Effect Of Juglone On Biological Behavior Of Human Melanoma Cells&Preparation Of Juglone-loaded PLGA Nanoparticles And It’s Antitumor On Melanoma

Part1 Effect of Juglone on biological behavior of human melanoma cells Objective:To explore the effects of Juglone(Jug)on the biological behavior of malignant melanoma cell lines and the related mechanisms.Methods:The inhibition rate of Jug on A375 and A2058 malignant cells was determined by MTT method and the IC50 value was calculated.Flow cytometry was used to detect the effect of Jug on the cycle and apoptosis of malignant melanoma cells.Western blot method was used to detect the effect of Jug on the expression of apoptosis-related proteins Bax,Bcl-2 and CyclinD1.Scratch test and Transwell chamber test to detect the effect of Jug on invasion and migration of melanoma cells.Results:Jug significantly inhibited the proliferation of A375 and A2058 cells,and there were significant time and concentration dependent effects.The IC50 values of 24h were:3.31μg/mL and 2.81μg/mL;48h IC50 values were:2.33μg/mL,2.16μg/mL.Jug can induce apoptosis of A375 and A2058 malignant cells by increasing the expression of proapoptotic protein Bax and decreasing the expression of anti-apoptotic protein Bcl-2.Jug can arrest the A375 and A2058 malignant cell cycle in the G0/G1 phase by affecting the expression of the cycle-associated protein CyclinD1.Jug can significantly reduce the invasion and migration ability of A375 and A2058 cells.Part2 Preparation of Juglone-Ioaded PLGA nanoparticles and it’s antitumor on melanomaObjective:To evaluate the anti-tumor effect and biological safety on A375 malignant melanoma cell line in vivo and in vitro,the Jug-PLGA nanoparticles were prepared and their physicochemical properties were investigated.Methods:Jug-PLGA-NPs were prepared by emulsification and volatilization method,and their particle size,encapsulation efficiency,drug loading rate and in vitro release characteristics were investigated.Fluorescence microscopy was used to observe the uptake of PLGA-NPs in vitro.The small animal live imager was used to observe the distribution of PLGA-NPs in the BALB/c tumor-bearing nude mice.MTT method was used to compare the inhibitory effect of Jug-PLGA-NPs and Jug on the proliferation of A375 malignant cells,and the apoptosis rate was detected by flow cytometry.The subcutaneous tumor model of A3 75 nude mice was constructed to evaluate the antitumor effect of Jug and Jug-PLGA-NPs.The visceral tissue was observed after sacrifice,and CyclinD1 protein immunohistochemistry was detected and analyzed.Results:The prepared Jug-PLGA-NPs had an average particle size of 149.6 nm,an entrapment efficiency of 68.39%,and a drug loading rate of 5.07%,and had good sustained-release characteristics.PLGA-NPs have good penetration and targeting properties in cellular uptake in vitro and drug distribution in vivo.Different concentrations of Jug-PLGA-NPs could significantly inhibit the proliferation and promote apoptosis of A375 cells,and they were significantly time-dependent(P<0.05),and slightly superior to the equivalent concentration of Jug at 48h.In the animal experiment,both Jug and Jug-PLGA-NPs showed good anti-tumor effect,while the nano-particle group had better anti-tumor effect and better tolerance in mice.Conclusion:The Jug-loaded PLGA nanoparticles are easy to prepare,have good drug release,tumor targeting and anti-tumor ability in vitro and in vivo,and the biosafety is acceptable.It provides a new pharmaceutical dosage form for the future clinical application of Jug.