degalactotigonin, a natural compound from solanumnigruml ...osteosarcoma cell viability in vitro....

Cancer Therapy: Preclinical

Degalactotigonin, a Natural Compound fromSolanumnigrumL., InhibitsGrowthandMetastasisof Osteosarcoma through GSK3b Inactivation–Mediated Repression of the Hedgehog/Gli1PathwayZhiqiang Zhao1,2,3, Qiang Jia4,5, Man-Si Wu6,7, Xianbiao Xie1,3, Yongqian Wang1,3,Guohui Song1, Chang-Ye Zou1, Qinglian Tang1, Jinchang Lu1, Gang Huang1,3, Jin Wang1,3,De-Chen Lin2,8, H. Phillip Koeffler2,8, Jun-Qiang Yin1,3, and Jingnan Shen1,3

Abstract

Purpose: Agents extracted from natural sources with antitumorproperty have attracted considerable attention from researchersand clinicians because of their safety, efficacy, and immediateavailability. Degalactotigonin (DGT), extracted from Solanumnigrum L., has anticancer properties without serious side effects.Here, we explored whether DGT can inhibit the growth andmetastasis of osteosarcoma.

Experimental Design:MTT, colony formation, and apopto-sis assays were performed to analyze the effects of DGT onosteosarcoma cell viability in vitro. The migration and inva-sion abilities were measured using a Transwell assay. Animalmodels were used to assess the roles of DGT in both tumorgrowth and metastasis of osteosarcoma. Gli1 expression andfunction were measured in osteosarcoma cells and clinicalsamples. After DGT treatment, Gli1 activation and the phos-phorylation status of multiple cellular kinases were measuredwith a luciferase reporter and phospho-kinase antibodyarray.

Results: DGT inhibited proliferation, induced apoptosis, andsuppressed migration and invasion in osteosarcoma cells. DGT,injected intraperitoneally after tumor inoculation, significantlydecreased the volume of osteosarcoma xenografts and dramati-cally diminished the occurrence of osteosarcoma xenograftmetas-tasis to the lungs. Mechanistically, DGT inhibited osteosarcomagrowth and metastasis through repression of the Hedgehog/Gli1pathway, whichmaintainsmalignant phenotypes and is involvedin the prognosis of osteosarcoma patients. DGT decreased theactivity of multiple intracellular kinases that affect the survival ofosteosarcoma patients, including GSK3b. In addition, DGTrepresses the Hedgehog/Gli1 pathway mainly through GSK3binactivation.

Conclusions: Our studies provide evidence that DGT cansuppress the growth and metastasis of human osteosarcomathroughmodulation of GSK3b inactivation–mediated repressionof theHedgehog/Gli1 pathway.ClinCancer Res; 24(1); 130–44.�2017AACR.

IntroductionOsteosarcoma is the most common primary malignant bone

tumor in childhood and adolescence. Current treatment strate-gies, which consist of multiagent chemotherapy and aggressivesurgery, have significantly improved the 5-year survival rate ofpatients with osteosarcoma from 10% to 70% over the past 30years (1). However, despite some advances in the treatment ofosteosarcoma, therapies have not changed significantly in the pastfew years. The survival rate for patients with metastatic osteosar-coma is still less than 20% (2, 3). Therefore, further research iswarranted to identify effective agents anddevelopnew therapeuticstrategies with less severe side effects for the treatment of thisdeadly disease.

Over the past three decades, agents derived from naturalsources have gained considerable attention from researchers andclinicians because of their safety, efficacy, and immediate avail-ability (4). They have long been used in traditional Chinesemedicine and show multiple biological activities, including anti-cancer, anti-inflammatory, and neuroprotective properties (5, 6).One of the most successful natural agents is artemisinin

1Department of Musculoskeletal Oncology, the First Affiliated Hospital of SunYat-Sen University, Guangzhou, China. 2Department of Medicine, Cedars-SinaiMedical Center, Los Angeles, California. 3Guangdong Provincial Key Laboratoryof Orthopedics and Traumatology, the First Affiliated Hospital of Sun Yat-SenUniversity, Guangzhou, China. 4Guangzhou City Polytechnic, Guangzhou, China.5Institute of Biology, Guizhou Academy of Sciences, Guiyang, China. 6School ofTraditional Chinese Medicine, Jinan University, Guangzhou, China. 7State KeyLaboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-SenUniversity, Guangzhou, China. 8Cancer Science Institute of Singapore, NationalUniversity of Singapore, Singapore.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Z. Zhao, Q. Jia, and M. Wu contributed equally to this article.

Corresponding Authors: Jingnan Shen, The First Affiliated Hospital of Sun Yat-Sen University, Zhongshan2 Road, Guangzhou 510080, China. Phone: 8602-0873-35039; Fax: 8620-8733-2150; E-mail: [email protected]; JunqiangYin, [email protected]; and Man-Si Wu, School of Traditional ChineseMedicine, Jinan University, Guangzhou, China. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0692

�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 24(1) January 1, 2018130

on October 18, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 26, 2017; DOI: 10.1158/1078-0432.CCR-17-0692

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-0692&domain=pdf&date_stamp=2017-12-9

http://clincancerres.aacrjournals.org/

(qinghaosu), with an antimalarial effect, which is considered atrue gift from old Chinesemedicine by Youyou Tu (7). Because ofthe great contribution of artemisinin, the founder, Youyou Tu,received the 2015 Nobel Prize in Physiology or Medicine. How-ever, natural agents have not been popularly accepted, primarilydue to their poorly defined molecular mechanisms. One suchlittle investigated agent is degalactotigonin (DGT), which isisolated from Solanum nigrum L. The extractions of SolanumnigrumL have anticancer properties in breast cancer, colon cancer, andprostate cancer while leaving normal cells unharmed (8–11).Thus far, one study has shown growth-inhibitory effects of DGTon a cervical cell line (HeLa) and contraceptive potential butprovided no detailed insights into the underlying molecularmechanisms (12). The goal of this study was to determine therange of growth and metastasis repression of DGT against oste-osarcoma cells both in vitro and in vivo, and to investigate how thedrug mediates these activities.

The Hedgehog (HH) signaling pathway is crucially involved inthe development of several cancers, such as glioma, colon cancer,gastric cancer, multiple myeloma, myeloid leukemia, and rhab-domyosarcomas (13–16). The HH signaling is mainly dependentonGli transcription factor family (Gli1, Gli2, andGli3), which aredownstream effectors. Specifically, Gli1 is the principal transcrip-tional effector that regulates gene expression in response to HHsignaling activation. Moreover, overexpression of Gli1 is a cred-ible indicator of poor prognosis in most solid malignancies,irrespective of intracranial tumors (17). However, its clinicalsignificance and biological function in osteosarcoma growth andmetastasis remains unclear.

Here, we investigated the potential of DGT to inhibit growthand metastasis at the cellular level and in animal models todemonstrate that DGT may be a promising natural agent fortreating patients with osteosarcoma. Moreover, we propose thatone of the mechanisms of action of DGT is through inducingGSK3b inactivation–mediated repression of the HH/Gli1 path-way, which is important in the development of osteosarcoma.

Materials and MethodsCell culture

All cell lines were obtained from ATCC except U2OS/MTX (amethotrexate-resistant derivative of the U2OS human osteosar-

coma cell line was provided by Dr. M. Serra, Instituti OrtopediciRizzoli, Bologna, Italy), ZOS, and ZOS-M (from human osteo-sarcoma patient with primary tumor and metastasis). All of theosteosarcoma cell lines were grown in DMEM (Invitrogen) with10% FBS (Invitrogen) at 37�C and 5% CO2. The hMSCs weregifted by Dr. Fangang Meng (The First Affiliated Hospital of SunYat-Sen University, Guangzhou, China) and cultured as shown(18). All cell lines used in this study were authenticated usingshort tandem repeat profiling when this project was initiated, andthe cells have not been in culture for more than 1 month.

Compounds and reagentsDGT (MW:1035.1; >98% purity) was extracted from Solanum

nigrumL. byQ. Jia andpurchased fromSigmaAldrich (catalog no.:39941-51-0). Antibodies against P21 (#2947S), CyclinD1(#2978S), p-H2AX (#9718S), H2AX (#2595S), Gli1 (#2643S),Akt (#2920S), p-Akt(#4060S), ERK (#4348S), p-ERK (#4370S),GSK-3b (#9315S), phospho-Ser9-GSK-3b (#9323S), PARP(#9532S), and cleaved PARP (#5625S) were purchased from CellSignaling Technology. Antibodies against CyclinA1 (sc-53233),CyclinB (sc-166152), GAPDH (sc-32233), and tubulin (sc-73242) were purchased from Santa Cruz Biotechnology. Anti-bodies against Gli1 (ab217326), smo (ab38686), PTCH1(ab53715), and SHH (ab53281) were purchased from Abcam.The reagents for IHC analyses were obtained fromDako Cytoma-tion. The human Gli1 plasmid was purchased from youbia.

Cell viability assayOsteosarcoma cells were seeded in 96-well plates at a density of

4,000 cells per well. They were treated with different concentra-tions of DGT for the indicated times, and the cell viability wasmeasured by MTT assay as described previously (19).

Colony formation assayBriefly, osteosarcoma cells were plated in triplicate at 500 cells

per well in 12-well plates in Fig. 1C with or without DGT and2,000 cells per well in 6-well plates for in Fig. 6D. All the cells werecultured for 12 days. Then, cell clones were washed three timeswith PBS, fixed inmethanol for 10minutes, and dyed with crystalviolet for 10minutes at room temperature. Afterward, the dyewaswashed off and colonies that contained more than 50 cells werecounted.

Cell-cycle and apoptosis assaysOsteosarcoma cells were treated with DGT for 48 hours and

were subsequently collected as described previously (20). Cell-cycle analysis was performed by propidium iodide staining (Sig-ma-Aldrich) for DNA content and followed by flow cytometricanalysis. The cells were stained with both propidium iodide andAnnexin V (BD Biosciences) and assayed on a LSRII flow cyt-ometer (BD Biosciences) for the apoptosis assay. All flow cyto-metry data were analyzed using FlowJo software (Tree Star).

Hoechst 33258 staining and caspase-3 activity assayCells were seeded at 50% confluency in 6-well plates, and after

overnight incubation, the cells were treated with vehicle or DGTfor 48 hours. Then, the standard procedures as previouslydescribed were performed for Hoechst 33258 staining and cas-pase-3 activity assay (19).

Translational Relevance

Despite impressive advances in systemic therapies, patientswith metastatic osteosarcoma still have a poor overall survivalrate. Thus, safe and effective agents are required todevelopnewtherapeutic strategies for treatment of this deadly disease.Recent efforts have identified numerous natural compoundsthat have potential antitumor activity. Here, using both in vitroand in vivo osteosarcoma models, we show that degalactoti-gonin, a natural compound from Solanum nigrum L., caninhibit the growth andmetastasis of osteosarcoma.We furthershow for the first time that themolecularmechanism is relatedto GSK3b inactivation–mediated repression of the Hedgehog/Gli1 pathway, which is involved in the prognosis of osteosar-coma patients. These observations support further clinicaldevelopment of this drug as a cotreatment strategy.

Antitumor Potential of Degalactotigonin in Osteosarcoma

www.aacrjournals.org Clin Cancer Res; 24(1) January 1, 2018 131




Immunofluorescence analysisTomeasure the p-H2AX expression in control andDGT-treated

osteosarcoma cells, an immunocytochemical analysis was per-formed. Briefly, U2OS cells were plated on culture slides (Costar)and treatedwith orwithoutDGT for 48hours. Then, sampleswerefixed using 4% paraformaldehyde solution for 15 minutes atroom temperature and extracted with buffer containing 0.5%Triton X-100 for 5 minutes. The cells were then incubated withprimary antibodies overnight. After three washes in PBS, thesamples were incubated with secondary antibody at room tem-perature for 1 hour. Cells were then counterstained with DAPI tovisualize nuclear DNA and examined using an Olympus confocalimaging system (Olympus FV100).

Western blottingWestern blotting was carried out as described earlier (19).

Briefly, the cells were lysed in RIPA buffer containing proteaseinhibitor and phosphatase inhibitor cocktails (Thermo FisherScientific). The nuclear protein was isolated according to theprotocol provided by the Nuclear Protein Extraction Kit (ThermoFisher Scientific). Then, equal amounts of proteinwas resolved on

10% SDS-PAGE and transferred to a PVDF membrane (Milli-pore). Membranes were incubated with primary antibody over-night at 4�C. Membranes were washed with TBST and incubatedwith horseradish peroxidase–conjugated secondary antibody.Proteins were visualized using ECL detection reagents (Beyo-time Co.).

Cell transfectionSmall interfering RNAs against the following genes were syn-

thesized by RiboBio.GSK3b (sense 50-CUCAAGAACUGUCAAGUAATT-30, antisense:

50-UUACUUGACAGUUCUUGAGTT-30); AKT, (sense 50-UGC-CCUUCUACAACCAGGATT-30, antisense 50-UCCUGGUUGUA-GAAGGGCATT-30); b-catenin (sense, 50-GCAGUUGUAAACUU-GAUUATT-30, antisense, 50-UAAUCAAGUUUACAACUGCTT-30).The shRNA targeting Gli1 was constructed as below. Briefly, thetargeting sequence (Gli1 shRNA #1, CTTTGATCCTTACCTCCCA;Gli1 shRNA#2, AGCTCCAGTGAACACATAT) was inserted intopLKO.1-puro gifted by Tiebang Kang (Sun Yat-Sen UniversityCancer Center, Guangzhou, China) following the protocol provid-ed by Addgene. Targeting siRNAs or shRNA were transfected into

Figure 1.

DGT inhibits proliferation in osteosarcoma cells. A, Molecular structure of DGT. B, Osteosarcoma cells were seeded in 96-well plates, and after 24 hours, theywere treated with a range of DGT concentrations for 72 hours. The viable cells were measured using MTT assays, and IC50 values were calculated. C, DGT reducescolony formation of osteosarcoma cells. Cells were treated with the indicated concentrations of DGT for 24 hours. Cells were then washed, allowed to formcolonies for 12 days, stained with crystal violet, and then counted. D, DGT inhibits osteosarcoma cell (U2OS/MTX, HOS, MG63, and ZOS) viability in a time- anddose-dependent manner. Osteosarcoma cells were treated with different concentrations of DGT for the indicated times, and the viability of the cells wasmeasured using MTT assays (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Zhao et al.

Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research132




osteosarcoma cells at 30% to50%confluence in 6-well plates usingLipofectamine 2000 following the instructions (Invitrogen).

RNA extraction and qRT-PCRTotal cellular RNA was extracted using the TRIzol reagent

(Invitrogen) according to the manufacturer's instructions. RNAwas reverse transcribed to produce cDNA and real-time PCRamplification was performed as described previously (19). Theprimer sequences we used are shown in Supplementary Table S1.

Luciferase assayOsteosarcoma cells were seeded in 24-well plate and cotrans-

fected with 200 ng Gli luciferase reporter and 5 ng pRL-TK Renillaluciferase construct (Promega) perwell using Lipofectamine 2000(Invitrogen). After 24 hours, the cells were treated with DGT orsiRNA (or Licl). Then, the cells were analyzed after an additional48 hours according to the Dual-Luciferase Assay System protocol(Promega).

In vitro migration and invasion assaysThe migration and invasion of osteosarcoma cells were exam-

ined using 24-well Boyden chambers with 8-mm inserts coatedwithout (migration) or with Matrigel (invasion) as describedpreviously (21). Generally, the osteosarcoma cells were treatedwith vehicle or DGT, and then, the cells were collected formigration and invasion assays. A total of 4 � 104 cells per wellwere plated on the inserts and cultured at 37�C in the upperchambers without serum. After 24 hours, the cells on the lowersurface of the filter were fixed, stained, and examined under amicroscope. The average number of migrated cells from fiverandom optical fields (�100 magnification) and triplicate filterswas determined.

IHC stainingIHC staining was performed as described previously (21).

Briefly, the primary antibody was diluted at a ratio of 1:50, andthe slides were incubatedwith primary antibody overnight at 4�C.Detection of the primary antibody was achieved by incubationwith a secondary antibody (Envision; Dako) for 1 hour at roomtemperature. The slides were stained with 3,3-diaminobenzidineafter washing in PBS again. Studies of patient sample wereapproved by the Institutional Review Board of the Sun Yat-SenUniversity.

Proteome profiler arrayThe proteome profiler array (R&D Systems) was performed

according to the manufacturer's protocol. Briefly, the cell lysateswere incubated with activated arraymembranes overnight at 4�C.Each array membrane was washed 3 times with 1� wash bufferand incubated with the diluted antibody cocktail for 2 hours atroom temperature. The array membranes were then washed 3times with 1� wash buffer and further incubated with streptavi-din–horseradish peroxidase for 30 minutes at room temperature.Each array membrane was then washed 3 times with 1� washbuffer, and finally, the membranes were exposed to X-ray filmfollowing chemiluminescent detection.

Animal experimentsThe animal experiments were approved by the Institutional

Review Board of Sun Yat-Sen University. Athymic nude (nu/nu)

mice, 5 to 6 weeks of age, were purchased from Shanghai SlacLaboratory Animal Company Limited. U2OS/MTX cells (1� 106

cells in 200 mL of PBS) were injected subcutaneously near thescapula of the nude mice. After 8 days, the mice were randomlyseparated into three groups. For the osteosarcoma xenograftgrowth of orthotopic animal model, ZOS cells were used asdescribed previously (22). After 14 days, the mice were randomlyseparated into the three groups. In both of the models, the firstgroup, the control, was treatedwith vehicle (DMSO inwater). Theother two groups were treated with DGT dissolved in the vehicle(either 150 or 300 mg/kg) every day. All the groups received thedrug through intraperitoneal injection. The resulting tumors weremeasured with a caliper every 4 days. The U2OS/MTX tumorvolume was calculated using the formula V ¼ 1/2 (width2 �length), and the ZOS tumor volume was calculated using theformula V¼ 4/3p [1/4 (D1þD2)]2. At the end of the experiment,the animals were sacrificed via cervical dislocation, and the tumorweights were measured after careful resection.

For the osteosarcoma metastasis model, mice were injectedwith 2� 106 143B cells (in 100 mL of PBS) into the lateral tail vein(6 mice/group). The mice were treated with DGT (300 mg/kg)every day and monitored 3 times per week for evidence ofmorbidity associated with pulmonary metastases. After 6 weeks,themice were sacrificed, and the lungs were harvested, fixed in 4%paraformaldehyde, and embedded in paraffin. To quantify thenumber of pulmonary metastatic lesions, sequential 3-mm thicksections of whole lungs were obtained. The sections were stainedwith H&E to identify the metastases using light microscopy asdescribed previously (23).

Statistical analysesAll data are presented as statistical plots generated using

GraphPad Prism 5. The differences between two groups weredetermined using two-tailed t tests. The differences among threeormore groupswere determined using one-wayANOVA followedby two-tailed t tests. Kaplan–Meier analysis of tumor patients andthe log-rank test were performed for comparison of the survivalcurves according to the Gli1 level. P < 0.05 was consideredstatistically significant.

ResultsDGT inhibits proliferation and affects cell-cycle progression inosteosarcoma cells

DGT (molecular weight, 1,035.1 Da), the major bioactiveconstituent of Solanum nigrum L., and its chemical structure isshown in Fig. 1A. To explore the effect of DGT on osteosarcomacells, 9 cell lines were treated with various doses of DGT for 72hours. The calculated IC50 values ranged from 12.91 to 31.46mmol/L (Fig. 1B). However, the IC50s of hfob1.19 (239.4 mmol/L)and hMSCs (536.8 and 537.2 mmol/L) are much more thanosteosarcoma cell lines (Supplementary Fig. S1). The antiproli-ferative activity of DGT was further evaluated with a colonyformation assay. Colony number and colony size were reducedin U2OS, HOS, andMG63 cells treated with DGT compared withcontrols (Fig. 1C).Moreover, DGT suppressed the proliferation ofU2OS/MTX, HOS, ZOS, and MG63 cells in a dose- and time-dependent manner. At concentration as low as 5 mmol/L, DGTinhibited cell proliferation after 3 days of treatment (Fig. 1D).Significant inhibition of cell growth was observed after 1 day at aconcentration of 40 mmol/L. These results indicate that DGT






exhibits antiproliferative activity against osteosarcoma cellsin vitro.

We examined the effect of DGT on cell-cycle progression inU2OS and MG63 cells using propidium iodide staining. Thepercentage of U2OS and MG63 cells in G2–M increased by8.6% and 15%, respectively, and the percentage of cells inG0–G1 decreased by 15.17% and 17.52%, respectively (Fig.2A). To further investigate the molecules affected by DGT, weexamined the levels of CyclinD1, which is related with G0–G1 toS-phase transition, and P21, which is a potent inhibitor of cell-cycle progression. The results showed that the expression ofCyclinD1 was decreased and P21 was increased at RNA andprotein levels, but DGT had little effect on CyclinA1 and CyclinB(Fig. 2B and C). These results indicate that DGT can induce cell-cycle G0–G1 decrease and G2–M arrest in osteosarcoma cell lines.Previous studies have demonstrated that a G2–M arrest is fre-quently the result of DNA damage, and phospho-H2AX is amarker of DNA double-stranded breaks and can therefore beused to monitor DNA repair (24). Therefore, we performed aphospho-H2A.X immunostaining analysis of U2OS cells andfound that the proportion of phospho-H2A.Xþ cells and thedistribution of phospho-H2A.Xþ foci per nucleus were obviouslyincreased (Fig. 2D). These results suggest that DGT treatment notonly affected cell-cycle progression but also inducedDNAdamagein osteosarcoma cells.

DGT induces apoptosis in osteosarcoma cellsIncreased phospho-H2A.X expression in the nucleus indicates

that cells repair the damaged DNA, which usually results in celldeath or apoptosis (25). Therefore, we sought to determinewhether treatment with DGT could induce osteosarcoma cellapoptosis. U2OS/MTX and ZOS cells were treated with DGT for48 hours at different concentrations, and Western blotting wasused to assess the expression of cleaved PARP, a marker ofapoptosis, which was increased after exposure to DGT (Fig.3A). As shown in Fig. 3B, caspase-3 activity assays were alsoperformed by extracting cellular protein from DGT-treatedU2OS/MTX and ZOS cells, and these assays indicated that cas-pase-3 activity was significantly increased (e.g., caspase-3 activityin U2OS/MTX and ZOS cells treated for 48 hours with 40 mmol/Lwas approximately 3.44 and 4.12-fold greater compared withuntreated control cells). Osteosarcoma cell apoptosis induced byDGT was further analyzed with Hoechst 33258 staining and flowcytometry assays to detect the morphologic changes and analyzethe proportion of apoptotic cells. Brighter blue staining andmoremorphologic changes were found in nuclear chromatin of MG63,U2OS/MTX, and ZOS cells following the DGT treatments for48 hours. Typical morphologic characteristics of apoptosis, suchas reduction in nuclear size, cell pyknosis, and chromatin con-densation, weremore easily observed in DGT-treated cells than incells treated with vehicle (Fig. 3C). Annexin V/PI staining of thesecells demonstrated a significant increase in apoptotic cells fol-lowing DGT treatment (Fig. 3D). Conclusively, all the aboveresults indicated that the cytotoxicity of DGT occurs throughinduction of apoptosis in osteosarcoma cells.

DGT reduces the growth of osteosarcoma xenografts in nudemice

Subsequent to finding that DGT inhibits proliferation andinduces apoptosis of osteosarcoma cells in vitro, we further eval-uated its effectiveness in inhibiting the growth of osteosarcoma in

nude mice. First, U2OS/MTX cells were subcutaneously injectedinto nude mice until a tumor volume of approximately 200 mm3

was reached. Themice were randomly separated into three groups(control, dose 1, and dose 2). The dose 1 group received 150 mg/kg of DGT every day, and the dose 2 group received 300mg/kg ofDGT every day. At the termination of the study, statistical analysisof tumor growth revealed a significant reduction in tumor size inmice treated with DGT. The mean volumes of the tumors were1,273.78 mm3 for the control group, 802.24 mm3 for the dose 1group (P < 0.01), and 540.03 mm3 for the dose 2 group (P <0.001; Fig. 4A). The average tumor weights were 1.248 g for thecontrol group, 0.754 g for the dose 1 group (P < 0.001), and 0.597g for the dose 2 group (P < 0.001; Fig. 4B). Next, we investigatedwhether DGT could inhibit osteosarcoma growth using an in vivoorthotopic osteosarcomamodel, as described in theMaterials andMethods section. As shown in Fig. 4C, notably, the growth ofDGT-treated groups was much slower than that of the controlgroup, consistent with the tumor growth curve. At the 42nd day,themean volumes of the tumors were 928.37mm3 for the controlgroup, 731.30 mm3 for the dose 1 group (P < 0.05), and 549.41mm3 for the dose 2 group (P<0.01). The data also show that thereis a concentration-dependent effect of DGT on tumor volume. Inaddition, in both of the two models, the average body weights ofthemice were not significantly different between the DGT-treatedgroups and the control group, andnoobvious pathologic changeswere observed in vital organs (heart, liver, kidney, and lung) asdetected by hematoxylin and eosin (H&E) staining (Supplemen-tary Figs. S2 and S3). H&E staining also showed that bonedestruction around the tibia was more obvious in the controlgroup (Fig. 4D). We found that DGT could induce apoptosis inosteosarcoma cells in vitro. Here, we analyzed the level of apo-ptosis in tumor tissue using a TUNEL assay, and the resultsshowed that DGT could induce apoptosis in vivo (Fig. 4E).Collectively, these results demonstrate that DGT possesses anti-tumor properties and can induce apoptosis in human osteosar-coma cells in vivo.

DGT suppresses osteosarcoma cell metastatic potential both invitro and in vivo

Tumor growth, metastasis, and invasion are key processesduring tumor development and progression, and we found thatDGT could inhibit the growthof osteosarcoma.Wenextwanted toassess whether DGT could reduce the migratory and invasiveproperties of osteosarcoma cells. Notably, DGT treatment of143B cells significantly reduced cell invasion (Fig. 5A and B) andmotility through Matrigel (Fig. 5C and D). Importantly, suppres-sion of cellmigration and invasionwas not due to reduced overallcell number, as equal numbers of cells were reseeded into thewells after the pretreatment period, andmigrationor invasionwasassessed within 24 hours. To investigate the ability of DGT toprevent osteosarcoma lung metastasis in vivo, we injected 143Bcells into the tail vein of nude mice as described previously (26).In addition, the mice were randomly separated into two groupsafter 2weeks; the control group received vehicle every day, and thetreatment group received 300 mg/kg of DGT every day. After 5weeks, the lungs were harvested, and micrometastases were ana-lyzed. Consistent with the in vitro results, DGT treatment dramat-ically diminished the occurrence of osteosarcoma xenograftmetastasis to the lungs, as indicated by the number of metastaticnodules in the lung and the wet lung weight (P < 0.001) (Fig. 5Eand F; Supplementary Fig. S4). Collectively, these results

Zhao et al.





Figure 2.

The effect of DGT on cell-cycle progression in osteosarcoma cells. A, Representative cell-cycle analysis of U2OS and MG63 cells treated with vehicle or DGTas indicated for 48 hours using flow cytometry; the length of each cell-cycle phase was calculated. B, DGT affects the mRNA levels of the cell cycle–related genesCyclinD1 and P21. Osteosarcoma cells were treated with the indicated concentrations of DGT for 48 hours, and total RNA was extracted and examined todetermine mRNA levels using quantitative RT-PCR after normalization to GAPDH expression. C, Western blot analyses were performed using the indicatedantibodies, including CyclinD1, P21, CyclinA1, CyclinB1, and p-H2A.X. D, Confocal fluorescence microscopy was performed to examine p-H2A.X in U2OScells after treatment with vehicle or 20 mmol/L DGT for 48 hours (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






demonstrate that DGT inhibits osteosarcoma metastasis in vitroand in vivo.

DGT inhibits osteosarcoma growth and metastasis throughrepression of the HH/Gli1 pathway

TheproteinGli1 functions as adownstream transcription factorof HH signaling and plays a pivotal role in cellular growth andmetastasis of many types of tumors (27–29). However, thefunction of Gli1 in osteosarcoma remains unclear. First, to inves-tigatewhether Gli1 is overexpressed in osteosarcoma cell lines, wecompared the protein expression in osteosarcoma cell lines and

hfob1.19 cells. We found that most of the osteosarcoma cell lineshave higher expression of Gli1, but other related genes of HHsignaling, such as PTCH, Smo, and SHH, were not overexpressed(Fig. 6A). The increased expression ofGli1 has been reported to beclinically correlated with unfavorable overall prognosis in mostsolidmalignancies (28, 30–32).WealsodeterminedwhetherGli1expression was associated with the clinical outcome of patientswith osteosarcoma. In total, 71 patient samples were collected forIHC using Gli1 antibody. Among them, Gli1 protein was stronglyexpressed in 66.2% of the samples (Supplementary Fig. S5A andS5B), and Kaplan–Meier survival analysis indicated that patients

Figure 3.

DGT induces apoptosis in osteosarcoma cells. A, DGT induces PARP cleavage in osteosarcoma cells. Whole-cell extracts from treated cells were analyzed byWestern blotting using the indicated antibodies. B, Caspase-3 activity assays were performed in U2OS/MTX and ZOS cells after treatment with DGT using theindicated dose for 48hours.C,Hoechst staining showed typical apoptoticmorphology changes after 20mmol/LDGT treatment for 48 hours inMG63, U2OS/MTX, andZOS cells. D, Cells were treated with DGT for 48 hours and then subjected to apoptosis analysis using flow cytometry and Annexin V/PI staining (error bars,SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Zhao et al.





with high Gli1 expression levels had worse overall survival thanthose with low expression levels (Supplementary Fig. S5C).

We subsequently investigated whether Gli1 affected thegrowth and metastasis of osteosarcoma. We stably knockeddown Gli1 (using two different shRNAs against Gli1) in oste-osarcoma cell lines and demonstrated that Gli1 silencing in thecells reduced proliferation, sphere formation, migration, andinvasion (Fig. 6B–F). In addition, inhibition of Gli1 decreasedthe expression of HH target genes, including Gli1, Sox2, OCT4,PTCH1, and Myc, and increased the expression of P21 (Fig.6G). These data suggested that Gli1 expression is both neces-sary and sufficient to promote osteosarcoma cell proliferation,migration, and invasion.

To determine whether DGT prevents Gli1 transcription andexpression in osteosarcoma cells, Western blotting and a Gli1luciferase activity assay were performed. The protein level of Gli1

was decreased in a dose-dependent manner after treatment withDGT inU2OS andZOS cells (Fig. 6H). TheGli1 luciferase reporteractivity was decreased by 40% after treatment with DGT inosteosarcoma cells (Fig. 6I). In addition, the Gli1 target genes,including Gli1, PTCH1, and N-myc, were decreased in dose-dependent manner after treatment with DGT in U2OS cells(Supplementary Fig. S6). Next, we explored whether the inhib-itory effect of DGT on osteosarcoma metastasis and growthdepends on HH/Gli1 signaling. Osteosarcoma cells were trans-fected with Gli1 plasmid or empty vector. These cells were thentreated with or without DGT, and growth and metastasis wereanalyzed. As shown in Fig. 6J, treatment with DGT led to down-regulation of endogenous and exogenous Gli1 expression, andoverexpression of Gli1 enhanced the metastatic ability of ZOScells, which was partly recovered when combined with DGTtreatment. The abovementionedfindings indicated thatGli1 plays

Figure 4.

DGT inhibits the growth of osteosarcoma cells in nude mice. A and B, Examination of tumor volumes and weights to evaluate the effect of DGT (dose 1, 150 mg/kg;dose 2, 300 mg/kg) on U2OS/MTX cells in a xenograft model. The mice bearing U2OS/MTX cells were treated as described in Materials and Methods. Thetumor volumesweremonitored as indicated, and the tumors were excised and weighed on the 24th day. The data represent the mean� SD of the volume of tumorsfrom 6 mice. C, Use of DGT (dose 1, 150 mg/kg; dose 2, 300 mg/kg) to treat ZOS cells in an orthotopic mouse model as described in Materials and Methods.The tumor volumesweremonitored every 4 days, as indicated.D and E, The bone destruction caused by the tumor inCwas detected using H&E staining. E,ATUNELassay was performed to analyze apoptosis in the DGT-treated tumors shown in A and C (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






a key role inDGT-induced growth andmetastasis, which is a goodprognosis factor for osteosarcoma.

DGT decreases the activities of multiple intracellular kinasesthat affect the survival of osteosarcoma patients

It has been demonstrated that many kinases play key roles incancer cell survival and growth, and inhibiting these kinasescan reduce cancer growth and metastasis. To examine the effectsof DGT on intracellular signaling, we screened the phosphor-ylation status of multiple cellular kinases in U2OS cells treatedwith vehicle or DGT using a human phospho-kinase antibodyarray. As shown in Fig. 7A, the phosphorylation levels ofmultiple kinases were altered after treatment with DGT, includ-ing Akt, ERK, GSK3b, and mTOR, whose expression changed 3

to 15-fold. To confirm the result, we tested the changes in thesekinases using Western blotting in osteosarcoma cells, includingU2OS, MG63, HOS, ZOS, and 143B cell lines, treated with 20mmol/L DGT for 48 hours. We found that the expression ofphospho-Akt and phospho-ERK was decreased, and phospho-GSK3b was increased (Fig. 7B). To confirm the results observedin the Western blot analysis, we conducted IHC analyses of theselected proteins. The results indicated that the DGT-treatedtumor tissues expressed decreased Gli1, phospho-Akt, andphospho-ERK but increased phospho-GSK3b (SupplementaryFig. S7). Our previous studies regarding the role of GSK3bshowed that increased phospho-GSK3b induces apoptosis andinhibits the growth of osteosarcoma (22). In addition, the levelof b-catenin was also decreased, which is associated with

Figure 5.

DGT suppresses osteosarcoma migration, invasion, and metastasis. A and B, DGT inhibits the migration ability of osteosarcoma cells. The indicated cells weretreated with or without DGT (10 mmol/L, 20 mmol/L), and cell migration was determined as described in Materials and Methods. C and D, DGT inhibits theinvasion ability of osteosarcoma cells. The indicated cells were treatedwith or without DGT (10 mmol/L, 20 mmol/L), and cell invasionwas determined as described inMaterials and Methods. E and F, DGT inhibits osteosarcoma cell metastasis to the lungs in nude mice in vivo. DGT treatment (300 mg/kg) decreased the rateof lung metastasis after tail-vein injection of 143B cells (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).

Zhao et al.





Figure 6.

DGT inhibits osteosarcoma growth and metastasis through repression of the HH/Gli1 pathway. A, The Gli1 protein level was visualized in the indicatedcell lines using a Western blot analysis. B, The effect of Gli1 shRNA and Sox2 expression was confirmed by Western blot analysis. C–F, The effect of Gli1deletion using shRNA on colony formation, sphere formation, migration, and invasion was analyzed. G, HH target genes, including Gli1, Sox2, OCT4,Nanog, PTCH1, Myc, and P21, in Gli1-deleted U2OS and ZOS cells were measured using qPCR. H, The Gli1 protein level was visualized in U2OS and ZOS cellstreated with DGT. I, U2OS and ZOS cells were transfected with a luciferase reporter for Gli1, treated with or without DGT (20 mmol/L) for 48 hours,and subjected to luciferase assays as described in Materials and Methods. J, The impairment of Gli1 expression and migration ability induced byDGT was rescued in ZOS cells stably overexpressing Gli1 (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






osteosarcoma growth and metastasis (33–35). To determinewhether Akt, ERK, GSK3b, and b-catenin repression was drivingDGT cytotoxicity, U2OS cells were treated with DGT aftersiRNA-mediated knockdown of these proteins. The results ofMTT assays show that inhibition of b-catenin slightly decreasedcell viability. However, knockdown of Akt induced greaterosteosarcoma cell death, and similar effects were seen in cellswith GSK3b knockdown (Fig. 7C). Next, we induced the siRNA-mediated knockdown of these kinases and then treated thesecells with vehicle or DGT. When combined with DGT treat-ment, we found that the cells with siRNA-mediated knockdownof b-catenin exhibited more cell death compared with cellstreated with vehicle (Fig. 7D). This result suggested that Akt

and GSK3b play a more important role in the survival ofosteosarcoma cells. Together, these data strongly support theconclusion that inhibition of Akt/ERK- and GSK3b-mediatedapoptosis is an important mechanism of DGT-induced cellularcytotoxicity.

DGT downregulates Gli1 expression and activation mainly byblocking GSK3b activity

Gli1 can be regulated by specific serine/threonine kinases(34, 36–38), and we have demonstrated that DGT inhibitsGSK3b activity, which are known to be associated with oste-osarcoma progression and poor patient outcome. Therefore, wehypothesized that GSK3b activity is involved in DGT-induced

Figure 7.

DGT decreases the activities of multipleintracellular kinases that affect thesurvival of osteosarcoma patients.A, Cell lysates of control and DGT (20mmol/L)-treated U2OS cells wereapplied to the proteome profilerantibody array analysis as described inMaterials and Methods. The antibodyarray was composed of duplicate spotsfor each kinase on a single membrane.B, Cell lysates of control and DGT-treated U2OS, MG63, HOS, ZOS, and143B cells were analyzed with Westernblotting using various antibodies, asindicated. C, Cell viability was analyzedusing MTT assays in U2OS cellstransfected with siRNAs as indicated.D, U2OS cells transfected with siRNAswere treated with DGT (20 mmol/L), andcell viability was measured with MTTassays (error bars, SD; � , P < 0.05;�� , P < 0.01; �� , P < 0.001).

Zhao et al.





Gli1 repression in osteosarcoma. Consistent with this hypoth-esis, we treated osteosarcoma cells with siRNA and Licl (aspecific inhibitor targeting GSK3b), and the results showedthat blocking GSK3b downregulates Gli1 expression but notsmo and inhibits Gli1 luciferase activity in ZOS cells (Fig. 8Aand B). Furthermore, we investigated whether Gli1 also plays akey role in GSK3b-mediated osteosarcoma malignant beha-viors, because we have demonstrated that GSK3b prompts thegrowth of osteosarcoma by activating the NF-kB pathway. Wenext transfected GSK3b knockdown ZOS cells with vector orectopic Gli1 expression plasmid and assessed the changes inmalignant behaviors, including the cell viability, metastasis,and sphere formation. Our results showed that ectopic Gli1expression could partially increase cell viability, invasion, andsphere formation impaired by knockdown of GSK3b in ZOScells (Fig. 8C–H). In addition, DGT impaired the increased cellviability induced by ectopic Gli1 and GSK3b expression (Fig.8I). Thus, the inhibition of the Gli1 pathway induced by DGTprimarily involves repression of GSK3b activity.

DiscussionIn this study, we report the effect of the natural compoundDGT

from Solanum nigrum L. on osteosarcoma, which is the first steptoward new drug development according to the current challengeof osteosarcoma treatment. Numerous researchers have focusedon natural extracts owing to the success of artemisinin (qin-ghaosu) and arsenic (III) oxide (As2O3) in the clinic. Owing totheir safety, long-term use, and their ability to target multiplepathways, there is a renewed interest in understanding themolec-ular mechanisms underlying their activity. Our previous studieshave also provided evidence of some natural agents havingpotential anti-osteosarcoma activity, such as dihydromyricetin,cinobufagin, and bufalin (19, 39, 40). However, there is noevidence at the cellular level or in animal models for such aneffect of DGT on osteosarcoma progression. The study presentedhere indicated that DGT diminishes the growth and metastasis ofosteosarcoma, which may be a promising therapeutic strategyagainst osteosarcoma in vitro and in vivo without obvious sideeffects.

In this study, all the osteosarcoma cells were susceptible tothe cytotoxicity of DGT, and the ability of DGT to preventcolony formation and proliferation was remarkable and dosedependent. The results of the cell-cycle assay showed that DGTcould induce G2–M cell-cycle arrest and increase p21 levels inosteosarcoma cells. The histone H2A.X, which is the goldstandard for early detection of DNA damage, results in cell-cycle arrest and/or apoptosis (41). We found that DGT treat-ment not only caused significant DNA damage, as detected bythe change in thep-H2A.X level, but also induced apoptosis ofosteosarcoma cells in dose-dependent manner, as detected byPARP, caspase-3 activity, and Annexin V/PI staining. We alsoreported that DGT had antitumor potential in nude mice,including an orthotopic model, without severe side effects.Pulmonary metastasis is the primary cause of medical therapyfailure and death in osteosarcoma patients. Given that con-trolling metastasis is the key to improving survival, there is anurgent need to develop more effective approaches to suppresslung metastasis. Therefore, we investigated the effect of DGT onlung metastasis of osteosarcoma. In vitro and in vivo, we foundthat DGT inhibited the migration, invasion, and metastasis of

osteosarcoma cells. These results encouraged us to investigatethe potential anti-osteosarcoma mechanism of DGT, which willbe beneficial for developing clinical trials in the future.

The HH signal pathway is also considered to be cruciallyinvolved in the development and progression of many cancersbecause it is overactivated and correlated with growth andmetastasis (36, 42, 43). Activated Gli proteins, primarily Gli1,translocate into the nucleus and stimulate the transcription ofHH pathway target genes, including Gli1, PTCH1, and manysurvival-promoting molecules (44–46). Here, we demonstratedthat Gli1 is overexpressed in osteosarcoma cell lines and ispositively associated with cell survival and metastasis in oste-osarcoma and thus presents a promising target. In addition, theGli1 protein is strongly expressed in 66.2% (47/71) of clinicalsamples and is correlated with the prognosis of osteosarcomapatients. Most importantly, we found that DGT prevents theexpression of Gli1 in osteosarcoma cells in a dose-dependentmanner, and Gli1 luciferase reporter activity was decreased aftertreatment with DGT. When combining DGT with Gli1-over-expressing osteosarcoma cells, the growth and metastatic abil-ities prompted by Gli1 were partially reversed. These findingssuggest that Gli1 is a significant prognostic marker and thatDGT inhibits growth and metastasis by inactivating the HH/Gli1 pathway.

Because many kinases play key roles in cancer cell survivaland growth, to further determine the potential anti-osteosar-coma molecular mechanism of DGT, we assessed the phos-phorylation levels of multiple kinases using a proteome profilerarray in DGT-treated osteosarcoma cells, especially the changein GSK3b. Our previous study showed that GSK3b activity maypromote osteosarcoma tumor growth, and therapeutic target-ing of GSK3b may be an effective way to enhance the thera-peutic activity of anticancer drugs against osteosarcoma (22).Our results show that DGT inhibits activation of GSK3b byincreasing the phosphorylation level. Gli1 has been reported tobe activated by many kinases, such as AKT, MAPK/ERK, andmTOR/S6K1 (42, 47). In this study, we were able to show a linkbetween GSK3b and Gli1. Targeting GSK3b with siRNA or Licldownregulated Gli1 expression but not smo, and it inhibitedGli1 luciferase activity in osteosarcoma cells. In addition, Gli1plays a key role in GSK3b-mediated osteosarcoma malignantbehaviors, because ectopic Gli1 partially increased the viability,invasion, and sphere formation impaired by knockdown ofGSK3b in ZOS cells. On the basis of our results, we suggest thatDGT possesses antitumor activity due to its ability to affect Gli1expression and activation by blocking GSK3b. However, howDGT inhibits GSK3b activity and represses the HH/Gli1 path-way requires further research.

In summary, the results from our cell-based and in vivostudies support the apoptosis-inducing, antiproliferative,anti-invasive, and antimetastatic activities of DGT. The under-lying mechanism by which DGT exhibits anti-osteosarcomaactivity seems to be through inhibition of GSK3b/Gli1 activa-tion. The efficacy of the inhibition of growth and metastasis ofthe osteosarcoma xenograft at relatively low concentrationsstrengthens the therapeutic value of DGT. Because osteosarco-ma is an extremely aggressive type of cancer that lacks anytargeted therapies, this study provides strong evidence forevaluating the safety and efficacy of DGT in clinical studies,and it may be an excellent auxiliary drug for treating patientswith osteosarcoma.






Figure 8.

DGT inhibits the HH/Gli1 pathway primarily by blocking GSK3b activity in osteosarcoma cells. A and B, Western blotting analysis was used to detect Gli1protein in U2OS cells with GSK3b inactivation using siRNA or Licl. C and D,Gli1 luciferase reporter assayswere performed in U2OS cells treated with GSK3b siRNA orLicl. The U2OS cells were transfected with Gli1 luciferase reporter for 24 hours. The cells were treated with or without DGT (20 mmol/L) for 48 hours and thensubjected to luciferase assays as described in Materials and Methods E, Cell viability was analyzed in Gli1-overexpressing U2OS cells transfected withGSK3b siRNAs using MTT assays as indicated. F–H, Cell migration and sphere formation were analyzed in Gli1-overexpressing U2OS cells transfected withGSK3b siRNAs. I, Cell viability was analyzed in Gli1- or GSK3b-overexpressing U2OS cells using MTT assays and compared with that of vector-transfectedcontrol cells (error bars, SD; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).


Zhao et al.




Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe funders had no role in study design, data collection and analysis,

decision to publish, or preparation of the manuscript.

Authors' ContributionsConception and design: Z. Zhao, Q. Jia, M.-S. Wu, J.-Q. Yin, J. ShenDevelopment of methodology: M.-S. Wu, J.-Q. YinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Zhao, Q. Jia, M.-S. Wu, X. Xie, Y. Wang, D.-C. Lin,J. ShenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Zhao, G. Song, C.-Y. Zou, Q. Tang, J. Lu, J. ShenWriting, review, and/or revision of the manuscript: Z. Zhao, M.-S. Wu, J. Shen

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Jia, G. Huang, J. Wang, J. ShenStudy supervision: H.P. Koeffler, J.-Q. Yin, J. Shen

AcknowledgmentsThis work was supported by grants from National Natural Science Founda-

tion of China (no. 81602356, 81560603, and no. 81472506); GuangdongNatural Science Foundation (S2013010016847), Sun Yat-Sen University Clin-ical Research 5010 Program (no. 200709), and Young teachers cultivatingproject of Sun Yat-Sen University (no. 14ykpy16).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMarch10, 2017; revised July 23, 2017; accepted September 20, 2017;published OnlineFirst September 26, 2017.

References1. Biermann JS, AdkinsDR, AgulnikM, Benjamin RS, BrigmanB, Butrynski JE,

et al. Bone cancer. J Natl Compr Canc Netw 2013;11:688–723.2. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival

rates from 1973 to 2004: data from the Surveillance, Epidemiology, andEnd Results Program. Cancer 2009;115:1531–43.

3. Sluga M, Windhager R, Pfeiffer M, Ofner P, Lang S, Dominkus M, et al.[Osteosarcoma and Ewing's sarcoma–The most frequent malignant bonetumors in children–therapy and outcome]. Z Orthop Ihre Grenzgeb2002;140:652–5.

4. Gupta SC, Prasad S, Sethumadhavan DR, Nair MS, Mo YY, Aggarwal BB.Nimbolide, a limonoid triterpene, inhibits growth of human colorectalcancer xenografts by suppressing the proinflammatorymicroenvironment.Clin Cancer Res 2013;19:4465–76.

5. Bai X, Cerimele F, Ushio-Fukai M, Waqas M, Campbell PM, GovindarajanB, et al. Honokiol, a small molecular weight natural product, inhibitsangiogenesis in vitro and tumor growth in vivo. J Biol Chem 2003;278:35501–7.

6. Zhang R, Gilbert S, Yao X, Vallance J, Steinbrecher K, Moriggl R, et al.Natural compoundmethyl protodioscin protects against intestinal inflam-mation through modulation of intestinal immune responses. PharmacolRes Perspect 2015;3:e00118.

7. Tu Y.The discovery of artemisinin (qinghaosu) and gifts from Chinesemedicine. Nat Med 2011;17:1217–20.

8. Jeong JB, JeongHJ, Park JH, Lee SH, Lee JR, Lee HK, et al. Cancer-preventivepeptide lunasin from Solanum nigrum L. inhibits acetylation of corehistones H3 and H4 and phosphorylation of retinoblastoma protein (Rb).J Agri Food Chem 2007;55:10707–13.

9. Lai YJ, Tai CJ, Wang CW, Choong CY, Lee BH, Shi YC, et al. Anti-canceractivity of solanum nigrum (AESN) through suppression of mitochondrialfunction and epithelial-mesenchymal transition (EMT) in breast cancercells. Molecules 2016;21:553.

10. Nawab A, Thakur VS, Yunus M, Ali Mahdi A, Gupta S. Selective cell cyclearrest and induction of apoptosis in human prostate cancer cells by apolyphenol-rich extract of Solanum nigrum. Int J Mol Med 2012;29:277–84.

11. Razali FN, Sinniah SK,HussinH, AbidinNZ, Shuib AS. Tumor suppressioneffect of Solanumnigrum polysaccharide fraction on breast cancer viaimmunomodulation. Int J Biol Macromol 2016;92:185–93.

12. Chakraborty D, Maity A, Jha T, Mondal NB. Spermicidal and contraceptivepotential of desgalactotigonin: a prospective alternative of nonoxynol-9.PLoS One 2014;9:e107164.

13. Santoni M, Burattini L, Nabissi M, Morelli MB, Berardi R, Santoni G, et al.Essential role ofGli proteins in glioblastomamultiforme.Curr ProteinPeptSci 2013;14:133–40.

14. Archer TC,Weeraratne SD, Pomeroy SL. Hedgehog-GLI pathway inmedul-loblastoma. J Clin Oncol 2012;30:2154–6.

15. Hahn H, Wojnowski L, Zimmer AM, Hall J, Miller G, Zimmer A. Rhabdo-myosarcomas and radiation hypersensitivity in a mouse model of Gorlinsyndrome. Nat Med 1998;4:619–22.

16. NilssonM,UndenAB, KrauseD,Malmqwist U, Raza K, Zaphiropoulos PG,et al. Induction of basal cell carcinomas and trichoepitheliomas in miceoverexpressing GLI-1. Proc Natl Acad Sci U S A 2000;97:3438–43.

17. Cheng J, Gao J, Tao K, Yu P. Prognostic role of Gli1 expression in solidmalignancies: a meta-analysis. Sci Rep 2016;6:22184.

18. Meng F, He A, Zhang Z, Lin Z, Yang Z, Long Y, et al. Chondrogenicdifferentiation of ATDC5 and hMSCs could be induced by a novel scaf-fold-tricalcium phosphate-collagen-hyaluronan without any exogenousgrowth factors in vitro. J Biomed Mat Res Part A 2014;102:2725–35.

19. Zhao Z, Yin JQ, Wu MS, Song G, Xie XB, Zou C, et al. DihydromyricetinactivatesAMP-activated protein kinase andP38(MAPK) exerting antitumorpotential in osteosarcoma. Cancer Prev Res 2014;7:927–38.

20. Sun H, Lin DC, Cao Q, Guo X, Marijon H, Zhao Z, et al. CRM1 inhibitionpromotes cytotoxicity in Ewing sarcoma cells by repressing EWS-FLI1-dependent IGF-1 signaling. Cancer Res 2016;76:2687–97.

21. Zhao Z,WuMS, ZouC, TangQ, Lu J, Liu D, et al. Downregulation ofMCT1inhibits tumor growth,metastasis and enhances chemotherapeutic efficacyin osteosarcoma through regulation of the NF-kappaB pathway. CancerLett 2014;342:150–8.

22. TangQL, Xie XB,Wang J, ChenQ,Han AJ, ZouCY, et al. Glycogen synthasekinase-3beta, NF-kappaB signaling, and tumorigenesis of human osteo-sarcoma. J Nat Cancer Inst 2012;104:749–63.

23. Su Y, Luo X, He BC, Wang Y, Chen L, Zuo GW, et al. Establishment andcharacterization of a new highly metastatic human osteosarcoma cell line.Clin Exp Metastasis 2009;26:599–610.

24. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechan-isms of mammalian DNA repair and the DNA damage checkpoints. AnnuRev Biochem 2004;73:39–85.

25. Jackson SP, Bartek J. The DNA-damage response in human biology anddisease. Nature 2009;461:1071–8.

26. Lu J, Song G, Tang Q, Zou C, Han F, Zhao Z, et al. IRX1 hypomethylationpromotes osteosarcoma metastasis via induction of CXCL14/NF-kappaBsignaling. J Clin Invest 2015;125:1839–56.

27. Inaguma S, Kasai K, Ikeda H. GLI1 facilitates themigration and invasion ofpancreatic cancer cells through MUC5AC-mediated attenuation of E-cad-herin. Oncogene 2011;30:714–23.

28. Marechal R, Bachet JB, Calomme A,Demetter P, Delpero JR, SvrcekM, et al.Sonic hedgehog and Gli1 expression predict outcome in resected pancre-atic adenocarcinoma. Clin Cancer Res 2015;21:1215–24.

29. Szkandera J, PichlerM, Absenger G, StotzM,WeissmuellerM, SamoniggH,et al. A functional germline variant in GLI1 implicates hedgehog signalingin clinical outcomeof stage II and III colon carcinomapatients. Clin CancerRes 2014;20:1687–97.

30. Wang Y, Sun C, Jiang J, Xie Y, Li B, Cui X, et al. GLI1 expression is animportant prognostic factor that contributes to the poor prognosis ofrhabdomyosarcoma. Histol Histopathol 2016;31:329–37.

31. Zhang J, TuK, YangW, Li C, Yao Y, Zheng X, et al. Evaluation of Jagged2 andGli1 expression and their correlation with prognosis in human hepato-cellular carcinoma. Mol Med Rep 2014;10:749–54.






32. Liu X, Wang X, Du W, Chen L, Wang G, Cui Y, et al. Suppressor of fused(Sufu) represses Gli1 transcription and nuclear accumulation, inhibitsglioma cell proliferation, invasion and vasculogenic mimicry, improvingglioma chemo-sensitivity and prognosis. Oncotarget 2014;5:11681–94.

33. Haydon RC, Deyrup A, Ishikawa A, Heck R, Jiang W, Zhou L, et al.Cytoplasmic and/or nuclear accumulation of the beta-catenin protein isa frequent event in human osteosarcoma. Int J Cancer 2002;102:338–42.

34. Wu J, Liao Q, He H, Zhong D, Yin K. TWIST interacts with beta-cateninsignaling on osteosarcoma cell survival against cisplatin. Mol Carcinog2014;53:440–6.

35. Zhang F, Chen A, Chen J, Yu T, Guo F. SiRNA-mediated silencing of beta-catenin suppresses invasion and chemosensitivity to doxorubicin in MG-63 osteosarcoma cells. Asian Pac J Cancer Prev 2011;12:239–45.

36. Stecca B, Mas C, Clement V, Zbinden M, Correa R, Piguet V, et al.Melanomas require HEDGEHOG-GLI signaling regulated by interactionsbetween GLI1 and the RAS-MEK/AKT pathways. Proc Natl Acad Sci U S A2007;104:5895–900.

37. Zhou J, Zhu G, Huang J, Li L, Du Y, Gao Y, et al. Non-canonical GLI1/2activation by PI3K/AKT signaling in renal cell carcinoma: A novel potentialtherapeutic target. Cancer Lett 2016;370:313–23.

38. Liang H, Zheng QL, Fang P, Zhang J, Zhang T, Liu W, et al. Targeting thePI3K/AKT pathway via GLI1 inhibition enhanced the drug sensitivity ofacute myeloid leukemia cells. Sci Rep 2017;7:40361.

39. Yin JQ, Wen L, Wu LC, Gao ZH, Huang G, Wang J, et al. The glycogensynthase kinase-3beta/nuclear factor-kappa B pathway is involved in

cinobufagin-induced apoptosis in cultured osteosarcoma cells. ToxicolLett 2013;218:129–36.

40. Xie XB, Yin JQ, Wen LL, Gao ZH, Zou CY, Wang J, et al. Critical role of heatshock protein 27 in bufalin-induced apoptosis in human osteosarcomas: aproteomic-based research. PLoS One 2012;7:e47375.

41. Fernandez-Capetillo O, Chen HT, Celeste A, Ward I, Romanienko PJ,Morales JC, et al. DNA damage-induced G2-M checkpoint activation byhistone H2AX and 53BP1. Nat Cell Biol 2002;4:993–7.

42. Wang Y, Ding Q, Yen CJ, Xia W, Izzo JG, Lang JY, et al. The crosstalkof mTOR/S6K1 and Hedgehog pathways. Cancer Cell 2012;21:374–87.

43. Das S, Harris LG, Metge BJ, Liu S, Riker AI, Samant RS, et al. Thehedgehog pathway transcription factor GLI1 promotes malignantbehavior of cancer cells by up-regulating osteopontin. J Biol Chem2009;284:22888–97.

44. Jiang J, Hui CC. Hedgehog signaling in development and cancer. Dev Cell2008;15:801–12.

45. Ruiz i Altaba A.Gli proteins and Hedgehog signaling: development andcancer. Trends Genet 1999;15:418–25.

46. Ng JM, Curran T. The Hedgehog's tale: developing strategies for targetingcancer. Nat Rev Cancer 2011;11:493–501.

47. Katoh Y, Katoh M. Integrative genomic analyses on GLI1: positive regu-lation of GLI1 by Hedgehog-GLI, TGFbeta-Smads, and RTK-PI3K-AKTsignals, and negative regulation of GLI1 by Notch-CSL-HES/HEY, andGPCR-Gs-PKA signals. Int J Oncol 2009;35:187–92.


Zhao et al.




2018;24:130-144. Published OnlineFirst September 26, 2017.Clin Cancer Res Zhiqiang Zhao, Qiang Jia, Man-Si Wu, et al.

Mediated Repression of the Hedgehog/Gli1 Pathway−Inactivation βInhibits Growth and Metastasis of Osteosarcoma through GSK3

.,Solanum nigrum LDegalactotigonin, a Natural Compound from

Updated version

10.1158/1078-0432.CCR-17-0692doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/09/26/1078-0432.CCR-17-0692.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/24/1/130.full#ref-list-1

This article cites 47 articles, 11 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/24/1/130To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-0692

http://clincancerres.aacrjournals.org/content/suppl/2017/09/26/1078-0432.CCR-17-0692.DC1

http://clincancerres.aacrjournals.org/content/24/1/130.full#ref-list-1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/24/1/130


degalactotigonin, a natural compound from solanumnigruml ...osteosarcoma cell viability in vitro....

Documents