Low-Intensity Therapeutic Ultrasound Effects on Melanoma Cells: An In Vitro Study

Therapeutic ultrasound has been widely investigated as a promising tool for oncological applications, particularly in sonodynamic therapy (SDT). Its biological effects strongly depend on acoustic parameters such as frequency, intensity, duty cycle, and exposure time. Depending on the conditions employed, low-intensity ultrasound may induce cellular damage or trigger biomodulatory responses affecting cellular metabolism, intracellular signaling, and proliferation. This study investigates the effects of low-intensity therapeutic ultrasound on melanoma and healthy skin cells. An indirect ultrasonic water-bath system was developed using a custom glass tank with a lower opening for positioning the ultrasound transducer. The water was heated to remove dissolved gases, cooled, and sonicated for 20 min at 2 W/cm² to further reduce residual gases. Irradiation was performed using a Sonopulse Aura device (Ibramed, Brazil) equipped with an 8 cm² effective radiating area transducer operating at 1 MHz. Custom adapters secured 96-well plates at the top of the tank, improving stability and standardizing the transducer-to-sample distance. This distance was determined using an indirect potassium iodide probe method for acoustic cavitation detection. Only central wells were considered for ultrasound-treated samples to minimize spatial variations in the acoustic field. SH-4 metastatic pigmented melanoma cells and normal human dermal fibroblasts (HDFn) were used as tumor and healthy cell models, respectively. Cells were seeded at 1 × 10⁴ cells per well and maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. Pulsed ultrasound was applied at a 50% duty cycle, intensities of 0.5, 1.0, and 1.5 W/cm², and an exposure time of 10 min. Cell viability was assessed by resazurin assay at 24 and 48 h after sonication. At 0.5 W/cm², HDFn cells showed viability above the control level, suggesting greater tolerance and a possible biomodulatory response, whereas SH-4 cells showed reduced metabolic activity. At 1.0 W/cm², both cell lines exhibited reduced viability, although values increased from 24 to 48 h, suggesting partial metabolic recovery. At 1.5 W/cm², HDFn cells were more strongly affected, while SH-4 cells maintained higher relative viability. Overall, cellular responses were dependent on cell type, ultrasound intensity, and post-treatment time.