5-Aminolevulinic Acid Mediated Photodynamic Therapy Of Nasopharyngeal Carcinoma (NPC)

Nasopharyngeal carcinoma(NPC)is very common in southeast of China,especially in Guangdong and Guangxi province,with a incidence up to 30/10~5.Most of the NPC patients are diagnosed in middle and advanced stage because of its early atypical symptoms,and the 5-yearl-survival rate is only 50%～70%after radiotherapy and chemotherapy,with severe complications. Thus other new method should be taken to improve the survival rate with less severe complications.Photodynamic therapy(PDT)has been applied since 1980s',and people more and more concerned about PDT because of it's well-selective,of less toxicity,rapid and reliable effectiveness.As the application of new photosensitizer,improvement of laser producing machine, PDT has become a new effective option to treat NPC.5-aminolevulinic acid(5-ALA)is one of prophotosensitizer,and then converted to HaematoporphyrinⅨ(PpⅨ)accumulating in for example tumor tissue.It is the PpⅨthat has a strong activity of photosensity.Our study is focused on feasibility of 5-ALA induced PDT in treating NPC in vivo or in vitro.Part One Cytotoxicity of 5-ALA induced PDT on NPC cell line of CNE2Objective:to investigate cytotoxicity of 5-ALA induced PDT on NPC cell line of CNE2,and intracellar metabolic procedure of 5-ALA to PpⅨ,toxicity of 5-ALA,and it's otipmal time and dose of cytotoxicity on CNE2.Method:1.Fluorescence Spectrophotometer was applied to detect the metabolic procedure of 5-ALA in tumor cells of CNE2.2.Laser Scanning Confocal Microscope was applied to detect the location of PpⅨafter it's precursor 5-ALA being intaked into CNE2 cells.3.MTT[3-(4,5-dimethy1 thiazol-2-y1)-2,5-diphenyl tetrazolium bromide] assay is applied to determine cytotoxicity of 5-ALA on CNE2,effects of laser on cells without photosensitier,effects of PDT dose,effects of time of incubation of 5-ALA,effects of serum on 5-ALA PDT.4.Flow cytometer is applied to detect cytotoxicity of 5-ALA PDT on CNE2.5.Light microscope and transmisson electromicroscope to detect the changes of CNE2 cells after 5-ALA PDT.Result:1.Emission of 635nm red light was detected in tumor cells by fluorescence Spectrophotometer after exposuring to 409nm violet light.and PpⅨmerely existed intracellarly;incubation of concentration of 2mmol/L 5-ALA with tumor cells can obtain highest cytotoxicity,amount of PpⅨproduced increased is proportion positively to the time of incubation of 5-ALA.2.Laser Scanning Confocal Microscope showed PpⅨmainly located on cell membrane and in cytoplasm.3.the result of MTT assay showed that no effects of incubation with only 5-ALA or exposure to laser on CNE2 cells growth(P＞0.1);under a certain concentration of 5-ALA and incubation time,PDT dose of 1J,2J,5J,8J is 35.3±4.6%,55.0±3.2%,92.0±6.2%,85.5±10.9%respectively;under a certain intensity of irradiation of laser,effectiveness of PDT was positively in proportion to time of incubation with 5-ALA,and reached maximum at the time point of 8h.Animal serum affected markedly on the cytotoxicity of 5-ALA PDT,which can be weakened by increasing photosensitier dose and intensity of laser exposure. 4.Flow cytometer showed that under different dose of 5-ALA,intensity of laser exposure and with or without serum,different apoptosis rate was detected: 2mmol/L 1J 62.3%,2mmol/L 2J 74.5%;2mmol.L 10%FBS 2J 11.1%; 4mmol/L 10%FBS 2J 28.8%;6mmol/L 10%FBS 2J 75.5%.5.Under the light microscope,CNE2 cells turned round and small.in minutes after PDT,many vacuoles can be detected in cytoplasm,with nuclear membrane getting thicker;8h after PDT,tumor cells began to break up into thin particles.Transmission electromicroscope displayed chromatin condensation and aggregation under the nuclear membrane,nuclear fragmentation and apoptosis body formation,and swelled mitochondrial was also detected.Conclusion:1.5-ALA-induced PpIX mainly accumulated in cell membrane and cytoplasm, 2mmol/L of 5-ALA is the optimal concentration of cytotoxicity,8h of incubation of 5-ALA got the highest cytotoxicity on CNE2.2.It suggested that incubation of 5-ALA without laser irradiation or laser irradiation without 5-ALA administration did not induced any damage to CNE2.Higher intensity of laser irradiation strengthens cytotoxicity,which peaked under energe of 5J.It seems that under a certain concentration of 5-ALA incubation and intensity of irradiation,the cytotoxicity effects could be enhanced when incubation time with 5-ALA was lengthened,which peaked at the time point of 8h;serum inhibited markedly the effect of 5-ALA PDT.3.It suggested that apoptosis is the main mechanism of 5-ALA cytotoxicity on CNE2 ceils,over high dose of 5-ALA or laser irradiationt could cause necrosis. Part Two Study on the efficacy of 5-ALA-PDT for the treatment of nasopharyngeal carcinoma transplanated in Nude miceObjective:establish model of nude mice bearing CNE2 cell to investigate the metabolic procedure in vivo,and inhibition of tumor growth by 5-ALA PDT administrated intratumorally or intraperitoneally.Methods:1.Developed CNE2 cell in vitro,inoculated the cells into right axilla of nude mice subcutaneously.2.Fluorescence Spectrophotometer was applied to detect metabolic process of 5-ALA in nude mice bearing tumor.3.Tumor cells inoculation:when tumor beared got 0.1～0.15cm3,nude mice were divided randomly into 4 groups,group A,D:were administrated with 20%100mg/kg 5-ALA intratumorly,group B:20%100mg/Kg intraperitoneally;group C:control.3～3.5 hours after inoculation,group A,B,D were exposured to irradiation of laser(100mw/100J,630nm),and then group D were killed 24 hours later;volume of tumors beared by group A, B,C were measured on 1,3,7,10,14 days after irradiation,and the 3 groups were all killed,tumors were retained and weighed.4.Tumor tissues of the 4 groups were detected under transmission electroscope, and were embedded in paraffin Wax,slices were prepared and studied pathologically.Result:1.All nude mice were transplanted with CNE2 successfully,tumor lesions all reached 0.1-0.15cm3 in 8～10 days after inoculation.2.5-ALA mainly accumulated in tumor tissue and liver of nude mice after administration,and then turned into PpⅨ.Concentration of PpⅨwas higher in liver than in tumor tissue,peaking 1 hour after 5-ALA injection,and then began to drop,reaching the level the same as that in tumor tissue;in tumor tissue,PpⅨwas detected 1 hour after 5-ALA administration,and gradually increased to summit at the time point of 3-4 hours,dropping thereafter;a little amount of PpⅨwas detected in tissue of paratumor,muscle,lung and kidney with little detected in the organ of heart.Ratio of amount of PpⅨin normal tissue(except for liver)to that in tumor tissue is 1:2～4.3.5-ALA-PDT did not cause any side effects in nude mice,tumor volume of the group treated was markedly bigger than the control groups;on the 14th day after PDT,tumor weight(gram)of group A,B,C3 was 1.353±0.204, 2.105±0.255,3.124±0.380,with significant difference(P=0.000).4.Under transmission electromicroscope,tumor cells from treated group shrinked,with many vacuoles in cytoplasm,and chromatin condensation and aggregation under the nuclear membrane;nuclear fragmentation and apoptosis body formation,swelled mitochondria was also seen.Necrotic cells were also seen.Pathological slices showed in the tissues being irradiated, blood vessels dilated with diffuse hemorrhage,many vacuoles in cytoplasm, multi-lesion of necrosis and apoptosis.On 14th days after PDT,mass coagulation necrosis lesions were detected under light microscope,with shrinked nucleius,even with nucleius remains,without pigmentation with haematoxyl.Conclusions:1.Model of nude mice bearing CNE2 was established successfully.2.5-ALA was distributed mainly in the tissue of liver and tumor after administration,and concentration of PpⅨpeaked 1 hour after injection of 5-ALA in liver,and 3-4 hours in tumor.3.5-ALA PDT is a safe technique in treating NPC,showing a remarkable cytotoxicity on CNE2 in our study,with better effects in group administrated intratumorly than in group administrated intraperitoneally.4.The results of transmission electroscope confirmed that 5-ALA PDT induced apoptosis and necrosis of CNE2 cells.Part three Effects of 5-ALA PDT on expression of relative genes in CNE2 cell line.Objective:to investigate expression of relative genes in transplated CNE2 cells after 5-ALA PDT.Methods:1.real-time quantitiy PCR was applied to detect shifts of VEGF,PCNA, Caspase3,Caspase8,Caspase9 gene in mRNA level.2.Immunohistochemistry technique was applied to detect expression and distribution of VEGF,PCNA protein.3.Western-blot was applied to detect expression of VEGFA,PCNA,Caspase9 protein and their state of activity.Results:1.In acute and chronic stage,level of expression of PCNA,Caspase9 mRNA was higher in treated group than that in control groups;2.Results of immunohistochemistry showed that,in acute stage(1 day after administration of 5-ALA-PDT),level of VEGFA was slightly higher in group administrated intraperitoneally than that in control,while that in group administrated intratumorly was slightly lower(P＞0.05).PCNA was down-regulated in tumor cells in acute stage for abnormal metabolism,and in chronic stage,PCNA expression in survival tumor cells of treated group was higher than that in control group,but p=0.386,there is no significant statistical difference.3.Results of Western-blotting showed that,VEGFA protein was down-regulated in chronic stage in group administrated intratumorly,while was up-regulated in acute stage;level of PCNA protein keeped stable in both acute and chronic stage;Caspase9 activated increased.Conclusion:1.VEGFA has no statistical difference in mRNA level;VEGFA protein was slightly up-regulated on 1st day(acute stage)and down-regulated remarkablly 14 days after PDT in chronic group,but there is no significant statistical difference between these four groups;2.Level of PCNA mRNA expression was higher in treated group than that in control group in both acute and chronic stage(P=0.000);total amount of PCNA protein did not change,but decreased in nuclear of tumor cells in acute stage,suggesting the shift in function of PCNA protein.3.Caspase9 mRNA expression was higher in treated group than that in control group,(P=0.021);amount of pro-Caspase9 protein dropped while Caspase9 activated increased.