5-aminolevulinic Acid Photodynamic Therapy In Inhibiting The Migration And Invasion Of A375 Melanoma Cells

Malignant Melanoma is a most common malignant tumor of skin mucous membrane and pigment membrane in clinical dermatology.It is also one of the malignant tumors with the fastest increasing incident and the highest mortality rate,which seriously threatens human health all around world.The primary melanoma usually originates from skin.The newly diagnosed patients are most with stageⅠorⅡmelanoma,but the five-year survival for patients appearing lymph node metastasis or distant metastasis will decrease sharply.Related study has shown the patient with lymph node metastasis or distant metastasis is an independent risk factor for poor prognosis.Therefore,prevention of primary metastasis is one of the important methods to improve the therapeutic effect of melanoma.Photodynamic therapy is an emerging therapeutic involving two steps:photosensitizers targeting to tumor cells and locally illumination with specific wavelength light to activate photosensitizers.Photosensitizers,such as 5-aminolevulinic acid,can produce reactive oxygen species leading to cytotoxic,including singlet oxygen,hydroxyl radicals and superoxide anions,and induce the destruction of cellular molecular biology through oxidative stress,leading to cell apoptosis or necrosis.Comparing with traditional treatment methods,ALA-PDT has many advantages of high selective and few complications.So it is widely used in the treatment of adenocarcinoma,non-melanoma and breast cancer,but the specific mechanism of the treatment of melanoma by ALA-PDT remains unclear.Epithelial-mesenchymal transformation is a highly conserved process in embryonic morphogenesis,which is highly related to the acquisition of mesenchymal characteristics of epithelial cells.EMT not only plays an important role in mesoderm formation,neural crest development and heart valve formation,but the invasion and metastasis of epithelial cell carcinoma.Therefore,we observe the effect of ALA-PDT on migration,invasion ability and EMT of melanoma A375 cell line,explore the possible mechanism of ALA-PDT in the treatment of melanoma.It can provide a theoretical basis for the clinical treatment method of melanoma.Methods1.CCK-8 method was used to detect the effect of different light intensity ALA-PDT on cell proliferation of melanoma A375 cell for 24h.2.The scratch test was used to detect the effect of different light intensity ALA-PDT on cell migration of melanoma A375 cell for 24h.3.Transwell was used to detect the effect of different light intensity ALA-PDT on cell invasion of melanoma A375 cell for 24h.4.Western Blot was used to detect the effect of different light intensity ALA-PDT on expression of epithelial mesenchymal transformation related protein E-cadherin,N-cadherin,Snail,Slug for 24h.5.Western Blot was used to detect the effect of different light intensity ALA-PDT on expression of TGF-β,Smad2,Smad3,PI3K/Akt and p38 in melanoma A375 cells after 24h.Results1.Treating with different light intensity(0J/cm~2,0.5J/cm~2,1.0J/cm~2,1.5J/cm~2,2.0J/cm~2,2.5J/cm~2)for 24h.ALA-PDT did not affect the cell proliferation of A375 cells when the light intensity was less than 1.5J.When the light intensity was 2.0J/cm~2 and 2.5J/cm~2,the cell proliferation decreased(9.31±1.16%)and(19.23±4.20%),respectively(p<0.01).2.The effect of ALA-PDT on the migration ability of melanoma A375 cells was detected by scratch experiment.After treatment with ALA-PDT under different light intensity(0J/cm~2,0.5J/cm~2,1.0J/cm~2,1.5J/cm~2)for 24h,the cell healing rate decreased by7.2%,13.7%,and 32.8%,respectively.In addition,the effect of ALA-PDT on invasion ability of melanoma A375 cells was detected by Transwell method.The cell invasion capacity decreased by 62.1%,78.1%,and 86.3%,respectively.3.After melanoma A375 cells treating with different light intensity ALA-PDT for 24h,Western Blot analysis showed that E-cadherin protein expression increased significantly and Snail protein expression decreased significantly,with significant difference,but no significantly difference in Slug protein expression.4.After melanoma A375 cells treating with different light intensity ALA-PDT for 24h,Western Blot analysis showed that TGF-β,Smad2,Smad3protein expression significantly decreased,and p-p38 and p-akt protein expression significantly increased,difference was statistically significant.Conclusions1.ALA-PDT inhibit the proliferation of melanoma A375 cells,showing a light-intensity dependent relationship.2.Low-power ALA-PDT can inhibit the migration and invasion of melanoma A375cells,showing a light-intensity dependent relationship.3.ALA-PDT can inhibit epithelial mesenchymal transformation of melanoma A375cells,and the mechanism may be related to the inhibition of TGF-β/Smad signaling pathway.