| BackgroundLung cancer remains one of the most common leading causes of cancer-related deaths worldwide because of its high incidence and mortality rates among all cance.Non-small-cell lung cancer(NSCLC),including squamous carcinoma,adenocarcinoma,and large-cell carcinoma,accounts for approximately 85%of all lung cancer cases.Chemotherapy is the main treatment for cancer because most patients have advanced disease at diagnosis.However,drug resistance and early metastasis limit the clinical efficacy of chemotherapy.Therefore,one promising approach to reduce lung cancer mortality is the identification of preneoplastic lesions and administration of chemopreventive agents to reverse,suppress,or arrest lung tumorigenesis.Wingless-type protein(Wnt)/?-catenin alterations,which are prominent in NSCLC,play critical roles in tumor prognosis and drug resistance.Thus,Wnt/?-catenin is an attractive target for a NSCLC prevention agent.Wnt binds to members of the Frizzled(FZD)family of receptors,forming a stable receptor complex among Wnt,FZD,lipoprotein receptor-related protein(LRP),dishevelled(Dvl),and scaffold protein axis inhibition protein(Axin).This complex phosphorylates Dvl,which inhibits the phosphorylation/proteolytic destruction of P-catenin.Hence,increasing cytoplasmic levels of ?-catenin resulted in ?-catenin migrating to the nucleus,complexing with members of transcription factors,and activating the transcription of a number of downstream members,including cyclin D1,Oct3/4,Sox2,Nanog,and MMP7.Sox2,Oct3/4,and Nanog are specifically expressed in cancer stem cells(CSCs),which help maintain CSCs.Moreover,the canonical Wnt/p-catenin signaling pathway promotes the epithelial-mesenchymal transition(EMT),which is crucial in the metastasis of NSCLC.Critical molecular features of EMT are the down-regulation of E-cadherin,which is a cell adhesion molecule in the plasma membrane of most normal epithelial cells,and up-regulation of N-cadherin and vimentin.The Wnt/?-catenin pathway is precisely controlled by a number of regulators,such as Drosophila naked cuticle and its two vertebrate orthologs NKD1 and NKD2,which have been shown to negatively regulate canonical Wnt signaling.Some potential chemopreventive agents,such as resveratrol,NO Non-steroid Anti-inflammatory Drugs[1],and dammarane-type triterpene sapogenin,for cancers that target the Wnt/?-catenin pathway have been reported.Thus,agents that target Wnt/?-catenin may have potential in cancer prevention and therapy.Moreover,some epidemiological studies on lung cancer risk worldwide have identified some potential dietary substances,such as green tea catechins,lycopene,soy isoflavones,pomegranate phenolics,selenium,vitamins A and D,curcumin,and resveratrol,as chemopreventive agents.However,completed clinical trials for NSCLC chemoprevention have routinely been negative.This phenomenon is partially due to low pulmonary bioavailability and unclear effects of most chemopreventive agents.Therefore,some traditional herbs with clear botanical,chemical,pharmacological,and therapeutic effects,may be excellent sources of cancer preventive substances.These herbs have been used clinically for decades.Artemisia annua L.has been used in traditional medicine for more than 2000 years to treat febrile symptoms associated with malaria.Artemisinin(ART),which is an active ingredient derived from A.annua L.,is a safe and effective FDA-approved and WHO-recommended primary treatment for malaria.Recent studies have suggested that ART exerts preferentially cytotoxic effects on NSCLC.Its two derivatives,dihydroartemisinin(DHA)and artesunate(ARTS),have also been demonstrated to exhibit antitumor effects toward various human cancers,such as lung carcinoma.The low toxicity of ART and its two derivatives,as well as their ability to kill parasites resistant to other anti-malarials,has been extensively examined because of their effects on NSCLC.ART,DHA,and ARTS in micromolar concentrations are cytotoxic to lung cancer cells.These agents can be administered either orally or rectally.ARTS and its active metabolite DHA are essentially eliminated completely 6 h after an intravenous injection(i.v.).High relative bioavailability of DHA following oral ART,DHA,and ARTS administration,as well as clinical outcomes comparable with those after i.v.,support the use of the oral formulation in the primary care setting.ART and its two derivatives may be excellent cancer preventive substances considering their safety,efficacy,bioavailability,and delivery pathway.ObjectivesIn the current study,we aimed to identify whether ART,DHA,and ARTS are potential preventive agents for NSCLC.We also explored the mechanism of Wnt/?-catenin for NSCLC prevention.We used two NSCLC cell lines,which are carcinoma in situ and metastasis,and a human tumor xenograft model to evaluate the preventive efficacy of ART,DHA,and ARTS on NSCLC.Furthermore,we investigated the influence of these substances on the Wnt/?-catenin pathway.Methods1.Cell linesThe human lung cancer cells(A549 and H1299)and bronchial epithelial cells(BEAS-2B)were purchased from ATCC.A549 and H1299 were cultured and maintained in RPMI-1640 medium supplemented with 10%FBS and 1%penicillin-streptomycin solution in a humidified atmosphere at 37 ? with 5%CO2.And BEAS-2B was cultured in BEGMTM Bronchial Epithelial Cell Growth Medium(Lonza,CH,GER).2.AnimalsFemal A/J mice,aged four to six weeks,were supplied from Jackson Laboratory.Record baseline weights and all groups received a single dose of BP(100 mg/kg body weight)in 0.2 ml of tricaprylin through intraperitoneal injection.Two week after the initiation of BP,all mice were randomized into five groups of chemical and herbal treated mice and gavaged with drug 5 times a week for 30 weeks and at the meantime.The gavage control group was also gaveged with 50%propanediol.The body weights of mice were measured every week for the duration of the study.Along weeks 30,all mice in different groups will be sacrificed for further analysis.All studies on animals were approved by the Guangzhou University of Chinese Medicine Animal Care and Use Committee(Guangzhou,China).Female Balb/c-nude mice(4-6 weeks,18-20 g),purchase from the Laboratory Animal Center of Sun Yat-Sen University(Guangzhou,China),were implanted subcutaneously with A459 cells(2 ×106,suspended in PBS)in each right flank.Tumor volume(V)was defined based on two dimensions(L,long diameter;W,wide diameter)measured by calipers,and calculated as formula:V =(L × W2)/2.When the tumors reached a mean volume of 50 mm3,all mice were randomized into following groups:control(sterilized coin oil),afatinib(5 mg/kg/d),ART(60 mg/kg/d),DHA(60 mg/kg/d),and ARTS(60 mg/kg/d).Body weights were recorded twice a week.Treatments were administered orally five times a week for four weeks.The nude mice were killed,and the tumors were segregated and measured.3.ImmunohistochemistryTumor tissue specimens were fixed in neutral formalin and embedded in paraffin after collection from the sacrificed mice.Tissue sections were cut and dewaxed,then incubated with 0.01 M natrium citricum for antigen retrieval.Then the slides were rinsed in phosphate-buffered saline and incubated overnight at 4? with diluted anti/?-catenin antibodies.Following steps were performed using the immunostaining kit(BOSTER Biological Technology)according to the manufacturer's instructions.4.Cell proliferation assayThe MTT assay was used to evaluate the cell viability.2 × 104 cells/ml of A549,H1299,and BEAS-2B were seeded into 96-well plates and cultured with different doses of ART,DHA,and ARTS for up to 48 h.At the end of treatment,0.5 mg/ml of MTT was added to the samples and incubated for 4 h.Then the supernatants were discarded and coloured formazan crystals dissolved with 150 ?l/well of DMSO,were read by an enzyme-linked immunosorbent assay reader.Three replicates were used for each treatment.5.Cell cycle analysis1 × 106 cells/ml of A549,H1299,and BEAS-2B were treated with vehicle or ART,DHA,and ARTS,for up to 48 h.For cell cycle analysis,cells were harvested,fixed with 70%cold ethanol,incubated with RNase,and stained with propidium iodide.Cell cycle distribution was analyzed by flow cytometry and Flow Jo software.6.Wound healingWound healing assay was conducted to examine the capacity of cell migration.Photomicrographs of initial wounds were taken using DMI 300B Leica at 100×magnification.Cells were treated with 0,7.5,15,or 30 ?M ART,DHA,and ARTS in RPMI 1640 media(1%FBS).Photomicrographs of final wounds were taken at 24 and 48 h.Initial and final wound sizes were measured using Image Pro Plus 6.0 software.7.TranswellInvasion analysis was performed by seeding A549 cells(1 x 105)treated with 7.5,15,or 30 ?M ART,DHA,and ARTS into the upper chamber of a Transwell apparatus coated with Matrigel and incubating the cells for 48 h.Cells were stained with crystal violet.The non-invasive cells were scraped off with cotton swabs,whereas the invasive cells stained with crystal violet were dissolved in DMSO for measurement.8.Western blotTotal proteins were extracted from cells after treatments.Protein samples were separated on 8%-12%SDS-polyacrylamide gel electrophoresis.Blots were immunostained with primary and secondary antibodies.?-Actin served as a loading control.Other detailed procedures were followed according to the literature.9.MMP activity assayMMPs activities were assessed using AmpliteTM Universal Fluorimetric MMP Activity Assay Kit,according to standard protocols.In brief,5,000 cells were seeded onto 24-well and then exposed to different concentrations(0,7.5,15,and 30 ?M)of ART,DHA,and ARTS for 48 h.Finally,50 ?L of supernatants from each well was added to a 96-well plate for detection.10.Wnt-5a siRNA transfectionA549 were plated into a 6-well plate with 1640 cell culture medium with 10%FBS.Wnt-5a siRNA and control siRNA were purchased from Santa Cruz Biotechnology(Santa Cruz,Biotechnology,USA).SiRNA transfection was performed according to the manufacturer's protocol.Seven hours post-transfection,cells were treated with different concentrations(0,7.5,15,and 30 ?M)of ART,DHA,and ARTS for 48 h.Cell lysates were analyzed for expression of Wnt5-a/b,?-catenin,Sox-2,Oct3/4,N-cadherin,E-cadherin proteins by western blotting,as described earlier.11.ImmunofluorescenceImmunofluorescence was performed on A549 cells seeded on 15 mm cofocal dish with 30 ?M ART,DHA,ARTS treating for 48 h and fixed in cold 4%paraformaldehyde.The cofocal dishes were incubated with primary antibodies.Detection of the primary antibodies was performed using 1:500 Alexa Fluor(?)conjugated secondary antibodies(Santa Cruz,Biotechnology,USA).The images were captured and analyzed using Leiaca TCS SP8 confocal microscope.The images were acquired using an identical acquisition time for all tissue sections.12.Data analysisData were expressed as the mean ± standard deviation(SD)of at least three independent experiments.Statistical significance of the data was determined by one-way ANOVA using SPSS software.Statistical significance was considered at p<0.05.ResulstsART,DHA,and ARTS suppressed cell viability and induced G1 arrest by inhibiting cyclin D1 in A549 and H1299The effect of ART,DHA,and ARTS on cell proliferation of NSCLC cells was investigated by conducting MTT and flow cytometry assays using A549 and H1299.These cell lines demonstrated sensitivity to varying degrees of ART,DHA,and ARTS treatments in 48 h MTT assays.However,all ART,DHA,and ARTS treatments did not exhibit effective growth inhibition on BEAS-2B up to a dose of 70 ?M.According to the difference among the sensitivity of ART,DHA,and ARTS,the inhibitory effects were DHA>ARTS>ART.IC50 was less than 200 ?M even in the ART group,which was the least responsive group.The cell cycle was analyzed to understand MTT effects.G1 phase accumulation was evident in all treatments.Accumulation of cells in the G1 phase was significantly enhanced with increasing concentrations of compounds.A549 and H1299 cells exhibited the greatest G1 phase accumulation,especially in ART treatment,with the increases of 17.63%± 0.67%(p<0.01)and 26.69%± 0.68%(p<0.01),respectively.The role of cyclin D1 in G1 phase arrest in ART,DHA,and ARTS treatments was tested using Western blot analysis to detect cyclin D1 expression.The results showed a dose-dependent down-regulation in the cyclin D1 protein expression in A549 and H1299 cells.ARTS was the most effective in inhibiting cyclin D1 protein compared with ART and DHA.Cell invasion and migration were inhibited by ART,DHA,and ARTSWound healing assay was used to evaluate migration in A549 and H1299 cells.ART,DHA,and ARTS significantly inhibited lung tumor cell migration in a dose-dependent manner.Among all three compounds,DHA at 30 ?M showed the most effective migration suppression with 27.1%± 3.52%compared with the control group(p<0.001).Additionally,transwell coated with matrigel was used to explore the invasive ability of lung tumor cells.Compared with abundant cells translocated to the underside of well in the control treatment,cell invasion activities were dose-dependently decreased by ART,DHA,and ARTS.Especially in ART treatments,cell invasion significantly decreased by 32.19%± 0.15%(p<0.001),58.77%± 1.79%(p<0.001),and 67.25%± 0,22%(p<0.001),respectively,with increased concentrations(7.5,15,and 30 pM).Meanwhile,matrix metalloproteinase(MMPs)activities were analyzed by MMPs activity assay kit to further determine the invasion capability of ART,DHA,and ARTS.All ART,DHA,and ARTS treatments could markedly inhibit MMPs activities in a dose-dependent manner.ART,DHA,and ARTS suppressed EMT and CSCsCSCs and EMT are two key factors affecting tumor metastasis,including migration and invasion.Whether the anti-metastasis effects of ART,DHA,and ARTS are associated with these two aspects were analyzed by Western blotting.Results showed that the expression levels of CSC markers including nanog,sox2,and oct3/4,as well as EMT-related proteins such as N-cadherin and vimentin,were all significantly inhibited by ART,DHA,and ARTS treatments,whereas those of E-cadherin either in A549 or H1299 cells increased.Among the three compounds,ART exerted the greatest inhibitory effects,especially in the expression of nanog and sox2 in A549 cells,and the percentages of decrease at 30 ?M were 65%± 0.26%(P<0.001)and 80%± 1.92%(p<0.001),respectively.Similarly,the protein levels of N-cadherin and vimentin were remarkably down-regulated by ART.However,in H1299 cells,most increased E-cadherin expression and decreased CSC markers were observed in ARTS treatments,indicating that the effective response of ART,DHA,and ARTS may slightly vary in tumor cells with different histological features.ART,DHA,and ARTS inhibited Wnt signalingWestern blots were employed to highlight the key regulatory factors on the Wnt pathway to detect whether the canonical Wnt signaling pathway is the mechanism in regulating EMT and CSCs in ART,DHA,and ARTS treatment.A cluster of altered proteins was associated with the inhibition of Wnt5-a/b in A549.Wnt5-a/b was highly reduced after ART,DHA,and ARTS treatments.LRP6 was reduced most significantly to 16.80%± 1.14%(p<0.01)after DHA treatment at 30 ?.Dv12 was elevated most markedly during ARTS treatment.Dvl2 decreased to 8.71%? 0.78%(p<0.01),6.50%±0.64%(p<0.01),and 4.69%±0.62%(p<0.01)under 7.5,15,and 30 ?M ARTS,respectively(p<0.01).In the assay,?-catenin,which decreased by 65%,was confirmed to have high selectivity to ARTS.Similar results were observed in H1299.Moreover,in those cell lines,ART,DHA,and ARTS treatments significantly decreased the protein of P-catenin accumulated either in cytoplasm or nucleus.Suppression of ART,DHA,and ARTS in proliferation and metastasis could partially depend on Wnt/p-catenin pathway inactivationTo determine whether the anti-tumor effects of ART and its derivatives depended on Wnt/?-catenin pathway,IWP-2,Wnt/[?-catenin pathway inhibitor and Wnt5a siRNA were used considering the important role of Wnt5-a/b and of which most protein reduction were observed previously.The expressions of Wnt5-a/b and?-catenin in A549 cells were dramatically suppressed by IWP-2,whereas those of NKD2 and Axin2 were not altered.However,pretreated with IWP-2,the G1 arrest induced by ART,DHA,and ARTS were enhanced in A549 cells.While interestingly,compared with IWP-2 treatment,no significant differences were observed in treatments of ART and its derivatives combined with IWP-2.Moreover,as expected,Wnt5-a/b knockdown consequently suppressed the protein expressions of sox2 and?-catenin but increased that of E-cadherin.However,with Wnt5a knockdown,ART,DHA,and ARTS still exerted anti-metastasis effect by decreasing sox2 while increasing E-cadherin.Therefore,with or without Wnt5a,the irreplaceable and sustained suppression of Wnt/?-catenin pathway occurred,suggesting that the anti-tumor effects of ART and its derivatives only partially depended on Wnt5-a/b inactivation.On the other hand,Axin2 and NDK2,two negative regulators in canonical Wnt/?-catenin signaling pathway,were simultaneously explored.In A549 cells,ART at 7.5,15,and 30 ?pM significantly increased the protein level of NKD2 by 132.38%±4.60%(p<0.001),198.83%± 12.25%(p<0.001),and 734.09%± 20.14%(p<0.001),respectively.Similar results were observed in H1299 cells.Additionally,the expressions of Axin2 were also notably increased by ART in A549 cells and by ARTS in H1299 cells.ART,DHA,and ARTS suppressed lung cancer growth in xenograft mice mainly through the Wnt/?-catenin pathwayA xenograft mouse model was employed to further explore the effects of ART,DHA,and ARTS.ART,DHA,ARTS,and afatinib did not cause significant body weight loss in mice.However,the tumor volume was smaller with oral administration of ART,DHA,and ARTS compared with the control group(p<0.05).Tumor weights were significantly reduced in the ART,DHA,and ARTS groups compared with that in the control group.IHC staining was employed to further confirm the mechanisms of ART,DHA,and ARTS on proliferation and metastasis.The results of IHC staining demonstrated reduced levels of ?-catenin from A549 xenograft mouse model in the ART,DHA,and ARTS groups.Furthermore,Western blots from tumor tissues were analyzed.P-Catenin proteins were significantly decreased by approximately 50%in ART,DHA,and ARTS-treated mice.The expression levels of Oct3/4,Sox2,and Nanog proteins from the ARTS groups were reduced to 5.10%± 0.25%(p<0.01),19.30%± 0.19%(p<0.01),and 7.92%± 0.15%(p<0.01)compared with those of the control group,respectively.By contrast,DHA reduced the expression of Oct3/4,Sox2,and Nanog proteins to 26.20%± 0.83%(p<0.01),37.33%± 3.8%(p<0.01),and 16%± 1.50%(p<0.01),respectively.The effects of ART in inhibiting the expression of Oct3/4,Sox2,and Nanog were less significant than those of ARTS and DHA.Similarly ARTS and DHA more significantly decreased the expression of vimentin and N-cadherin and more markedly increased E-cadherin than ART.Western blots were employed to explore the related proteins from tumor tissues of A549 xenograft mouse to further determine the effect of ART,DHA,and ARTS on the Wnt signaling pathway.DHA was more effective in reducing Wnt5-a/b,which decreased to more than tenfold.Furthermore,Western blots were employed to explore the two proteins from tumor tissues of A549 xenograft mouse.Both NKD2 and Axin2 increased significantly in the three treatments.Up-regulation was more remarkable in the ARTS group.ConclutionIn conclusion,ART,DHA,and ARTS are promising agents for the chemoprevention of NSCLC with clear effects and novel mechanism in NSCLC.We also demonstrated that multiple targets of the Wnt/?-catenin pathway were responsible for the suppression of tumorigenesis and metastasis in ART,DHA,and ARTS cancer prevention.Further studies should be initiated to test their efficacy in clinical trials. |
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