Study Of Rho-GTPase On Dendricity And Melanin Transportation In Human Melanocytes And B16 Murine Melanoma Cells

PartⅠ: Effects of human purified ET-1 on the dendricityvia RhoA and Rac1 in cultured human melanocytesObjective To investigate the endothelin (ET) -1 level secreted bykeratinocytes after 311nm narrow-band UVB irradiation and the effectof human purified ET-1 on the dendricity by regulating the activityof RhoA and Rac1 in cultured human melanocytes.Methods MTT test was performed to detect the keratinocyticviability at various doses of 311nm UVB using. ELISA method was usedto detect the IL-la,ET-1 and bFGF level secreted by humankeratinocytes after 311nm UVB irradiation. The dendricity in humanmelanocytes were observed by phase contrast microscope. Pull downassay was performed to detect the activity of GTP-RhoA and GTP-Rac1in human melanocytes before ET-1 treatment and after treatment withET-1 for 5 min, 10 min, 30 min and 60 min respectively.Results The results of cell viability revealed that 311nm UVB 25and 50 mJ/cm~2 irradiation had little influence on the cell viability,as compared to the control group. However, 311nm UVB 100 and 200mJ/cm~2irradiation significantly reduced the survival rate of cultruedhuman keratinocytes (P＜0.01).The concentrations of IL-la and ET-1were significantly elevated in supernatants of cultured human keratinocytes irradiated with 311nm narrow-band UVB 25 and 50mJ/cm~2,compared with control groups. The number of dendrites considerablyincreased, more than 3 dendrites(63.67±5.51%) in human melanocytestreated with ET-1 50pg/mL, compared to control cells only with twoor three dendrites. Pull down analysis revealed that GTP-Rac1 proteinsignificantly increased by 3 times after treatment with ET-1 50pg/mLfor 10 minutes, then decreased a little but still remained elevatedafter treatment for 30 min and 60 min, compared to control cells.The expression of GTP -RhoA fell into one-third after treatment withET-1 50pg/mL for 10 min, then became elevated and almost reached thebaseline level after treatment for 60 min.Conclusion ET-1 can promote dendrite formation via dual pathwaysof activating Rac1 and surpressing RhoA in human melanocytes.PartⅡ: Effects of 311nm UVB on F-actin rearrangement anddendricity via activating Racl in B16 murine melanoma cellsObjective To investigate the effects of 311nm UVB irradiation onF-actin rearrangement and dendrite formation by regulating theactivity of Rac1 in B16 mouse melanoma cells.Methods MTT method was used to detect the cell viability at variousdoses of 311nm UVB irradiation on B16 mouse melanoma cells. Themorphological changes in B16 melanoma cells were observed by phasecontrast microscope and the cell cytoskeleton F-actin microfilamentwas observed by laser scanning confocal microscope(LSCM). Pull downassay was performed to detect the activity of GTP-RhoA and GTP-Rac1in B16 melanoma cells before UVB irradiation (0 min) and at 15 min, 30 min, 60 min and 120 min after irradiation respectively.Results The results revealed that 311nm UVB 25,50 and 100 mJ/cm~2irradiation had little influence on the cell viability, compared tothe control group. However, 311nm UVB 200 and 300mJ/cm~2 irradiationsignificantly reduced the survival rate of B16 melanoma cells(P＜0.01) .The morphological changes in B16 melanoma cells wereconsiderably evident with gobular cell body and increased dendriteslike tree branch at 24 hours following irradiation with 311nm UVB100mJ/cm~2 (P＜0.01) , compared to non- irradiated cells with two orthree dendrites. LSCM revealed that F-actin appeared organized withnumerous clear stress fibers crossing the cytoplasm innon-irradiated cells. However, these stress fibers became obscureas actin was disassembled after 311nm UVB 100 mJ/cm~2 irradiation intime-dependent manner. This event could be observed as early as 30min following irradiation and became more evident and showed punctatespots at 6 h. Pull down analysis revealed that GTP-Racl proteinsignificantly increased at 15 min and reached twice at 30 minfollowing 311nm UVB 100 mJ/cm~2 irradiation, then decreased a littlebut still remained elevated at 60 min and 120 min, compared to controlcells. However, GTP-RhoA changed a little during 30 min following311nm UVB 100 mJ/cm~2 irradiation, then became elevated at 60 min andreached 1.6 times at 120 min, compared with control cells.Conclusion 311nm UVB can promote dendrite formation via activatingRacl in B16 mouse melanoma cells.PartⅢEffects of arbutin and lcariin on dendricity inmelanocytes and melanin transfer in co-cultrue of melanocytes and keratinocytesObjective To investigate the effects of arbutin and lcariin ondendricity in human melanocytes and melanin transportation inco-cultrue of human melanocytes and keratinocytes.Methods MTT method was used to detect the cell viability inco-cultrue of human melanocytes and keratinocytes after treatmentwith arbutin and lcariin . The morphology in human melanocytes wasobserved by phase contrast microscope and pull down assay wasperformed to detect the activity of GTP-RhoA and GTP-Racl in humanmelanocytes before treatment with arbutin (0 min)and after treatmentfor 15 min, 30 min, 60 min and 120 min respectively. Flow cytometrywas performed to detect melanosome transfer in co-cultrue modelofhuman melanocytes and keratinocytes after treatment with arbutin andlcariin.Results MTT revealed that arbutin at the concetration of 320-640mmol/L significantly reduced the cell viability and had littleinfluence at 40-160mmol/L, as compared to the control group. Lcariinsinificantly increased the cell viability, however , the cellviability reduced at high concetration of lcariin (120-240mmol/L) .Melanocytes treated with arbutin (160mmol/L) for 48h showed dendritelength was 110.67±7.37μm, compared to untreated cells with267±10.15μm. Meanwhile melanocytes treated with lcariin exibitedlittle dendrite change. GTP-RhoA protein increased in some degreeafter treatment with arbutin for 15 min and spiked by 30 min , thendecreased a little. However, GTP-Racl showed no change aftertreatment for 15 min, 30 min, 60 min and 120 min. Melanosome transferwas inhibited in co-cultrue model in a concentration-dependentmanner. Arbutin in concentration of 160mmol/L gave 37.15±3.87% inhibition of melanosome transfer in the co-culture model.Conclusion Arbutin could inhibit dendrite elogation by activatingRhoA in melanocytes, resulting in the surpression of melanin transferin cocultrue of melanocytes and keratinocytes.