Cytoplasmic signaling involved in sonoporation-induced apoptosis and mitosis repression of myeloid leukemia cells

摘要

Sonoporation is known to be a transient phenomenon that may disrupt the homeostasis of living cells. In this work, we showed that sonoporation may bear time-lapse impact on cellular viability through up-regulation of cytoplasmic signaling proteins related to apoptosis and cell-cycle arrest. Our experiments were done on HL-60 leukemia cells (10⁶ cells/ml), and sonoporation was induced via the use of 1% v/v microbubbles and 1-min. pulsed ultrasound exposure (0.5MPa peak negative pressure, 1MHz center frequency, 10% duty cycle, 1 kHz pulse repetition frequency). The transient nature of sonoporation in these cells was confirmed by performing scanning electron microscopy on selected cell samples that were fixed respectively after a few seconds into the ultrasound exposure and one minute after the end of exposure. Cytoplasmic signaling changes of these cells were studied at four post-sonoporation time points (4h, 8h, 12h, 24h) using western blot analysis. Five signaling proteins related to apoptosis and mitosis were analyzed in this work: 1) PARP (for DNA repair); 2) cleaved-PARP (fragments due to cleavage by pro-apoptotic caspase proteins); 3) Bcl-2 (inhibitor for mitochrondrial release of pro-apoptotic molecules); 4) Bax (complement of Bcl-2); 5) Cdc-2 (regulator for cell mitosis). Three key results were found from the cytoplasmic signaling analysis. First, PARP levels were reduced over the monitoring period whilst cleaved-PARP had increased in expression, and in turn they indicate that the cells' anti-apoptotic responses were dampened following sonoporation and pro-apoptotic caspase proteins were likely activated. Second, drop in Bcl-2 and rise in Bax were observed, and these suggest that the mitochondrion was involved in apoptotic signal transduction inside sonoporated cells. Third, Cdc-2 was seen to decrease, implying that mitosis was repressed in sonoporated cells.