Protective effect of autophagy in laser-induced glioma cell death in vitro.
OBJECTIVE
Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser-induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple-membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser-induced tumor cell death in vitro.


METHODS
The monolayers of U251 human glioma tumor cells were exposed to 532 nm laser light from a single mode frequency-doubled Nd-YVO4 laser. A flattened Gaussian radial profile of laser beam (0.5-4 W) was used to uniformly illuminate entire colony of cells for various amounts of time (15-120 seconds) in the absence of cell culture medium. The cells were grown for 24 hours and the cell viability was determined by crystal violet or MTT assay. The presence of autophagy was assessed after 16 hours by fluorescence microscopy/flow cytometric analysis of acridine orange-stained autophagolysosomes and Western blot analysis of the autophagosome-associated LC3-II protein. The concentration of the principal pro-autophagic protein beclin-1 was determined after 6 hours by cell-based ELISA.


RESULTS
The intracytoplasmic accumulation of autophagic vesicles, increase in LC3-II and up-regulation of beclin-1 expression were clearly observed under irradiation conditions that caused approximately 50% cytotoxicity. Post-irradiation addition of three different autophagy inhibitors (bafilomycin A1, chloroquine, or wortmannin) further increased the laser-induced cytotoxicity, without affecting non-irradiated cells.


CONCLUSIONS
These data indicate that beclin-1-dependent induction of autophagy can protect glioma cells from laser-mediated cytotoxicity.
Krmpot
Aleksandar J
AJ
Institute of Physics, University of Belgrade, 11080 Belgrade, Serbia. krmpot@phy.bg.ac.rs
Janjetovic
Kristina D
KD
Misirkic
Maja S
MS
Vucicevic
Ljubica M
LM
Pantelic
Dejan V
DV
Vasiljevic
Darko M
DM
Popadic
Dusan M
DM
Jelenkovic
Branislav M
BM
Trajkovic
Vladimir S
VS
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D000071186:Beclin-1; D015153:Blotting, Western; D049109:Cell Proliferation; D002470:Cell Survival; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005910:Glioma / Q000473:pathology*; D006801:Humans; D006979:Hyperthermia, Induced; D066298:In Vitro Techniques; D053685:Laser Therapy / Q000295:instrumentation / Q000379:methods*; D053844:Lasers, Solid-State; D008856:Microscopy, Fluorescence; D009336:Necrosis; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C512829:Becn1 protein, rat