L. Serpe¹, R. Canaparo², F. Foglietta², G.P. Zara², C. Ferretti² , M. Eandi², R. Frairia³

Dept. of Drug Science and Technology¹, Dept. of Anatomy, Pharmacology and Forensic Medicine², Dept. of Clinical Pathophysiology³, University of Torino, Italy

An attractive form of treatment for solid tumors is sonodynamic therapy based on the ability of ultrasounds to generate acoustic cavitation and to activate a tumor-localizing sonosensitizer agent such as porphyrin compounds like 5-aminolevulinic acid (ALA). High Energy Shock Waves (HESW), generated by a piezoelectric device, are able to induce acoustic cavitation, which results in a concentration of energy sufficient to generate a sonoluminescence emission, able to cause electronic excitation of porphyrins by energy transfer and initiate a photochemical process resulting in cytotoxic reactive oxygen species (ROS). To this purpose, we have investigated the ability of HESW to activate ALA in human neuroblastoma SK-N-BE and SH-SY5Y cell lines.
Cell cytotoxicity was measured with WST-1 proliferation assay and cell death was evaluated by flow cytometric analysis. The relationship between sonodynamic treatment and production of ROS was evaluated by flow cytometric analysis with dichlorofluorescein diacetate. Furthermore, mRNA expression of different genes involved in apoptosis through ROS production was evaluated by quantitative SYBR Green real time RT-PCR and fluorescence microscopic examination was carried out to highlight ROS production and cell death.
We have identified different treatment schedule of ALA (50-300 μg/ml) and HESW (0.22-0.43 mJ/mm²; 500-1000 shots, 4 shots/sec) to get the best cytotoxic rate in the two cell lines studied. Briefly, sonodynamic treatment was able to induce a significant decrease of cell growth compared to untreated cells at 72 h in both SK-N-BE and SH-SY5Y cells up to 35% and 50%, respectively. Exposure of ALA pre-incubated cells to HESW was able to increase significantly ROS production with different onset and extent in SK-N-BE and SH-SY5Y cells and the apoptotic rate was significant increased at 24 h in both cell lines (p<0.01).
Our results show that HESW could be able to activate porphyrin compounds in neuroblastoma cell lines by acoustic cavitation obtaining a significant in vitro cytotoxicity through ROS production.