Induction of endoplasmic reticulum stress by sonoporation: linkage to mitochondria-mediated apoptosis initiation.

The use of cavitational means to create transient membrane pores on living cells (i.e., sonoporation) may potentially induce a broad range of downstream bio-effects that disrupt the functioning of various organelles. Here we observed that on HL-60 leukemia cells, sonoporation may induce endoplasmic reticulum (ER) stress on a time-lapse basis and, in turn, signal the mitochondria to commit a cell toward apoptosis. Our observations were derived from in vitro ultrasound exposure experiments performed on HL-60 cells in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio; 1-MHz frequency; 0.45-MPa in situ peak negative pressure; 100-cycle pulse length; 1-kHz pulse repetition frequency; 60-s exposure period). Using flow cytometry, we found that sonoporated cells exhibited a progressive loss of functional ER mass over a 6-h period. Also, post-exposure Western blot assays (between 0 and 24 h) revealed various indications of post-sonoporation ER stress: (i) upregulation of ER-resident enzymes responsible for catalyzing protein folding; (ii) activation of trans-ER-membrane stress sensors; (iii) increased expression of ER-induced regulatory proteins that mediate pro-apoptotic signals to the mitochondria. These results corresponded to flow cytometry observations that depicted a progressive depolarization of a sonoporated cell's mitochondrial outer membrane potential. They were also consistent with another Western blot assay that found, in sonoporated cells, a time-lapse increase of caspase-9 (a mitochondria-activated apoptosis initiator protein). Taken together, our findings indicate that sonoporation may upset ER homeostasis, and this may ultimately result in initiation of apoptosis.