Intervention Effects Of (-)-Epigallocatechin-3-gallate On Photodamage Induced By Ultraviolet B In Primary Human Melanocytes

BackgroundUltraviolet radiation of sunlight,mainly including UVA and UVB in human's daily expouse,is the overwhelming factor contributing to sunburn, photoaging and photocarcinogenesis development.UVA radiation, penetrating deeper into dermis,can cause immunosuppression and lead to indirect DNA damage by reaction with reactive oxygen species(ROS). UVE radiation,the most energetic mutagenic and carcinogenic component of solar radiation,can induce direct DNA damage,gene mutation,ROS formation and local or systemic immunosuppression.Under the same dose of radiarion,the photo damage efficiency of UVB is 800～1000 times higher than that of UVA.It was thought melanoma was mainly associated with UVA radiation,but more and more epidemiology data and animal experiments indicated that UVB radiation is also very important in melanoma development.UVB radiation is absorbed directly by DNA,and gives rise to the formation of dimeric photoproducts between adjacent pyrimidine bases,namely cyclobutane pyrimidine dimers(CPDs,＞75%), pyrimidine(6-4)pyrimidone photoproducts(6-4PP)and Dewar isomers of 6-4PPs.Nucleotide excision repair(NER)is the main mechanism of DNA damage repair in mammalian cells for the removal of those DNA photoproducts.If the damaged cells are not complete repaired,the remaining photoproducts could induce immunosuppression,and at last cause tumor formation.(-)-Epigallocatechin-3-gallate or EGCG,the major active component of Polyphenols derived from green tea,has widely beneficial health effects,including antioxidation,ROS scavenge and especially anticarcinogenic activity in various tumor models.Many studies have confirmed that EGCG can stimulate the removal or repair of UVB-induced CPDs,thus EGCG can prevent photocarcinogenesis in mice skin.Our lab group also has done a series of researches in the last few years,studying the intervention effects of EGCG on photoproducts induced by UVB in HaCaT cells,fibroblast and Langerhans cells;yet there was no such detection on melanocytes.ObjectiveTo observe the growth of human primary melanocytes in M-254 medium plus HMGS-2.To investigate the production and clearance of UVB-induced photoproducts and the intervention effects of EGCG on human primary melanocytes.Materials and Methods1.Cell cultureHuman primary melanocytes were separated from foreskins and cultured in the melanocyte growth medium(mixture of M-254 500ml and HMGS-2 5ml).The MCs in the exponential growth phase were plated in 6 or 96-well plate with an equal number(10⁴～10⁵ cells/well)for the next step.2.UVB irradiation and EGCG treatmentAccording to the experiment design,30mJ/cm² UVB irradiation was conducted and melanocytes were cultured by EGCG before or after UVB irradiation.3.Cell morphology observationUVB irradiation impacts on cell morphology were observed by light microscope.4.Cell proliferation assayCell proliferation rates were detected by MTT assay after MCs were incubated with variable concentrations of EGCG for 72 h.The concentrations of EGCG for the following study were determined.5.Detections of CPDsThe formation of CPDs were qualitatively examined by immunofluorescence and immunocytochemistry assay at 0.5h after irradiated by UVB.The production and clearance of CPDs were quantitatively analyzed by immunodotblot assays at 0.5 and 24 hours after the treatment of UVB and EGCG.6.Flow Cytometry cell cycle analysisThe influence of UVB or/and EGCG on apoptosis rate and cell cycle arrest ??was detected by flow cytometry.Results1.Cell cultureWhen cultured by melanocyte growth medium(mixture of M-254 500ml and HMGS-2 5ml),MCs were mixed by many keratinocytes initially,but after differential selection by trypsin digestion for the passages,the MCs of the third generation almost reached 100%purity and the 3^rd～8^thgeneration of MCs developed 2～5 dendrites and behaved high proliferative activity.2.Cell morphology observation0.5h after irradiated by 30mJ/cm² UVB,no obvious cell morphological changes were observed.But 24h later,most MCs shrinked with shorter dendrites and loss of membrane integrity;several MCs condensed and their dendrites totally disappeared;some cells even detached from the culture plate.3.Cell proliferation assayLower concentration of EGCG(10 and 20mg/L)promoted the proliferation of cultured human MCs(P＜0.05),but there was no statistical significance between them.However,when MCs were incubated with EGCG over the 40 mg/L concentration,the proliferating activity was significantly decreased(P＜0.01);and most MCs showed shrinkage pictures with shorter dendrites under phase contrast microscope observation,both of which demostrated EGCG higher than 40mg/L suppressed cell growth. 30mJ/cm²UVB irradiation significantly reduced the proliferating activity of MCs(P＜0.05).The 10 and 20mg/L EGCG treatment can relieve this inhibitive effect,it suggested the possible photoprotective effects.Thus,10 and 20mg/L was considered as the EGCG concentration for the following research.4.Qualitative and quantitative analysis of CPDsPositive products were observed localizing in the whole nucleus of overall MCs when detected at 0.5h after UV irradiation by immunofluorescene microscopy and immunocytochemistry assay.Meanwhile,the control group showed negative results.When quantitatively examined by immunodotblot assay,the CPDs formations at 0.5h in UVB group, UVB+10mg/L EGCG group and UVB+20mg/L EGCG group were 100%, 101.23%and 103.31%,which has no statistical significance;but the remains of CPDs at 24h after irradiation showed statistical significance among 83.03%,75.09%,11.55%,respectively(P＜0.05).5.Flow Cytometry cell cycle analysisUVB irradiation increased apoptosis rate from 8.31%to 50.62%,and induced G₁ arrest.Treatment of 20mg/L EGCG reduced apoptosis rate to 39.26%,but arreat more MCs cells in G₁ phase for DNA repair. ConclusionsHuman primary melanocytes growed well when cultured in Medium 254 plus HMGS-2.EGCG can facilitate the clearance of photoproducts,but had no effect on the CPDs formation.