| Melanoma is one of the most aggressive and deadly malignant tumor and one of the most rapidly increasing malignant tumor.Although the incidence of melanoma in China is relatively low,in recent years,the number of new cases can get twice,The annual growth rate is 3-6%.In addition to the definite effect of surgical resection of early melanoma lesions,there are many therapeutic methods for melanoma,including immunotherapy and individualized targeted therapy.But the clinical outcome is still very regrettable.The main reason is the resistance of melanoma to conventional radiotherapy and chemotherapy.Because the clinical treatment of advanced melanoma is very limited,and once the disease worsens,the tumor growth is too fast to be stopped,it is easy to occu r systemic metastasis,the possibility of the final cure is very low,seriously affecting people's health and psychological bear.It is the focus of current research to find an effective new adjuvant therapy for melanoma to inhibit its proliferation and invasion.Photodynamic therapy is a new model for the treatment of malignant tumor in recent years.Due to selective uptake of photosensitizer through local lesions and appropriate wavelength of light,target cell apoptosis and necrosis are caused by photose nsitizer mediated involvement with oxygen molecules and oxidative damage.It has been widely used in the treatment of esophageal cancer,skin cancer,cervical cancer and other tumors,and is expected to become a conventional adjuvant therapy for a variety of malignant tumors.Experimental studies have found that PDT may be an effective adjunct to the treatment of melanoma in the future.However,its effect on the proliferation and invasion of melanoma is rarely reported,and the specific mechanism is still unclear,so further research is needed.In this respect,To indicate 5-ALA PDT effect on melanoma cell migration and invasion ability,we used the melanoma cells B16-F10 and A375 for research.Firstly,this study observed the photosensitizer 5-ALA could sensitization PDT which affect melanoma cell proliferation,and discuss its relevant mechanism and provide theoretical basis for photodynamic therapy of melanoma.I.Inhibitory effects and mechanism of ALA-PDT on migratory and invasive abilities of melanoma cells A375(human)and B16-F10(mouse)1.ALA-PDT inhibited the migration and invasion of melanoma cells A375 and B16-F10 in vitro: The migration rate of B16-F10 and A375 cells treated with 2.4j and 4.8j ALA-PDT for 12 h and 24 h was significantly lower than that of the blank control group(p<0.001).At the same time,Transwell assay was used to detect the invasion capacity of melanoma cells.It was found that after 24 h of treatment with 2.4j and 4.8j ALA-PDT,the number of B16-F10 and A375 cells passing through Matrigel was less than that of A375 cells in the blank control group(p<0.001).2.After ALA-PDT treatment,A375,B16-F10 autocrine inflammatory factor m RNA was down regulated: the results of real-time quantitative PCR and ELISA showed that the expression levels of TGF-?,IL-1,IL-8,IL-6 and TNF-? were significantly decreased(p < 0.05),while the expression levels of IL-1,IL-8,IL-6 and TNF-? were not significantly changed in the two cell lines,but the level of TGF-? m RNA was down regulated in different degrees in the two cell lines treated with photodynamic therapy.3.Down-regulation about autocrine inflammatory factor proteins of A375 and b16-f10 after ALA-PDT treatment: 2.4J and 4.8J ALA-PDT treated B16-F10 and A375 cells for 12 h and 24 h respectively,and then detected the levels of IL-1,IL-6,IL-8,TNF-?,TGF-? protein in A375 and B16-F10 autocrine inflammatory cells by ELISA.It was found that only TGF-? protein,which was dose-dependent on the energy and time of photodynamic therapy,was down regulated in different degrees(P < 0.05).4.PDT combined TGF-?,there was no significant difference in the abilities of migration and invasion: 10 ng/ml TGF-? was added back for reverse validation.There was divided into four groups: blank control group,TGF-? group,4.8J PDT group,4.8J PDT+TGF-? group.The wound-healing assay was used to detect that TGF-? reversed PDT and inhibited migration of A375 and B16-F10(P < 0.05);Transwell chamber test was used to detect that TGF-? reversed PDT and inhibited invasion of A375 and B16-F10(P < 0.05).II.ALA-PDT inhibits the proliferation of melanoma cell line B16-F10(mouse)and its mechanism1.Photodynamic effect mediated by photosensitizer 5-ALA is more obvious when 5-ALA increases the dose and prolongs the action time(1)B16-F10 cells were treated with 5-ALA concentration dependent(0,1.25,2.5,5,7.5,10 mmol/L)and action duration(2h,4h,24h).CCK-8 assay was used to detect the proliferation of B16-F10 cells treated with 5-ALA at different doses and at different times,but there was no significant effect on its proliferation(P > 0.05).(2)The melanoma cells(B16-F10)were treated with 5-ALA concentration dependent(0,2.5,5,10 mmol/L)and time-consuming(4 hours,24 hours),irradiated by 635 nm Red light.The proliferation of B16-F10 cells was detected by CCK-8.It was found that ALA-PDT could significantly inhibit the proliferation of B16-F10 cells(P < 0.05),and it was time-dependent and dose-dependent.2.5-ALA can inhibit the autophagy of melanoma cells by reducing the expression of autophagy related genes and proteins(1)RT-qPCR was used to detect the up-regulated expression of ATG3,ATG5,ATG7,LC3,Beclin-1 mRNA in melanoma cells(B16-F10)in concentration dependent(0,2.5,5,10 mmol / L)and for 4 hours and 24 hours,and the expression of p62 m RNA was down regulated(P < 0.05);(2)Western blot showed that the expression of ATG3,ATG5,ATG7,LC3,Beclin-1 protein increased(P < 0.05),while the expression of p62 protein decreased(P < 0.05);3.Effect of 5-ALA sensitized PDT on the proliferation of melanoma cells induced by rapamycin(1)Melanoma cells(b16-f10)were treated with 5-ALA(0mmol/L),5-ala(10mmol/L),RAPA(2.5ug/ml)and RAPA(2.5ug/ml)+ 5-ALA(10mmol/L)for 24 h.After exposure to red light at 635 nm,CCK-8 showed no significant difference in proliferation of RAPA+ ALA-PDT compared with the 5-ALA(0mmol/L)group(P>,0.05).(2)There were 4 groups of 5-ALA(0mmol/L),5-ALA(10mmol/L),RAPA(2.5ug/ml)and RAPA+ 5-ALA.After 24 h of treatment with them,the expression of LC3 in RAPA+ 5-ala co-treated group was decreased(P<0.05)and the expression of P62 protein was increased(P<0.05).Confocal focus was used to detect the formation of autophagos omes,and the expression of autophagosomes in RAPA+ 5-ala co-treated group was decreased compared with that in RAPA group alone(P<0.05).In conclusion,photodynamic therapy can significantly inhibit the migration and invasion of B16-F10 and A375 cells in vitro,which may be related to its inhibition of TGF-? autocrine in melanoma cells.At the same time,PDT inhibited the proliferation of B16-F10 more obviously when the dose of photosensitizer 5-ALA was increased,and autophagy inhibiting mediated by 5-ALA may be involved in sensitizing PDT against proliferation of B16-F10. |
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