Mechanisms Of Dihydrocapsaicin Inhibits Melanoma Cell Proliferation And Metastasis In Vitro And In Vivo

Part One Dihydrocapsaicin inhibits the proliferation,migration and invasion of melanoma cells in vitroObjective:To investigate the effects of dihydrocapsaicin(DHC)on the proliferation,migration and invasion of melanoma cells in vitro and its potential molecular mechanisms.Methods:1.The effects of DHC on the proliferation of melanoma cells were evaluated by MTT and EdU staining assays.2.Melanoma cells were treated with DHC.The changes of cell cycle were observed by flow cytometry,and the expression of cyclin D1,c-Myc and cyclin A2 were detected by western blotting and qRT-PCR.3.The effects of DHC on the migration and invasion of melanoma cells were evaluated by wound healing and transwell assays,and the expression of?-catenin,MMP2 and MMP7 were detected by western blotting and qRT-PCR.Results:1.MTT assay showed that the viability of melanoma cells was decreased after DHC treatment,and significant decreases in EdU positive rates were observed in DHC treated cells.2.The percentage of cells in S-phase was markedly elevated after DHC treatment.In addition,the expression of cyclin D1,c-Myc and cyclin A2 was dose-and time-dependently down regulated.3.The wound healing of melanoma cells were significantly delayed by DHC.Transwell assays showed that the cellular migration and invasion was hindered by DHC.The expression of?-catenin,MMP2 and MMP7 proteins were remarkably decreased in a dose-and time-dependent manner in DHC treated cells.The m RNA expression of MMP2 and MMP7 were also decreased after DHC treatment,while?-catenin m RNA was barely changed.Summary:1.DHC inhibits the proliferation of melanoma cells in vitro,which may achieved by inducing S-phase cell cycle arrest through down regulating the expression of cyclin D1,c-Myc and cyclin A2.2.DHC inhibits the migration and invasion of melanoma cells in vitro,and its mechanism may be through down regulating the expression of?-catenin,MMP2 and MMP7.Part Two Dihydrocapsaicin inhibits the proliferation and metastasis of melanoma cells in vivoObjective:To investigate the effects of DHC on tumor growth and metastasis of melanoma in vivo.Methods:1.Soft agar colony formation assay was performed to evaluate the effect of DHC on the tumorigenicity of melanoma cells in vitro.2.An A375 xenograft NOD/SCID mice model was established.Tumor size and weight of the mice administered with DHC were compared with those from the DMSO group.The effect of DHC on the expression of Ki-67 was observed by immunohistochemistry(IHC).3.An A375 tumor metastasis NOD/SCID mice model was established,and the effect of DHC on lung metastasis of the cells was observed by H&E pathology.Results:1.Smaller and fewer colonies were generated by DHC treated cells in comparation with the control group.2.Tumor size and weight of the mice administered with DHC were smaller than those from the DMSO treated mice,and the expression of Ki67was significantly reduced in DHC treated tumors.3.Fewer metastases were found in the lungs of A375 metastasized mice under DHC treatment compared with the DMSO group.Summary:1.DHC inhibits the tumorigenesis of melanoma cells in vitro.2.DHC inhibits melanoma growth in NOD/SCID mice.3.DHC inhibits lung metastasis of melanoma cells in NOD/SCID mice.Part Three Dihydrocapsaicin inhibits melanoma cell proliferation and metastasis via down-regulating?-catenin pathwayObjective:To study the status of?-catenin in DHC inhibiting the proliferation and metastasis of melanoma cells,and the mechanism by which DHC regulates?-catenin.Methods:1.A375 and MV3 melanoma cell lines stably overexpressing?-catenin(OE-?-catenin)or empty vector(OE-Vector)were established and treated with DHC.Cell viability and proliferation were observed by MTT and EdU staining assays.2.Melanoma cells OE-?-catenin/Vector were treated with DHC.The changes in cell cycle were observed by flow cytometry,and the expression of cyclin D1,c-Myc and cyclin A2 were detected by western blotting and qRT-PCR.3.Cellular migration and invasion of melanoma cells OE-?-catenin/Vector were verified by transwell assay,MMP2 and MMP7 levels were detected by western blotting and qRT-PCR.4.A375^OE-?-catenin and A375^OE-Vector xenograft NOD/SCID mice model were established.Tumor size and weight of the mice administered with DHC were compared with those from the DMSO group.The expression of Ki-67 in xenografts was observed by IHC staining.5.A375^OE-?-catenin and A375^OE-Vector tumor metastasis NOD/SCID mice model were established,and the effect of DHC on lung metastasis of the cells was observed by H&E pathology.6.The molecular mechanism of DHC regulating?-catenin was explored by protein turnover and ubiquitination assays.Results:1.MTT assay showed that?-catenin overexpression could reduce the inhibition of DHC on the viability of melanoma cells.EdU staining showed that the decreased EdU positive rate induced by DHC was partially reversed by?-catenin overexpression.2.Overexpression of?-catenin reduced the S-phase cell cycle arrest caused by DHC.The DHC-induced decrease of cyclin D1,c-Myc and cyclin A2 were partially rescued after?-catenin was up-regulated.3.Under DHC treatment,the number of OE-?-catenin melanoma cells passing through the membrane of transwell was more than that of OE-Vector cells.Additionally,the DHC-induced decrease of MMP2 and MMP7 were partially rescued after?-catenin was up-regulated.4.Under DHC treatment,the mice xenografted with A375^OE-?-catenin cells developed tumors larger in volume and weight compared with A375^OE-Vectorcells.Furthermore,the decreased expression of Ki67 in DHC treated tumors was partly rescued by?-catenin overexpression.5.A375^OE-?-catenin cells formed more metastases in lungs of the mice under DHC treatment,compared with the A375^OE-Vector cells.6.DHC accelerated the degradation of?-catenin protein and up-regulated the expression of BTRC in melanoma cells.Ubiquitination assay confirmed that DHC significantly increased the ubiquitination level of?-catenin.Summary:1.Overexpression of?-catenin attenuates the anti-proliferative effect of DHC on melanoma cells.2.Overexpression of?-catenin attenuates the inhibitory effect of DHC on the migration and invasion of melanoma cells.3.Overexpression of?-catenin attenuates the inhibitory effect of DHC on the growth and lung metastasis of melanoma in NOD/SCID mice.4.DHC could enhance the expression of BTRC and accelerating the ubiquitination degradation of?-catenin in melanoma cells.Conclusions:1.DHC inhibits the proliferation,migration and invasion of melanoma cells in vitro by regulating cell cycle progression and the expression of migration and invasion related proteins.2.DHC can effectively inhibit the growth and lung metastasis of melanoma in mice model.3.DHC may play an anti-melanoma effect by down-regulating?-catenin signaling pathway.4.The mechanism of DHC regulates?-catenin in melanoma cells may be through enhancing the expression of E3 ubiquitin ligase BTRC,thereby promoting the ubiquitination degradation of?-catenin.