Study On The Role Of Nitric Oxide In5-aminolevulinic Acid Photodynamic Therapy-induced Apoptosis In Human Skin Squamous Cell Carcinoma A431Cells

Objective:To explore the effects of Nitric oxide in5-aminolevulinic acid photodynamic therapy (5-aminolevulinic acid-Photodynamic Therapy, ALA-PDT)-induced apoptosis in human skin carcinoma A431cells.Method:1. A431cells were cultured and the cell proliferation was detected by CCK-8assay.2. A431cells were cultured with the optimal concentration of1mM ALA and the optimal incubation time5h. The630nm red light with different light doses of2J/cm2and4J/cm2were illuminated on the A431cell. Phospholipid binding protein V (Annexin V)/propidium iodide (PI) double staining by flow cytometry was used after ALA-PDT at different time points (4h,12h,20h,24h) to detect the apoptosis in human skin squamous cell carcinoma A431cells.3. The expression of iNOS in A431cells at different time points was detected by Western Blot assay.4. The level of NO was detected by the Griss assay with A431cell culture supernatant which were collected at4h,12h,20h,24h after ALA-PDT.5. iNOS inhibitor1400w and the exogenous NO donor SNP were applied while ALA-PDT with the optimal light dose and the best observation point from previous experiment. The A431cell proliferation were detected by CCK-8assay.The morphological changes of apoptosis was observed with inverted phase contrast microscope and HE staining was used to detect the acidophila of the cells. Phospholipid-binding protein V (Annexin V)/propidium iodide (PI) double staining was used to detecte ALA-PDT-induced skin squamous cell carcinoma apoptosis of A431cells by flow cytometry.Results:1. We have successfully cultured A431cells, observed the normal cell form and drawn the survival curve.2. The flow cytometry showed that the apoptosis rate of A431cells in ALA-PDT group increased as time increased, and much higher in the light dose4J/cm2than2J/cm2in a time and dose dependent maner, which was of significant difference compared with other control groups (p<0.05). The necrosis of A431cell was not obvious with no significant difference (p>0.05).3. Western blot showed that the expression of iNOS increased as time increased in the light dose2J/cm2ALA-PDT group,which was significantly different compared with the control groups.4. The level of NO detected by Griss method increased as time increased in the ALA-PDT treatment group, and the light dose4J/cm2group produced more amount of NO than2J/cm2group in a time and dose dependent manner. It was statistically different when compared with other control groups (p<0.05).5. After iNOS inhibitor1400w and the exogenous NO donor SNP were given, the cell survival rate in ALA-PDT group,1400w combined ALA-PDT group, SNP combined ALA-PDT group and SNP alone group was significantly lower compared with ALA alone group, light alone group and blank control group, which was statistically significant (p<0.05). The cells survival rate of1400w combined ALA-PDT group was higher than ALA-PDT group, while that was lower in SNP combined ALA-PDT group than ALA-PDT group and SNP alone group,which was statistically significant (p<0.05).6. A Morphological observation was detected by inverted phase contrast microscope after given iNOS inhibitor1400W and exogenous NO donor SNP.It showed that cells shrinkage and round were seen in the1400w combined ALA-PDT group, ALA-PDT group and SNP combined ALA-PDT group, compared to cells in SNP alone group, ALA alone group, light alone group,1400w alone group and blank control group.The cells shrinkage and round were more obvious in SNP combined ALA-PDT group than the1400w combined ALA-PDT group and ALA-PDT group. ALA-PDT group was followed SNP combined ALA-PDT group,and1400w combined ALA-PDT group was relatively weak.7. HE staining under light microscope showed that cells eosinophilic staining enhancements were more obvious in1400w combined ALA-PDT group, ALA-PDT group and SNP combined ALA-PDT group than the SNP alone group, ALA alone group, light alone group, SNP alone group and the control group. And SNP combined ALA-PDT group showed the most obvious cells eosinophilic staining enhancements, which likeed scattered leaves. This group was followed by the ALA-PDT group and1400w combined ALA-PDT group was relatively weak.8. Flow cytometry showed that the rate of apoptosis in ALA-PDT group,1400w combined ALA-PDT group and SNP combined ALA-PDT group were higher significantly than that in ALA alone group,light lone group,1400w alone group and the control group (p<0.05).Conclusions:1. ALA-PDT can inhibit the proliferation of human skin squamous cell carcinoma A431cells. And Small doses of ALA-PDT mainly led to the cell death of squamous cell carcinoma A431through apoptosis in a certain amount of time and dose dependent manner. 2. A431cell line itself can release a small amount of NO and NO production in the process of ALA-PDT treatment increased in a certain amount of time and dose dependent manner.NO production can be limited by its upstream iNOS synthase, whose amount increased can lead to an increased release of endogenous NO, which can be released into the extracellular.3. Exogenous administration of NO donors to release NO plays a collaborative role with ALA-PDT in the ALA-PDT-induced apoptosis of A431cells.