Direct analysis of the actin-filament formation effect in photodynamic therapy.

Photodynamic therapy (PDT) is a method in which a photosensitizer is administered in vivo and irradiated with light to generate reactive oxygen species (ROS), thereby causing the selective death of cancer cells. Since PDT is a noninvasive cancer treatment method with few adverse effects, it has attracted considerable attention and is increasingly used. In PDT, there are two dominant processes based on the actin filament (A-filament) formation effect: the destruction of cells by necrosis and vascular shutdown. Despite the importance of its fine control, the mechanism of the reaction process from the generation of reactive oxygen by photoinduction inducing the formation of A-filament and its polymerization to form stress fibers (S-fibers) has not yet been clarified because, for example, it has been difficult to directly observe and quantify such processes in living cells by conventional methods. Here, we have combined atomic force microscopy (AFM) with other techniques to reveal the mechanism of the A-filament and S-fiber formation processes that underlie the cell death process due to PDT. First, it was confirmed that activation of the small G protein RhoA, which is a signal that induces an increase in A-filament production, begins immediately after PDT treatment. The production of A-filament did not increase with increasing light intensity when the amount of light was large. Namely, the activation of RhoA reached an equilibrium state in about 1 min: however, the production of A-filament and its polymerization continued. The observed process corresponds well with the change in the amount of phosphorylated myosin-light chains, which induce A-filament polymerization. The increase in the elastic modulus of cells following the formation of S-fiber was confirmed by AFM for the first time. The distribution of generated A-filament and S-fiber was consistent with the photosensitizer distribution. PDT increases A-filament production, and when the ROS concentration is high, blebbing occurs and cells die, but when it is low, cell death does not occur and S-fiber is formed. That is, it is expected that vascular shutdown can be controlled efficiently by adjusting the amount of photosensitizer and the light intensity.