| Objective: Malignant melanoma(MM)is a kind of highly aggressive dermatological malignancy with a poor prognosis which originated in the neural crest cell.Over the past decades,the incidence of malignant melanoma is increasing,becoming the leading cause of shin cancer.Currently,the main method is surgical removal of the melanoma,but the onset of the melanoma is hidden,most have occurred when transfered to the patient,missing the best surgical treatment period.So far,the standard chemotherapeutic approaches used in the treatment have been disappointing,as well as a variety of treatments,including surgery,still not have a breakthrough.Therefore,it is time to investigate some novel drugs with high efficiency and minimal toxicity for metastatic melanoma.Identification of effective strategies for the treatment of metastatic melanoma remains an urgent need.Antibiotics are common durgs with low toxicity and price but high effectiveness.They have been suggested to be drug candidates for cancer therapy in recent years.As one of the third-generation tetracycline antibiotic,tigecycline is similar to the structure of tetracycline drugs,which is firstly approved for antibacterial treatment in clinic by FDA.Reports have shown that tetracycline and their derivatives,such as minocycline and doxycycline had antitumoral activity.As analogues of tetracycline,tigecycline is used in the adjuvant therapy on tumor,such as acute leukemia,prostate cancer and the treatment of tumor surgery,basing on its function of antibacterial and anti-inflammatory function.In recent years,some studies have shown that tigecycline has obvious effect in inhibiting tumor cell proliferation in gastric cancer、glioma and myeloid leukemia,but the effect on melanoma and its mechanism is not yet clear.Therefore,in order to provide more theoretical basis that tigecycline can act as a clinical drug candidate in the treatment of metastatic melanoma,we tried to investigate the molecular biology effect and mechanism antitumour effect of tigecycline on malignant melanoma,through a variety of experiments involving cell proliferation,cell cycle arrest,cell migration/invasion in vitro,colony formation in soft agar and the tumor growth of tumor-bearing mice in vivo.Methods:1 The change on the proliferation capacity of melanoma cells after treated with tigecycline(0μmol/L、2μmol/L 、5μmol/L、10μmol/L)was determined by the method of cell counting、MTT.BrdU immunofluorescence was used to reveal the effect of tigecycline on the growth of A375 and MV3 cells.2 The effect of tigecycline on cell cycle was detected by PI staining and flow cytometry.In order to explore the mechanism of the cycle arrest induced by tigecycline,Western Blot assay was performed to assess the cell cycle-related protein levels in A375 and MV3 cells,respectively after treated wih tigecycline(0μmol/L、2μmol/L 、5μmol/L、10μmol/L).3 The effect of tigecycline on cell migration and invasion was detected by wound-healing assay and transwell assays after treating with DMSO or tigcycline for 24h、48h、72h.In order to explore the mechanism of the cell migration and invasion induced by tigecycline,Western Blot assay was performed to assess the EMT-related protein levels in A375 and MV3 cells,respectively after treated wih tigecycline(0μmol/L、2μmol/L 、5μmol/L、10μmol/L).4 The effect of tumorigenic capacity of tigecycline on A375 and MV3 melanoma cells was verified by soft agar assay and the tumor formation in vivo of mice.5 The effect of overexpressed p21 infected with the lentivirus system influence the cell proliferation,colony formation retardation,cell cycle as well as migration and invasion after treated wih tigecycline.Results:1 Both the cell counting and MTT assay demonstrated tigecycline could inhibit the proliferation of A375 and MV3 cells at a concentration(0μmol/L,2μmol/L,5μmol/L,10μmol/L)-dependent effect significantly(P<0.05).BrdU immunofluorescence staining further confirmed that compared to the control group,the percentage of BrdU-positive cells of the group treated with tigecycline reduced significantly(P<0.05).2 The PI staining and the flow cytometry show there is a significant increase in the proportion of cells in the G0/G1 phases after treated with tigecycline,compared to the control group(P<0.05).Western Blot assay of the cell cycle-related protein levels showed that the expression levels of Cyclin E and CDK2 as well as p21 were decreased in tigecycline treated cells in a doseand time-dependent manner.3 The wound-healing assay and transwell assays show the tigecycline groups significantly suppressed the cell migration and invasion abilities compared to the control group in a time-dependent manner(24h、48h、72h)(P<0.05).Western Blot assay of the EMT-related protein levels showed that the expression levels of E-Cadherin increased,as well as expression levels of Vimentin decreased in tigecycline treated cells in a dose-and time-dependent manner.4 Soft agar colony assay showed that compared with the control group,tigecycline inhibited clone formation significantly(P<0.05).The study of A375 and MV3 melanoma cells in vivo xenografted in the female BALA/c nude mice model showed that the volume and weight of xenograft tumor were remarkable decreased after treated with tigecycline,compared with control group(P<0.05).5 Overexpression of p21 infected with the lentivirus system showed that compared to tigecycline-treated vector groups,p21 overexpressing groups promoted cell proliferation,boosted cell migration and invasion and formed more colonies in soft agar in tigecycline-treated cells,while had no significance changes compared with DMSO-treated vector groups.Conclusion:1 Tigecycline dramatically inhibited cell proliferation and induced cell cycle arrest at G0/G1 phase.At the same time,tigecycline suppressed cell invasion and migration through preventing epithelial-mesenchymal transition(EMT)process.In addition,tigecycline also significantly blocked tumor growth in vivo.Tigecycline may be an effective and promising therapeutic agent for melanoma treatment.2 p21 play an important role in tigecycline-induced proliferation inhibition,colony formation retardation,cell cycle arrest as well as migration and invasion suppression.Overexpression of p21 significantly rescued the tigecycline-induced proliferation inhibition,colony formation retardation,cell cycle arrest as well as migration and invasion suppression.Tigecycline inhibited proliferation,cell cycle and migration/invasion of human melanoma cell lines were p21-dependent,p21 is expected to be used as a key target genes for the treatment of melanoma. |
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