DNA Double-Strand Breaks Induced by Cavitational Mechanical Effects of Ultrasound in Cancer Cell Lines

Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage

Ultrasound-induced cell killing in MES-SA and MES-SA/DX5 cells 24 hr post exposure to different acoustic intensities.

<p>(a) Cell viability assessed by WST-8 and cell counting assay. Asterisks (*) indicate the statistical significance of the difference between the absolute percentages obtained from WST-8 and cell counting assays at one intensity for one cell line. (b) Flow cytometric analyses for FITC-labelled Annexin V and PI staining. Vertically-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (−) cells, whereas horizontally-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (+) cells in comparison to control. Data points are presented as mean ± SEM.</p

Dual treatment protocols.

<p>Cell survival following dual treatment protocols with doxorubicin (1 µM) and ultrasound (0.4 W/cm²). (a) Simultaneous treatment protocols. (b) Sequential treatment protocols. Data points are presented as mean ± SEM. Asterisks (*) denote the statistical significance of changes in cell survival following (Dox-US) protocol compared to (US 48) and (Dox 48) for each cell line. Following (US-Dox) treatment protocol, the cell survival of MES-SA cells decreased significantly compared to (US 48) but was insignificantly increased compared to (Dox 48). In MES-SA/DX5 cells, cell survival was unchanged. However, (US-Dox) protocol showed consistently large difference between the percentages obtained from WST-8 and cell counting assays indicating the presence of cells contributing to viability below the size threshold for detection by the cell counter.</p

Fluorescent microscopy.

<p>Pictograms of MES-SA (a & b) and MES-SA/DX5 (c & d) cells 24 hr post sonication at 0.4 W/cm². Cells were stained simultaneously with Hoechst 33342 and Alexa flour-488 – conjugated wheat germ agglutinin (WGA) in 2% paraformaldehyde (PFA)/PBS for 20 min followed by immediate observation. Cells show nuclear budding of genomic content (d) that is occasionally translocated through the cytoplasm (b) and emerges to form a small cell (Raju cell) (a). Two distinct forms of nuclear budding can be identified in treated MES-SA cells bearing similarity to neotic cytokinesis described by Rajaraman et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048291#pone.0048291-Rajaraman1" target="_blank">[33]</a>, namely, (a) sequential cytokinesis type-2 perinuclear and (b) sequential cytokinesis type-1 pericellular, whereas the first form only was observed in treated MES-SA/DX5 cells. Open-head arrows indicate nuclear budding; closed-head arrows indicate the surrounding membranes of emerging cells. (e & f) Sonicated MES-SA cells showing multiple cytokinesis captured at the fourth (D4) and fifth (D5) days post exposure, respectively. (g) Control MES-SA cell with spontaneous cytokinesis. Cells (e–g) resemble in morphology neotic mother cells shown by Sundaram et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048291#pone.0048291-Sundaram1" target="_blank">[31]</a>. Bars, 10 µm.</p

The extent of histone H2AX phosphorylation in MES-SA and MES-SA/DX5.

<p>Cells were fixed 15 min after exposure to ultrasound at different intensities. Cells were assayed flow cytometrically. Data points are presented as mean ± SEM. Asterisks (*) denote the statistical significance between MES-SA and MES-SA/DX5 at respective intensities.</p

Proliferation of MES-SA and MES-SA/DX5 cells over 7 days (D1–D7) after sonication.

<p>Ultrasound was applied at an intensity of 0.4 W/cm² on D0 (arrow). Cells were counted immediately after sonication and plated at a density of 1×10⁵ cells/dish. Data points are presented as mean ± SEM.</p