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Protective effect of autophagy in laser-induced glioma cell death in vitro
Aleksandar J Krmpot1, Kristina D Janjetovic, Maja S Misirkic
1Institute of Physics, University of Belgrade, 11080 Belgrade, Serbia. krmpot@phy.bg.ac.rs
Background And 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.
Study Design/Materials And 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.
Insights
Autophagy protects glioma cells from laser-induced death. Inhibiting autophagy increases laser-mediated cell death, suggesting a protective role for this cellular process in cancer therapy.
Area of Science:
- Oncology
- Cell Biology
- Biomedical Engineering
Background:
- Laser phototherapy shows promise for cancer treatment, but its cell death mechanisms are not fully understood.
- Autophagy, a cellular degradation process, plays a role in cell survival and death.
- Investigating autophagy's role in laser-induced tumor cell death is crucial for optimizing cancer therapy.
Purpose of the Study:
- To investigate the role of autophagy in laser-induced tumor cell death in vitro.
- To determine if autophagy induction influences glioma cell sensitivity to laser treatment.
Main Methods:
- U251 human glioma cells were exposed to 532 nm laser light.
- Cell viability was assessed using crystal violet and MTT assays.
- Autophagy markers (autophagolysosomes, LC3-II, beclin-1) were analyzed via fluorescence microscopy, flow cytometry, and Western blot.
Main Results:
- Laser irradiation induced autophagic vesicle accumulation and increased LC3-II and beclin-1 expression at cytotoxic doses.
- Autophagy inhibition (bafilomycin A1, chloroquine, wortmannin) significantly enhanced laser-induced cytotoxicity.
- Non-irradiated cells were unaffected by autophagy inhibitors.
Conclusions:
- Beclin-1-dependent autophagy induction protects glioma cells from laser-mediated cytotoxicity.
- Targeting autophagy could potentially enhance the efficacy of laser-based cancer therapies.
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