Abstract：Objective To investigate the role of caspase-3 in the apoptosis of squamous-cell carcinoma Colo-16 cells induced by 5-ALA-PDT.Methods Colo-16 cells in the logarithmic growth phase were divided into three groups: control group, 5-ALA photodynamic therapy (PDT) group, and caspase-3 inhibitor group (Z-DEVD-FMK). Control group was not given photosensitizer or light treatment. 5-ALA PDT group was exposed to 3-minute laser irradiation at a power density of 10 mW/cm2 and energy density of 2.5 J/cm2 after 4 hours of dark incubation with 0.4 mg/ml 5-ALA before being cultured for 12 hours. Caspase3 inhibitor group was treated in the same way as 5-ALA PDT group except that Z-DEVD-FMK at a final concentration of 40 μmol was added during incubation. Cells in each group were collected after experiment, the changes of intracellular caspase-3 fluorescence intensity were observed by immunofluorescence, and the percentage of positive cells and apoptosis rate of active caspase-3 were detected by flow cytometry.Results Immunofluorescence Results showed that there was weak green fluorescence in the cytoplasm of Colo-16 cells in control group and Caspase-3 inhibitor group, indicating a low expression of caspase-3, while green fluorescence was significantly enhanced in 5-ALA-PDT group, showing a significant increase of caspase-3 content in cytoplasm of Colo-16 cells. Flow cytometry Results showed that the respective percentage of active caspase-3 positive cells was (2.26±0.16)％,(32.16±1.31)％ and (3.36±0.31)％ in control group, 5-ALA PDT group and caspase-3 inhibitor group respectively. The difference was statistically significant (P<0.05). The apoptosis rate of control group, 5-ALA PDT group and caspase-3 inhibitor group was (2.35±0.22)％,(31.55±3.68)％ and (11.46±2.25)％, respectively. Paired comparison was made between the three groups, and the differences were statistically significant (P<0.01).Conclusions The apoptosis of Colo-16 cells induced by 5-ALA-PDT is closely related to the activation of caspase-3.
Dolmans D E,Fukumura D,Jain R K. Photodynamic therapy for cancer[J]. Nat Rev Cancer,2003,3(5):380-387.
[3]
Buytaert E,Dewaele M,Agostinis P. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy [J]. Biochim Biophys Acta, 2007, 1776(1): 86-107.
[4]
Garg A D,Nowis D,Golab J,et al. Photodynamic therapy: illuminating the road from cell death towards anti-tumour immunity [J]. Apoptosis, 2010, 15(9): 1050-1071.
[5]
Kim K H, Park J J. The effects of photodynamic therapy in upper-gastrointestinal malignant diseases [J]. Gut Liver, 2010, 4(Suppl1): S39-S43.
[6]
Tirapelli L F, Morgueti M, Cunha Tirapelli D P, et al. Apoptosis in glioma cells treated with PDT [J]. Photomed Laser Surg, 2011, 29(5): 305-309.
[7]
Castano A P, Demidova T N, Hamblin M R. Mechanisms in photodynamic therapy: part one-photo sensitizers, photochemistry and cellular localization [J]. Photodiagn Photodyn, 2004, 1(4): 279-293.
[8]
Yang L, Wei Y, Xing D, et al. Increasing the efficiency of photodynamic therapy by improved light delivery and oxygen supply using an anticoagulant in a solid tumor model [J]. Lasers Surg Med,2010,42(7):671-679.
[9]
Weiss A, den Bergh H V, Griffioen A W, et al. Angiogenesis inhibition for the improvement of photodynamic therapy: The revival of a promising idea [J]. Biochim Biophys Acta, 2012, 1826(1): 53-70.
[10]
Kessel D, Oleinick N L. Initiation of autophagy by photodynamic therapy [J]. Methods Enzymol, 2009, 453: 1-16.
[11]
Ji H T, Chien L T, Lin Y H, et al. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase [J]. Mol Cancer, 2010, 9(4): 91-101.
2017, Vol. 28 
