| Objective:Malignant melanoma is a kind of malignant tumor and has been found distant metastasis in diagnosis.Some research found that the epithelial mesenchymal transition(EMT)is associated with invasion and metastasis of melanoma.Other studies have shown that increased Cx43 express and gap junction inhibition of epithelial mesenchymal transition in malignant tumors.In tumor microenvironment,tumor associated macrophage through polarity transformation and the secretion of cytokines to promote epithelial mesenchymal transition.Gap junction was closely related to immune and increased phagocytic function of macrophage.The previous study found that dioscin increased Cx43 express and gap junction.We discussion that dioscin increased Cx43 affect the EMT and cancer stem cells in B16 cells and C57BL/6 mouse metastasis model.Then we explored that dioscin inhibit mouse melanoma and mechanism in the tumor associated macrophage.MethodsCell lines and reagentsThe murine melanoma B16 and RAW264.7,cultured in RPMI 1640 medium(penicillin,100 U/ml;streptomycin,100mg/ml;10%FBS),were incubated at 37? in 5%C02.Dioscin and retinoic acid(RA)were purchased from Chengdu Must Bio-technology Co.,Ltd.(Chengdu,China).Primary and corresponding secondary antibodies were obtained from Abcam,Inc.(Abcam,CA,USA)and Santa Cruz Biotechnology,Inc.(Santa Cruz,CA,USA).The over-expressed Cx43 plasmid(EX-A0334-LV155)was bought from GeneCopoeia Biotechnology(GeneCopoeia Biotechnology,MD,USA)while by which the mutated Cx43 plasmid(G21R)was synthesized.DAPI,Lucifer yellow,LPS and calcein-AM were purchased from Sigma-Aldrich(Sigma,MO,USA).DiI were obtained from Beyotime Biotechnology(Shanghai,China).Primers were synthesized from Genomics biological technology(Guangzhou,China).MTT assayB16 cells and RAW264.7 cells(5.0×13/we0l)were planted in 96-well plates,and treated with dioscin(1?M,2?M,4?M,8?M)for 24 h,48h,72h.After adding MTT reagent(0.5 mg/ml)for 4h incubation,the formazan was dissolved with DMSO.The OD value were read by Victor X3(PerkinElmer,MA,USA)at 490nm.Plasmids construction and transit cell transfectionAccording to the previous study,the mutated Cx43 plasmid(G21R,c61.G?A)was synthesized.For transit cell transfection,B16 cells(2×105)and RAW264.7(3×105)cells were seeded in 6-well plates,after incubation for 24 h,cells were transfected with 3 ?g over-expressed Cx43 and 3 ?g mutated Cx43 plasmids using Lipofectamine 2000(Invitrogen,Carlsbad,CA,USA).Transfectants were incubated in medium containing dioscin(1?M and 2?M)and RA(20 ?M)for indicated time and collected for subsequent analysis.Western blot analysisB16 cells(2×105 cells/well)were seeded in 6-well plates,and treated with dioscin(1 ?M and 2 ?M)and RA(20 ?M)for 48 h.The protein was centrifuged from cell extraction lysed by RIPA buffer(1%PMSF).According to the standard WB procedure,the blots were incubated with primary antibodies including anti-Cx43,anti-p-actin,anti-E-cadherin,anti-N-catenin,anti-Vimentin,anti-?-catenin,anti-Snail,anti-Sox-2,anti-Nanog,and anti-OCT3/4 for overnight at 4 ?.Then after incubated with the corresponding secondary antibodies for 1h,the density of band was examined by ECL chemiluminescence detection regents(PE,CA,USA).Quantitative real-time PCRTotal RNA was extracted by TRIzol(Invitrogen,CA,USA)according to the manufacturer's instructions.cDNA was generated by reverse transcription kit(TaKaRa,Dalian,China).SYBR Green real-time PCR were carried out in ABI 7500 fast system.Transwell and wound healing assaysB16 cells(2×104 cells/well)were cultured in the upper chamber with low-serum medium contained dioscin and RA,respectively,while high-serum medium(20%FBS)was added to the lower chamber.After 48 h of incubation at 37 ?,cells were fixed with 4%paraformaldehyde,and stained with 0.1%crystal violet for 15 min.Then dissolved the crystal violet in DMSO,and the OD value were detected by Victor X3(PerkinElmer,MA,USA)at 600 nm.For wound healing assays,pretreated B16 cells with dioscin and RA,respectively until reached 80%confluency,scratched in the center of well,and took images at 0 and 24 h using microscope(Leica,Jena,Germany).MMPs activity assayAccording to the standard protocol,we detected MMPs activity by AmpliteTM Universal Fluorimetric MMP Activity Assay Kit(AAT Bioquest,CA,USA).In brief,B16 cells(2×105/well)were seeded in 6-well plate and treated with dioscin(1 ?M and 2 ?M)and RA(20 ?M)for 48 h.The supernatant were collected for fluorimetric detection.ImmunofluorescenceAccording to the protocol described previously,the immunofluorescence assays were performed with anti-Cx43(Abeam,1:500),anti-E-cadherin and anti-N-cadherin antibodies(Santa Cruz,1:200),respectively.Incubated with the corresponding PE/FITC conjugated secondary antibodies(Santa Cruz,1:200),then stained the nucleus with 0.1%DAPI for 30min,after that,the images were detected by confocal microscopy(Leica,Jena,Germany).Gap junction dye transport assay According to the previous study,the Gap junction dye transport assayswere performed.The donor cells were labeled with Calcein-AM and DiI,while the dye transporting ratio between donor cells and recipient cells was captured by confocal microscopy(Leica,Jena,Germany)after 6 h of co-incubation.The recipient cells,who generated fluorescence were detected by FACS Aria ?(BD Biosciences,Auckland,New Zealand)and analyzed using Flow Jo 7.6.1 software(Tree Star,Inc.,OR,USA).SL/DL assayAccording to the previous reported protocol,the scrape loading/dye transfer(SL/DT)were performed.Briefly speaking,after washing the dioscin pre-treated B16 cells,0.05%Lucifer yellow was added into the center of scrape for approximately 1 min,and then rinsed immediately with PBS.Fixed the cells in 4%paraformaldehyde and examined by fluorescence microscopy(Leica,Jena,Germany).Flow cytometry analysisRAW264.7 cells(3.0×105/well)were cultured in 6-well plates,and treated with dioscin(1 ?M and 2 ?M)and LPS(1 ?g/ml)for 48h.According to manufacturer's protocol,suspended cells and incubated with PE-conjugated anti-CD68,PE-cy7-conjugated anti-CD86,PE-conjugated anti-F4/80,Alexa Fluor488-conjugated anti-NOSi,FITC-conjugated anti-CD206 and FITC-conjugated anti-CD209 antibodies,respectively(eBioscience,CA,USA).The fluorescent dyes were detected by FACS Aria III(BD Biosciences,Auckland,New Zealand)and analyzed by Flow Jo 7.6.1 software(Tree Star,Inc.,OR,USA).Co-culture,conditioned medium,and ELISAB16 cells were co-cultured with RAW264.7 cells in the presence of dioscin(1?M and 2 ?M)or LPS(1 ?g/ml)for 48 h.RAW264.7 cells were stained with Dil(red)and B16 cells were stained with CFSE(green)for 30 min,then RAW264.7 cells co-cultured with B16 cells for another 4 h and equivalent phase contrast images were taken by confocal microscopy(Leica,Carl Zeiss,Jena,Germany).For phagocytic analysis,B16 cells also co-cultured with RAW264.7 cells transfected with Cx43 and mCx43 for 48 h.Then,B16 cells were stained with CFSE(green)for 30 min,and images were taken.According to the previous study;RAW264.7 cells were pretreated with dioscin(1?M and 2 ?M)and LPS(1 ?g/ml)for 48h.After change to drug-free medium for 24h,the tumor associated macrophage conditioned medium(TAM-CM)was collected for following B16 cells culture(another 48h).According to the manufacturer's instructions,the expressions of IL-6,IL-1?,and TNF-? were measured by the corresponding ELISA kits(eBioscience,CA,USA).Animal experimentAll animal studies were approved by Animal Care and Use Committee(IACUC)in Guangzhou University of Chinese Medicine.Female C57BL/6 mice(6-8 weeks)were purchased from animal center of Guangzhou University of Chinese Medicine.For experimental metastasis model,after suspended B16 cells(1.5×106/ml)and injected(i.v.)into C57BL/6,gavaged the mice with dioscin(30 mg/kg/d and 60 mg/kg/d)for 18 days,then separated the metastasis lung from sacrificed mice.For spontaneous metastasis model,injected(s.c.)B16 cells(2×106/ml)into C57BL/6,then gavaged the mice with dioscin(30 mg/kg/d and 60 mg/kg/d).When the tumor volumes reached 1000mm3,removed the tumor and continually gavaged mice for another 30 days.At the endpoint,tissues were collected for further analysis.Body weight was recorded twice a week.Results:Dioscin significantly enhanced the expression and communicative function of Cx43 through activing RAR pathway in B16 melanoma cellsPreliminary MTT assay results suggested that dioscin significantly suppressed cell proliferation of B16 in a dose-and time-dependent manner.Meanwhile,considering the accumulated toxicity of dioscin observed,dioscin at 1 ?M and 2 ?M with respective cell viability inhibition of 8.65 ± 0.86%and 20.19 ± 0.84%at 48h were chosen for following studies.Compared to the control group,dioscin treatments remarkably increased the protein and gene expressions of Cx43,which is similar to that in RA positive control treatment.Moreover,in the recipient B16 cells,the increasing fluorescent density of calcein-AM indicated that the gap transporting function of Cx43 was also notably enhanced by dioscin.Simultaneously,dioscin at 1 ?M and 2 ?M dramatically elevated the fluorescence in recipient cells by 23.56 ± 0.67%and 26.63 ± 0.15%,respectively.Furthermore,compared to control group,dioscin remarkably activated RAR pathway in a dose-dependent manner through increasing the expressions of RAR?,RAR?,and RAR? while inhibiting that of RXR?.Dioscin remarkably inhibited EMT of B16 melanoma cellsBy conducting wound healing and transwell assays,we found that dioscin(1?M and 2?M)and RA(20?M)dose-dependently reduced migration and invasion capacity of B16 melanoma cells.Meanwhile,the MMP activities were also significantly suppressed by dioscin with reduction of 74.72 ± 1.27%and 69.55± 0.95%,respectively at 1 ?M and 2 ?M.Furthermore,after dioscin treatments,the expressions of EMT-associated proteins such as snaill,vimentin,N-cadherin,and ?-catenin were notably decreased while that of E-cadherin was increased as expected.Specifically speaking,the protein expression of E-cadherin could be induced by dioscin up to 3 fold;meanwhile,dioscin at 2 ?M remarkably reduced the expression of ?-catenin and N-cadherin by 60.48± 2.42%and 97.13 ± 1.94%,respectively.At the same time,in a dose dependent manner,the markers for cancer stem cells(CSC)including sox-2,oct3/4 and nanog were remarkably inhibited by dioscin in B16 cells.Despite the most reduction observed in RA positive control group,dioscin at 2 ?M significantly decreased the protein of sox-2 and nanog with the decline of 65.85 ± 2.87%and 39.99 ± 2.51%.Highly expressed functional Cx43 significantly inhibited,while non-functional Cx43 enhanced the tumor malignancy of B16 cells.To further determine whether the expression or the function of Cx43 induced by dioscin was directly related to the metastasis capacity and malignancy of tumor cells,the overexpressed Cx43 transfected cells(Cx43-LV155 and mutated Cx43(G21R))were established.Compared to the Cx43-LV155 plasmid,the mutated Cx43(G21R)plasmid,generated by changing the nucleotide G to A at position 61,could lead to functional deficiency while still overexpressed Cx43.After confirming the transfection efficiency by anti-Cx43 IHC staining and WB,the function of Cx43 was determined by SL/DT assay.Compared to the control group,overexpressed Cx43 could significantly increase the intercellular fluorescence transition,whereas in mutated Cx43 transfected cells,due to loss of transporting function,the fluorescence could not be diffused.Furthermore,we found that the overexpressed Cx43 significantly inhibited the migration and invasion abilities of B16 cells with respective reduction of 18.5 ± 0.71%and 26.00 ± 2.76%.On the contrary,without signal transporting function,the overexpressed Cx43 strikingly increased the metastasis capacity in mutated Cx43 transfection by 11.4 1.27%,21.45 ± 2.40%,respectively.Similarly,the overexpressed Cx43 dramatically reduced MMP activities by 39.18 ± 1.91%while mutated Cx43,due to functional deficiency,conversely inhibit MMP activities by 19.40 ± 2.20%.Dioscin dramatically reversed EMT promotion induced by non-functional Cx43overexpression in B16 cells To further assess the effects of dioscin on malignancy promotion induced by Cx43 functional deficiency,the expressions of EMT markers in mutated Cx43(G21R)were analyzed after dioscin and RA treatments.Compared with the control group,the overexpressed Cx43 could stimulate the gene and protein expressions of E-cadherin while suppress that of N-cadherin,?-catenin,snaill,and vimentin in Cx43-LV155 transfected cells.Contrarily,with the vanished transporting function of Cx43,the gene and protein levels of E-cadherin were significantly decreased.Simultaneously,regardless of Cx43 highly expression,the expressions of N-cadherin,?-catenin,snaill,and vimentin were reduced in mutated Cx43(G21R)transfection.These results suggested that EMT capacity of B16 cells was remarkably enhanced by non-functional overexpressed Cx43.However,unexpectedly,these enhancements were significantly reversed by dioscin through reducing the EMT associated markers such as N-cadherin,-catenin,snaill,and vimentin in the mutated Cx43(G21R)transfected cells.Dioscin enhanced the phagocytosis of macrophage via inducing Cx43 overexpressionMTT assay was conducted to explore the cytotoxicity of dioscin in RAW264.7 cells.Compared to the control group,dioscin(1-8 ?M)treatments could not alter the cell viability of RAW264.7 cells.Considering the equivalent concentration of dioscin used in treating B16 cells,dioscin(1 ?M and 2 ?M),with no visible cytotoxicity at 48h,were set for following studies.The polarization of macrophages stimulated by dioscin was analyzed by FACS,while the expressions of TNF-?,IL-1?,and IL-6 were also detected by ELISA.In F4/80+RAW264.7 cells,dioscin notably promoted M1 polarization through inducing the NOSi+cells from 5.98 ±0.18%in control group to 16.5 ± 0.49%and 41.6 ±0.57%,respectively in dioscin treatments(1 ?M and 2?M).Simultaneously,the F4/80+CD86+ and F4/80+CD68+ cells were also remarkably enhanced by dioscin.On the contrary,compared to the control group,dioscin significantly suppressed M2 phenotype biomarkers(e.g.CD206 and CD209)in F4/80+ RAW264.7 cells.Furthermore,the cytoplasmic expressions of TNF-?,IL-1?,and IL-6,hallmark cytokines highly secreted in M1 macrophages,were dose-dependently increased by dioscin.To further investigate the consequent of M2-to-Ml phenotype transition induced by dioscin,the phagocytosis of RAW264.7 cells was analyzed by confocal microscope.Dioscin(1 ?M and 2 ?M)and LPS(1 ?g/ml)could significantly enhanced the phagocytic capacity of RAW264.7 cells when co-cultured with B16 cells.At the meantime,comparing with the control group,dioscin at 1 ?M and 2?M remarkably increased the protein expression of Cx43 by 220.50 ± 4.90%and 268.00 ± 4.24%,respectively in RAW264.7 cells.To determine whether the Cx43 overexpression induced by dioscin was related to the phagocytic stimulation,the B16 cells were co-cultured with Cx43-LV155 and mutated Cx43 transfected RAW264.7 cells,respectively.Compared to the control group,with the Cx43 overexpression,the phagocytosis of RAW264.7 cells was significantly enhanced.However,when Cx43 loss of transporting function,the phagocytic activity of macrophages was contrarily decreased in mutated Cx43 transfectedRAW264.7 cells.Dioscin notably inhibited EMT of B16 cells through promoting inflammation cytokines secretion of macrophagesAfter culturing B16 cells with the conditioned medium of RAW264.7 cells pretreated with dioscin(1?M and 2?M)and LPS(1?g/ml),the EMT capacity of B16 cells was examined.Compared to the control treatment,the migration and the invasion ratio were remarkably reduced by dioscin-pretreated TAM-CM.Specifically,dioscin at 2 ?M decreased the migrated and invasive capacity of B16 cells by 56.04 ±1.82%and 64.75±1.48%,respectively.Furthermore,by employing western blot,the protein expressions of Cx43,EMT-associated proteins,and CSCs markers were further analyzed.We found that the supernatants,collected from dioscin-pretreated RAW264.7 cells,notably increased the protein express of Cx43 and E-cadherin,while decreased that of snail1,vimentin,and N-cadherin in a dose-dependent manner.Similarly,CSCs markers were also significantly inhibited by dioscin-pretreated TAM-CM in B16 cells after conditional culture.Dioscin remarkably suppressed tumor growth and metastasis in vivoTumor metastasis includes multiple steps,such as spontaneously dissemination from primary tumors,invasion into blood vessels,attachment in the metastasis tissues,and ultimately colony formation.To recapitulate this comprehensive progression and systematically explore the effect of dioscin on melanoma metastasis in vivo,two classical animal models,"experimental metastasis model" and "spontaneous metastasis model",were used in our current study.In spontaneous metastasis model,dioscin treatments(30mg/kg and 60mg/kg)significantly decreased the tumor volumes and tumor weights,without resulted in severe body weight loss.Furthermore,the lung metastatic node number was also remarkably reduced by dioscin with reduction of 60.88±1.67%and 34.62±3.02%,respectively at 30mg/kg and 60mg/kg.Subsequently,dioscin notably increased the expressions of E-cadherin and Cx43 while simultaneously decreased that of N-cadherin in tumor tissues.Moreover,to further investigate the correlation between Cx43 overexpression and tumor metastasis,the experimental metastasis model was established.Similarly,dioscin,either at 30mg/kg or 60mg/kg,could not cause observed toxicity in vivo.However,the metastasis node number was dramatically reduced by 70.13 ± 1.67%and 36.36 ± 2.02%,respectively in dioscin treatments(30mg/kg and 60mg/kg).In addition,immunohistochemical result also indicated that dioscin significantly enhanced the abundant expression of Cx43,as well E-cadherin in metastasis tissues.These findings suggested that dioscin exerted anti-tumor and anti-metastasis potentialities in vivo,and the Cx43 overexpression,negatively correlated with the EMT-associated markers,was closely related to its antitumor effectiveness.Conclusion:1.Dioscin inhibited epithelial mesenchymal transition through upregulation Cx43.Dioscin increased Cx43 might through RAR receptor signaling.2.Dioscin increased phagocytic function through up-regulated Cx43.Then dioscin increased M2-to-M1 phenotype transition and promoted secretion of inflammation cytokines.RAW264.7 cells supernatant inhibit EMT and CSCs through up-regulated Cx43.3.In vivo,dioscin reduced tumor growth and number of lung metastasis node.Furthermore,dioscin inhibited EMT by up-regulated Cx43. |
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