In Vitro And In Vivo Anti-uveal Melanoma Activity Of JSL-1,a Novel HDAC Inhibitor

Background: Uveal melanoma(UM)is the most common primary intraocular tumor in adults.The tumor can be classified into the choroid,ciliary body and iris melanoma according to its origin.However,the choroid is the most common location of patients.High metastasis rate is the major hallmarks of uveal melanoma and the main cause of death in UM patients,approximately 50% of patients ultimately develop liver metastasis.Because of lackage of effective therapies,the prognosis of UM patients with liver metastasis is very poor.The median overall survival of patients with metastasis is between 2 to 15 months.Enucleation,radiotherapy and chemotherapy are options for the treatment of UM patients.Chemotherapeutic agents include vinblastine,cis-platinum and fotemustine.However,all these therapeutics have limited improvement of event-free survival in UM patients.Therefore,novel targeted-therapeutic agents to treat uveal melanoma are extremely needed.JSL-1 is a novel HDAC inhibitor.Inhibition of HDAC activities by HDAC inhibitors can result in hyperacetylation of histones and chromatin remodeling,which leads to the transcription of genes involved in cell growth,differention and apoptosis.Numerous studies have shown that HDAC inhibitors exert potently anti-tumor activities in vitro and in vivo.Moreover,HDAC inhibitors exhibit low toxicity to the normal tissues.The HDAC inhibitor Suberoylanilide hydroxamic acid has been approved by the FDA as a drug to treat cutaneous T cell lymphoma.Currently,the study of HDAC inhibitors on uveal melanoma is rarely reported in the world.Therefore,to give an insight into the investigation of the anti-tumor activities of JSL-1,a novel HDAC inhibitor,provides theoretical and clinical practical basis for the treatment of uveal melanoma.Objective: 1.To explore the anti-tumor activities of the novel HDAC inhibitor JSL-1 in uveal melanoma and elucidate its mechanism.2.To provide theoretical and clinical practical basis for the development of effective agent for the treatment of uveal melanoma.Methods: 1.The protein levels of acetyl histone H3,H4 and the tumor suppressor p53 were analyzed by western blotting.The transcription levels of the p53 downstream molecules p21,Puma and Noxa were detected by q RT-PCR.2.The proliferation of UM cells 92.1,Mel270,Omm1 and Omm2.3 treated with JSL-1 were detected by MTS assay and soft agar colony fomation assay.3.The effects of JSL-1 on apoptosis and mitochondrial membrane potential in the UM cells 92.1,Mel270,Omm1 and Omm2.3 were analyzed by flow cytometry,and the protein levels of specific-apoptosis proteins,related-apoptosis proteins and the release of AIF and Cytochrome C in the Cytoplasm were detected by western blotting.4.The migration and invasion ability of uveal melanoma cells 92.1,Omm2.3 were evaluated by wound-healing and transwell assay,the protein levels of the Matrix metalloproteinases MMP-2 were detected by immunoblotting.5.The self-renew and replating abilities of uveal melanoma cells 92.1,Mel270,Omm1 and Omm2.3 were analyzed by tumor sphere formation assay.The percentage of ALDH+ cells were detected by flow cytometer.To explore the mechanism,the protein levels of ?-catenin in the nuclear and cytoplasm were detected by western blotting.6.The xenograft model of NOD-SCID mice was used to evaluate the anti-tumor activities of JSL-1 in vivo.Results: 1.JSL-1 dose-dependently increased the protein levels of acetyl histone H3,H4 and p53.And the transcription levels of p53 downstream molecules p21,Puma,Noxa were upregulated by JSL-1 in uveal melanoma cells Mel270 and Omm1.2.The proliferation of UM cells 92.1,Mel270,Omm1 and Omm2.3 were inhibited by JSL-1 in a dose dependent manner,the value of IC50 in 92.1,Mel270,Omm1,Omm2.3 cells were 90 n M,39 n M,53 n M and 83 n M,respectively.Also,JSL-1 dose-dependently inhibited thecolony formation of UM cells 92.1,Mel270,Omm1 and Omm2.3 in the solft agar.3.JSL-1 induced apoptosis in UM cells 92.1,Mel270,Omm1 and Omm2.3 in a dose-and time-dependent manner,JSL-1 reduced the protein levels of anti-apoptosis proteins XIAP,Bcl-XL,Bcl-2,Survivin and increased the expression of pro-apoptotic protein Bim.JSL-1 broke the balance of mitochondrial membrane potential,caused apoptosis inducing factor(AIF)and cytochrome C releasing into the cytoplasm.4.JSL-1 obviously suppressed migration and invasion of UM cells 92.1 and Omm2.3.The mechanism is involved in the downregulation of MMP-2 expression.5.JSL-1 evidently inhibited the melanosphere formation and serially replating capability of UM cells 92.1,Mel270,Omm1 and Omm2.3.Additionally,JSL-1 also decreased the percentage of ALDH+ cells with Wnt/?-catenin signalling suppressed.6.JSL-1 inhibited the growth of UM cells Omm1 in the xenograft NOD-SCID mice.Conclusions: 1.JSL-1 inhibits the activities of HDAC in UM,which leads to the acetylation of histone H3,H4 and p53.2.JSL-1 can effectively inhibit the proliferation of UM cells.3.JSL-1 induces apoptosis in the UM cells through the intrinsic pathway.4.JSL-1 suppresses migration and invasion of UM cells.5.JSL-1 inhibits the self-renewal ability of UM cells in association with the classical Wnt/?-catenin pathway blocked.6.JSL-1 strongly inhibits the growth of uveal melanoma cells in the xenograft NOD-SCID mice.