Ultrasonic low-energy treatment: a novel approach to induce apoptosis in human leukemic cells.
OBJECTIVE
We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity.


METHODS
Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose.


RESULTS
The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism.


CONCLUSIONS
The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.
Lagneaux
Laurence
L
Laboratoire d'Hématologie Expérimentale, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium. laurence.lagneaux@bordet.be
de Meulenaer
Eric Cordemans
EC
Delforge
Alain
A
Dejeneffe
Marielle
M
Massy
Martine
M
Moerman
Carine
C
Hannecart
Baudouin
B
Canivet
Yves
Y
Lepeltier
Marie Françoise
MF
Bron
Dominique
D
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D002470:Cell Survival; D053938:DNA Fragmentation; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000032:analysis; D018922:HL-60 Cells / Q000737:chemistry / Q000000981:diagnostic imaging / Q000473:pathology; D006639:Histidine / Q000494:pharmacology; D006801:Humans; D017665:Hydroxyl Radical; D007425:Intracellular Membranes / Q000000981:diagnostic imaging; D020014:K562 Cells / Q000737:chemistry / Q000000981:diagnostic imaging / Q000473:pathology; D007938:Leukemia / Q000473:pathology*; D008353:Mannitol / Q000494:pharmacology; D008563:Membrane Lipids / Q000032:analysis; D008928:Mitochondria / Q000000981:diagnostic imaging; D009363:Neoplasm Proteins / Q000032:analysis; D014411:Neoplastic Stem Cells / Q000737:chemistry / Q000000981:diagnostic imaging* / Q000473:pathology; D018384:Oxidative Stress; D010718:Phosphatidylserines / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D026082:Singlet Oxygen; D014410:Tumor Stem Cell Assay; D014464:Ultrasonic Therapy; D014463:Ultrasonography; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D016166:Free Radical Scavengers; D008563:Membrane Lipids; D009363:Neoplasm Proteins; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D026082:Singlet Oxygen; D017665:Hydroxyl Radical; D008353:Mannitol; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione