ABG-001 at 1 mM,1.5876 0.158 (***); NGF + U0126, 0.7176 0.054 (###); ABG-001 + U0126, 0.1786 0.031($$$). ABG-001 induced phosphorylation of ERK1/2 in a (D) dose-and (E) time-dependent manner (the cells treated with each agent for 16 hours in dose-dependent experiment). (F) ABG-001–stimulated CREBphosphorylation. NGF was used as a positive control, and ERK and GAPDH antibodies were used as loading controls. ***Significantly different fromcontrol group at the same time point at P , 0.001. ###,$$$Significantly different from NGF or ABG-001–treated group at P , 0.001.

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involved in ABG-001–induced neurite outgrowth of PC12cells.Effects of ABG-001 on the PI3K/Akt Signaling Path-

way. PI3K is critical for NGF-dependent survival of sympa-thetic neurons, and Akt, a major effecter, has the importantrole of integrating various survival signaling cascades(Crowder and Freeman, 1998; Pierchala et al., 2004). Here,specific inhibitors of PI3K, namely, LY294002 and wortman-nin, were used to examine the effects of ABG-001 on thePI3/Akt signaling pathway.ABG-001–induced neurite outgrowth and the average

length of PC12 cell neuritis was markedly reduced by 20 mMLY294002 (Fig. 3, A and B). LY294002 attenuated thepercentage of neurite-bearing cells from an initial value of70.9 6 3.3% to 42.75 6 1.2% after the cells had been treatedwith 1 mM ABG-001 (Fig. 3C; Supplemental Fig. 3A).Next, we investigated Akt phosphorylation at the protein

level at multiple time points (Fig. 3D). Akt phosphorylationlevel increased with increase in treatment time in dose-dependent manner at ABG-001 concentrations of 0.0, 0.6, 1.0,and 1.2 mM. Furthermore, Akt phosphorylation was signifi-cantly decreased by LY294002 treatment (Fig. 3E). ABG-001–induced neurite outgrowth was also reduced by wortmannin(Supplemental Fig. 3B). The inhibitory effects of LY294002were stronger than those of wortmannin. These resultssuggest that PI3K/Akt may exert an important effect onABG-001–induced neuronal differentiation of PC12 cells.Effects of ABG-001 on the Insulin/IGF-1 Signaling

Pathway and PC12 Cell Differentiation. To determinethe target of ABG-001, we focused on receptors locatedupstream of PI3K. As expected, HNMPA(AM)3, the specificinhibitor of insulin receptor, slightly attenuated the per-centage of neurite-bearing cells from an initial value of69.46 4.0% to 59.76 4.3% after treatment with 1mMABG-001(Fig. 4A; Supplemental Fig. 4A). However, the percentage of

neurite-bearing cells was reduced from an initial value of60.9 6 5.5% to 41.9 6 3.7% after treatment with the IGF-1inhibitor AG1024 (Fig. 4B; Supplemental Fig. 4B).Changes in IGF-1 receptor phosphorylation were fur-

ther observed after treatment with ABG-001. As shown inFig. 4, D and E, ABG-001 induced dose-dependent en-hancements in IGF-1 receptor phosphorylation, whichpeaked at 10 minutes; these enhancements were signifi-cantly reduced by AG1024. Furthermore, application of thetyrosine kinase IGF-1 receptor inhibitor T9576 led to asignificant decrease in the percentage of neurite outgrowthfrom PC12 cells from 83 6 2.3% to 42.6 6 2.3% (Fig. 4C;Supplemental Fig. 4C).To determine whether the IGF-1 signaling pathway is the

primary mechanism by which ABG-001 affects PC12 cells, weexamined the effects of IGF-1 on neurite outgrowth of thesecells. As expected, IGF-1 significantly enhanced the neuriteoutgrowth of PC12 cells, just as ABG-001 and NGF did. Thisneurotrophic effect of IGF-1 on PC12 cells could also beinhibited by the IGF-R inhibitor T9576 (Fig. 4F). Theseresults suggest that ABG-001, like IGF-1, targets the IGF-1receptor and regulates the expression of downstream genes toinduce NGF-like effects in PC12 cells.Effects of Knockdown IGF-1 Receptor by siRNA on

Neurite Outgrowth after Treatment with ABG-001. Tofurther verify that ABG-001 targets the IGF-1 receptor, wefirst used FAM-siRNA to determine the best siRNA trans-fection concentration. Photomicrographs of PC12 cells aftertransfection with FAM-siRNA are displayed in Fig. 5, A andB. Approximately 90% of the PC12 cells produced fluorescenceat 120 nM. Therefore, we used this dose to transfect IGF-1receptor siRNA into PC12 cells for 6 hours and then treatedwith 1 mM ABG-001.The lengths of neurites induced by ABG-001 were signifi-

cantly reduced by IGF-1 receptor siRNA from 48.66 6 4.17 to

Fig. 3. (A) Effect of ABG-001 on the PI3K/Akt signaling pathway in PC12 cells. Morphologic changes in PC12 cells after treatment with ABG-001 andLY294002 for 48 hours: (a) Control (0.5% DMSO), (b) positive control (NGF 40 ng/ml), (c) 1 mM ABG-001, (d) 40 ng/ml NGF + 20 mM LY294002, and (e)1 mM ABG-001 + 20 mM LY294002. (B) The average neurite length for the control group was 0.190 6 0.042 (mm); NGF at 40 ng/ml, 1.576 6 0.276***;ABG-001 at 1 mM, 1.4866 0.231***; NGF + LY294002, 0.7176 0.054###; ABG-001 + LY294002, 0.1786 0.031$$$. (C) Effects of LY294002 on ABG-001–induced neurite outgrowth. (D and E) ABG-001–stimulated phosphorylation of Akt in a time- and dose-dependent manner (the cells treated with eachagent for 30 minutes in dose-dependent experiment). NGF was used as a positive control, and Akt and GAPDH antibodies were used as loadingcontrols. ***Significant difference compared with control group at P , 0.001. ###,$$$Significant difference compared with NGF or ABG-001–treatedgroup at P , 0.001.

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28.22 6 1.37 mm (Fig. 5, C–E). The percentages of neuriteoutgrowth and the gene expression level of IGF-1 receptorwere also significantly decreased after treatment with IGF-1RsiRNA (Fig. 5, F and G). Furthermore, the total protein levelof IGF-1 receptor and phosphorylation of IGF-1 receptor wereboth significantly reduced by treatment with siRNA againstIGF-1 receptor (Fig. 5H). At the same time, phosphorylation ofAkt and ERK were significantly lowered by siRNA againstIFG-1 receptor (Fig. 6F). These results are consistent with ourearlier observations that ABG-001 acts through the IGF-1receptor.Effects of ABG-001 on the PKC, PLC, PKA, JNK, p38,

Ras, Raf, and NGF Signaling Pathways and PC12 CellDifferentiation. Because PKC is located downstream ofPI3K, we investigated the function of PKC in ABG-001–induced neurite outgrowth using various PKC inhibitors(Gö6983, GF109203X, and Ro318220). Ro318220 and Gö6983showed appreciable inhibitory effects (Fig. 6, A and B,Supplemental Fig. 1B). The percentage of neurite-bearingcells decreased from an initial value of 70.1 6 2.8% to 36.0 67.9% after treatment with Ro318220 (P, 0.01). Likewise, thepercentage of neurite-bearing cells decreased from an initialvalue of 55.1 6 3.0% to 23.0 6 7.9% after treatment withGö6983 (P , 0.05). Furthermore, a low concentration of NGFcould augment the effects of ABG-001 on neurite outgrowth ofPC12 cells (Fig. 6, C and D). These results show that PKC isinvolved in PC12 cell differentiation induced by ABG-001, but

NGF signaling is not involved in the neurite outgrowth ofPC12 cells induced by ABG-001.In addition, inhibitors of PLC (U73122 and U73343), PKA

(H-89), JNK (SP600125), p38 MAPK (SB203580), Raf (AZ628),and Ras (S3131 [sulindac sulfide], farnesylthiosalicylic acid)were used to investigate whether these signaling pathwaysare involved in neurite outgrowth mediated by ABG-001 inPC12 cells. The results showed that none of these inhibitorshad a significant effect on the neurite outgrowth induced byABG-001. These results verify that the signaling pathwaysassociated with these proteins are not involved in the NGF-mimicking effects of ABG-001.

DiscussionABG-001 (Fig. 1A) is a leading compound derived from the

neuritogenic compound gentisides. It possesses a long alkylchain of 14 carbons, two close hydroxy groups on the benzenering, and an ester linkage between the ring and alkyl chain.Results from experiments aimed at investigating itsstructure-activity relationships showed that the length ofthe alkyl chain, number and position of hydroxyl groups onthe benzene ring, and the type of linking group between thebenzene ring and the alkyl chain had distinct effects on itsneuritogenic activities (Luo et al., 2011).NGF is known to induce both neurite outgrowth and

survival by binding and phosphorylating the TrkA receptor

Fig. 4. (A, B, and C) Effect of insulin/IGF-1 inhibitors on the neurogenic effects of ABG-001 in PC12 cells. Cells were pretreatedwith 10mMHNMPA(AM)3,0.1 mM AG1024, 0.1mM T9576, specific inhibitors of insulin, and IGF-1 inhibitors for 30 minutes, followed by addition of 1 mM ABG-001. (D) Time- and(E) dose-dependent changes in phosphorylation of the IGF-1 receptor (the samples were obtained after treatment with ABG-001 for 5minutes). NGFwasused as a positive control, and IGF-1 receptor and GAPDH antibodies were used as loading controls. (F) Effects of IGF-1 and T9576 on the neuriteoutgrowth of PC12 cells. ***Significantly different from the NGF-treated group at the same time point at P , 0.001. ###Significantly different from theABG or IGF-1–treated groups at the same time point at P , 0.05 or P , 0.01.

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in cell membranes (Chao, 2003). Therefore, we used the TrkAreceptor inhibitor K252a to examine whether the TrkAsignaling pathway is involved in the neurogenic effects ofABG-001. The results indicated that K252a had no inhibi-tory effect on the neurogenic function of ABG-001 (Fig. 1D;Supplemental Fig. 1A), which suggests that the TrkAreceptor is not involved.MEK and ERK1/2 have important functions in controlling

gene transcription events leading to proliferation or differen-tiation of PC12 cells in response to NGF (Traverse et al., 1992;Rubinfeld and Seger 2005). NGF is known to promote PC12cell survival via ERK1/2 signaling (Vaudry et al., 2002). In thepresent study, ABG-001 was confirmed to take part in neuriteoutgrowth of PC12 cells (Fig. 2, A–C) and to induce ERK andCREB phosphorylation (Fig. 2, D–G). Similar results wereobtained when PD98059 [2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one] was used (Supplementary Fig. 2A). Theseresults suggest that neurite outgrowth of PC12 cells aftertreatment with ABG-001 involves at least in part theMEK/ERK pathway. In addition, we found the early phase ofCREB phosphorylation (Supplemental Fig. 5), but phosphor-ylation of ERK at the same timewas not observed (unpublisheddata). At this point, it is possible that other signaling pathwaystake part in the regulation of ABG-001 NGF-mimicking effects.The Ras/MAPK (Ras/ERK1/2), PLC-g, and PI3K/Akt sig-

naling pathways (Kaplan and Miller 1997; Kusunoki et al.,2008) are important for the neurogenic effects of NGF.

Specific inhibitors of Ras, PLC, and PI3K/Akt, includingS3131, farnesylthiosalicylic acid, U73122, U73343, LY294002,and wortmannin, were used in our study. Interestingly, onlyPI3K inhibitors were found to inhibit the neurogenic effectsof ABG-001 (Fig. 3, A–C) and the phosphorylation of Aktby ABG-001 (Fig. 3, C and D). These results indicate thatPI3K/Akt-mediated signaling plays an important role inthe neurogenic effects of ABG-001.Insulin/IGF-1 exerts important growth-promoting effects

by activating the PI3K/Akt signaling pathway (Cui andAlmazan 2007). The biologic actions of IGF-1 are mediated bythe IGF-1 receptor, a member of the receptor tyrosine kinasefamily that induces dimerization and activates severaldownstream pathways to transmit proliferative signals afterextracellular stimulations (Chitnis et al., 2008; Annenkov2009; Bibollet-Bahena and Almazan 2009). Our results showthat ABG-001 induces IGF-1 receptor phosphorylation andthat AG1024, T9576, and IGF-1 receptor siRNA inhibit PC12cell differentiation induced by ABG-001 (Figs. 4–5). Theseresults suggest that the NGF-mimicking effects of IGF-1 onPC12 cells may be mediated via ABG-001 binding to IGF-1receptor and thereby activating PI3K and MAPK signalingcascades to induce neuritogenic activities. However, PC12 celldifferentiation induced by ABG-001 was not completely in-hibited by AG1024, T9576, or siRNA against the IGF-1 receptor.This suggests that other receptors may also contribute to theNGF-mimicking effects of ABG-001.

Fig. 5. Effect of IGF-1 receptor siRNA on the neurogenic effects of ABG-001 in PC12 cells. Microphotograph of PC12 cells after transfection with (A and B)FAM-siRNA and (C and D) IGF-1 receptor siRNA. (E) Change of neurite length in PC12 cells after treatment with IGF-1 receptor siRNA. (F) Geneexpression of IGF-1 receptor after treatment with IGF-1 receptor siRNA and ABG-001. (G) Neurite outgrowth percentage of PC12 cells after treatment withIGF-1 receptor siRNA and ABG-001. (H)Western blot analysis for IGF-1 receptor after treatment with siRNA of IGF-1 receptor. Cells were transfected withlipofectamine 2000 and 120 nM IGF-1 receptor siRNA for 6 hours and then treated with 1 mM ABG-001. The ABG-001 treatment group was used asa positive control. **,***Significantly different from the ABG-001–treated group at the same time point at P, 0.01, P, 0.001. #Significantly different fromnegative siRNA-treated group at the same time point at P , 0.05.

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Some studies have suggested that aside from the threesignaling cascades described herein, the stress-activatedprotein kinase (SAPK)/JNK pathway also affects differentia-tion (Gelderblom et al., 2004). Therefore, we investigated thepossible involvement of the SAPK/JNK pathway in regulatingthe NGF-mimicking effects of ABG-001 using a specificinhibitor SP600125 and the P38 inhibitor SB203580. Differ-entiation induced by ABG-001 was only weakly inhibitedwhen PC12 cells were treated with SP600125 and SB203580(unpublished data). These data suggest that the SAPK/JNKpathway does not participate in the neurogenic effects ofABG-001.Previous reports also indicated that cAMP and PKC are

involved in neuronal differentiation (Hansen et al., 2000;Kolkova et al., 2000). Therefore, the effects of cAMP-dependent protein kinase A (PKA) and PKCwere investigatedto explore other possible signaling pathways regulating theABG-001–induced neurite outgrowth of PC12 cells. Theinhibitory effects induced by PKC inhibitors were statisticallysignificant (Fig. 6, A and B), but those induced by H-89, a PKAinhibitor, were not (unpublished data).Taking these findings together, we propose that the

neuritogenic effect of ABG-001 is mediated by a signaling

cascade that follows this order: IGF-1 receptor, PI3K, Akt-PKC,ERK, CREB (Fig. 6, E and F).Notably, the effective concentrations of some protein

inhibitors (K252a, AG1024, T9576, and Ro318220) used inour study were higher than those described in other reports(Tapley et al., 1992; Alessi, 1997; Wen et al., 2001; Linderet al., 2007). In test experiments, we used the reportedconcentrations to investigate whether they have inhibitoryeffects on homologous proteins. These inhibitors did not produceinhibition of neurite outgrowth in PC12 cells (unpublished data).We note that a few other studies also used higher concentrationsof these inhibitors (Yang and Wang, 2009; Pandya and Pillai,2014). It is possible that differences in the purity or sources ofinhibitors account for the apparent discrepancy in the concen-trations at which these inhibitors are effective.In conclusion, we demonstrated that ABG-001–induced

time- and dose-dependent phosphorylation of IGF-1 receptor,Akt, ERK, and CREB in PC12 cells. Moreover, inhibition ofIGF-1 receptor, PI3K, PKC, and ERK activation by AG1024,LY294002, Ro318220, and U0126 inhibitors, respectively,blocked the observed phosphorylation effects. In addition,siRNA against IGF-1 reduced the neurite outgrowth ofPC12 cells induced by ABG-001 and IGF-1, like ABG-001,

Fig. 6. (A and B) Effect of PKC inhibitors on the neurogenic effects of ABG-001 in PC12 cells. (C and D) Effect of NGF on the neurogenic effects ofABG-001 in PC12 cells treated for 24 hours. (E) Proposed mechanism of ABG-001 in induction of neurite outgrowth in PC12 cells. ABG-001 inducesneurite outgrowth through activation of the IGF-1 receptor, PI3K/Akt-PKC, and MAPK/ERK dependent pathways in PC12 cells (Figs. 2–6). (F) Thechange in Akt and ERK phosphorylation after treatment with siRNA against IGF-1 receptor. Cells were pretreated with 0.1 mM Ro318220 and 5 mMGö6983, specific inhibitors of PKC, for 30 minutes and then administered 0.3 or 1 mM ABG-001. NGF was used as a positive control. **Significantlydifferent from the control group at the same time point at P , 0.01. #,##,$$Significantly different from the NGF or ABG-001–treated group at the sametime point at P , 0.05 or P , 0.01.

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hadNGF-mimicking effects. These results indicate that IGF-1receptor-mediated MAPK activation is essential for ABG-001–induced neurite outgrowth in PC12 cells.

Authorship Contributions

Participated in research design: Qi, Osada.Conducted experiments: Tang, Gao, Cao.Contributed new reagents or analytic tools: Chen.Wrote or contributed to the writing of the manuscript: Xiang,

Kawatani.

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Address correspondence to: Jianhua Qi, Institute of Medicinal Chemistry,College of Pharmaceutical Sciences, Yu Hang Tang Road 866, Hangzhou310058, People’s Republic of China. E-mail: qijianhua@zju.edu.cn or LanXiang, Institute of Medicinal Chemistry, College of Pharmaceutical Sciences,Yu Hang Tang Road 866, Hangzhou 310058, People’s Republic of China.E-mail: lxiang@zju.edu.cn

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