Primary Research For Photodynamic Therapy In Malignant Melanoma Treatment

Objective: Photodynamic therapy(PDT)is a special therapy applying photodynamic reaction to clinical treatment. It is a new therapy which is divided into two steps. First, a kind of photosensitizer or a precursor of photosensitizer (e,g 5-ALA or its derivates)which can produce endogenous photosensitizer is needed to administered. Second, exposed to laser with property wavelength and through a series of photochemistry reactions, photosensitizer is activated from ground state to excited state, leading to produce considerable active oxygen (mainly singlet oxygen), which will cause the inreversible damage of tumor. Nowadays, researchers have done a great deal of work in applying photodynamic therapy to treat tumors. But there are only a few reports on using photodynamic therapy to cure malignant melanoma. In order to provide the experimental evidence for further investigating the treatment of MM with PDT, we treated the malignant melanoma cell line A-375 with chlorin e6 and 5-ALA in vitro and in vivo, and observed the photodynamic effects caused by chlorin e6 and 5-aminolevulinic acid on malignant melanoma,Methods: (1) Experiments in vitro: Cultured human malignant melanoma A-375 cells were incubated in a medium containing various concentrations(2,4,7.5,15,30,60μmol/L) of chlorin e6 for 12h or precursor of photosensitizer(5-ALA) for 6h respectively, then illuminated with different light doses (2, 5, 10 and 20 J/cm~2) of a semiconductor laser (wavelength 652 nm; energy density of 15.6mW/cm~2 )for PDT. After incubated for 12h, the survival rates of human malignant melanoma A-375 were analyzed by MTT assay. (2) Experiments in vivo: we collected the cells of human malignant melanoma and inoculated the cells to abdominal part of the nude mice subcutaneously, according to 2 × 10~6 per mouse. After successful inoculation, the tumor beared nude mice were divided into 4 groups (the blank group, 5-ALA-PDT group, Ce6-PDT group and combinative PDT group). Each group contains 6 tumor-beared ones. During the experiment, the blank group was treated only with illumination and without PDT, the 5-ALA-PDT group was treated topically with of10% ALA in DMSO (60%) for 2h, the Ce6-PDT group was administered by the phosphate buffered saline solution of Ce6(7.5mg/kg) for lh, the combinative PDT group was treated topically with of 10% ALA in DMSO (60%) for lh, then in addition, administered by the phosphate buffered saline solution of Ce6 (7.5mg/kg) for 1 more hour. After these treatments each tumor-beared nude mouse was illuminated with semiconductor laser (wavelength 652 nm; light doses 100J/cm2). 2 weeks later, we killed all the nude mice, weighted the tumor, observed the metastasisd and sent the tumor, the livers, the spleens, the lungs, the kidneys for hisopathological examination.Results: ?The cytotoxcity of Ce6 on human malignant melanoma A-375 was little when there was no irradiation and the concentrations of chlorin e6 were less than 15 umol/L (p>0.05),but if the concentrations of chlorin e6 were more thanl5 umol/L,even if there were no irradiation,it can affect the livability of human malignant melanoma A-375 cells(p<0.01). While cultured human malignant melanoma A-375 cells were incubated in a medium containing 5-ALA,the effect on the melanoma A-375 was limited(p>0.05); ?Both 5-ALA induced protoporphyrin IX PDT and Ce6-PDT can kill the human malignant melanoma A-375 effectively in vitro. Higher phototoxicity was obtained with higher dye and/or laser doses. Among the various concentrations of chlorin e6(or 5-ALA) and laser doses, the livability of human malignant melanoma A-375 exist prominent discrepancy(p<0.01). But more laser doses than 10J/cm2 and more higher dye than 1.5mmol/L with 5-ALA, the phototoxicity caused by protoporphyrin IX do not become higher obviously; ?The experiments in vivo show, compared with the blank group, 5-ALA-PDT, Ce6-PDT and combinative PDT can inhibit the growth of the tumor effectively.(/?<0.01), 5-ALA-PDT has less ability in inhibitting the growth of the tumor than Ce6-PDT and combinative PDT; ?No side effects cause by phototoxicity on the skin of nude mice were found during the experiment. We also acquired that PDT can not affect the metastasis of melanoma.Conclusions: CDThe cytotoxcity of Ce6 on human malignant melanoma A-375 was little in low concentrations^ 15 umol/L),but in higher concentrations it can affect the livability of cells, 5-ALA do not effect livability of cells notablely even in higher concentration. ?Ce6-PDT and protoporphyrin IX PDT which was induced by 5-ALA can kill the human malignant melanoma A-375 cells effectively in vitro. Higher phototoxicitywas obtained with higher dye and/or laser doses. ?Both 5-ALA-PDT and Ce6-PDT can inhibit the growth of the tumor effectively in vivo. Both of them have little side effect. The Ce6-PDT seems more effective. Furthermore, the effect on inhibitting the growth of the tumor of the 5-ALA-PDT and Ce6-PDT can not be added up. @5-ALA induced protoporphyrin IX PDT and Ce6-PDT can not inhibit the metastasis of melanoma.