Time-dependent effects of low-intensity pulsed ultrasound on apoptosis and autophagy in malignant melanoma stem cells

Cancer stem cells (CSCs) in malignant melanoma contribute to therapeutic resistance and tumour recurrence. While low-intensity pulsed ultrasound (LIPUS) has been proposed as a non-invasive strategy to induce cell death, its effects on CSC-specific apoptotic and autophagic responses remain unclear. This study aimed to explore the time-dependent effects of LIPUS on apoptosis and autophagy in CD133+ melanoma CSCs and CD133− non-stem melanoma cells. Human melanoma cells (CHL-1) were sorted via FACS into CD133+ and CD133− populations. Cells were exposed to LIPUS (1 MHz, 20% duty cycle, 1 W/cm²) for 1, 5, and 10 min. Protein expression levels of Caspase-3, Caspase-8, mTOR, and LC3 were evaluated via immunofluorescence and quantified by image-based analysis. Both cell populations showed significant increases in Casp3, Casp8, mTOR, and LC3 intensities following LIPUS application. Notably, CD133+ cells exhibited delayed but sustained increases in Casp3 and LC3 expression, while CD133− cells responded more rapidly. mTOR activity demonstrated distinct temporal dynamics between the two groups, suggesting differential modulation of autophagy-related pathways. LIPUS triggers temporally distinct apoptotic and autophagic responses in melanoma CSCs and non-stem cancer cells. These findings suggest a potential therapeutic avenue to selectively disrupt CSC survival mechanisms using mechanical stimulation.