Research On Mechanism Of Apoptosis Induced By 5-Aminolevulinic Acid-Based Photodynamic Therapy On A375 Cells And A431 Cells

Objective: To investigate the killing effect and mechanism of 5-aminolevulinic acid-mediated photodynamic therapy on human melanoma A375 cells and human epidermoid carcinoma A431 cells. Methods: Human melanoma A375 cells and human epidermoid carcinoma A431 cells were employed in present study. A375 cells and A431 cells were incubated in different concentration of ALA. The killing effects of ALA-PDT on A375 cells and A431 cells was detected by MTT colorimetric assay. TdT-mediated dUTP nick end labeling(TUNEL) method was performed to detect the proportion of apoptosis cells. The protein expression level of Bcl-2, Bax, Caspase3, Caspase8 and Caspsase9 were determined by Western blot. Lastly, Cytochrome c(Cyt-c) subcellular localization was compared by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT. Results: The results of MTT colorimetric assay showed that ALA-PDT presented significant anti-proliferation effects on both A375 cells and A431 cells in a dose and time dependent manner. When incubated in 0.4mmol/L ALA and at 4h after ALA-PDT, the proliferation inhibition of A375 cells reached greatest degree. Meanwhile, when incubated in 0.6mmol/L ALA and at 8h after ALA-PDT, the proliferation inhibition of A431 cells reached greatest degree. A375 cells were more sensitive to PDT than A431 cells. The phenomena of apoptosis were observed in ALA-PDT-cells by TUNEL assay. The apoptosis rates of A375 cells increased to11.79%, 44.84%, 63.75%, 75.33% and 90% respectively at 0.5h, 1h, 2h, 4h and 6h after ALA-PDT. The apoptosis rates of A431 cells increased to 10%, 25%, 55.5%, 61.54% and 70% respectively at 1h, 2h, 4h, 6h and 8h after ALA-PDT. Western blot discovered that apoptosis induced by ALA-PDT involved up-regulation of Bcl-2 protein and down-regulation of Bax protein obviously. It was observed that the protein expression of cleaved-Caspase3, 8, and 9 protein and cleaved-PARP increased at 0.5h, 1h and 2h after ALA-PDT in A375 cells. It occurred in A431 cells at 1h, 2h and 4h after ALA-PDT. Immunohistochemical localization of Cytochrome c found that Cyt-c was released from mitochondrian into the cytosol in apoptosis cells. Conclusion: ALA-PDT could significantly inhibit proliferation of both A375 cells and A431 cells. And PDT could also induce apoptosis in the two cells. The apoptosis induced by ALA-PDT was related to the Caspase-dependent death-receptor pathways and Cytochrome C-dependent mitochondrial pathways.