The use of ultrasound in cancer therapy has become an important issue [
- Barnett S.B
- ter Haar G.R
- Ziskin M.C
- Nyborg W.L
- Maeda K
- Bang J
Current status of research on biophysical effects of ultrasound.
Umemura S, Kawabata K, Sasaki K, Yumita N, Umemura K, Nishigaki R (1996) Recent advances in sonodynamic approach to cancer therapy. Ultrason Sonochem 3-S:187
High intensity focused ultrasound—potential for cancer treatment.
]. Ultrasound has been used in conjunction with hyperthermia, and photo-, radio-, and chemotherapy [
Synergism between hyperthermia, ultrasound and γ irradiation.
]. Malignant cells are known to be more susceptible to these combined methods than their normal counterparts [
Distinct sensitivity of normal and malignant cells to ultrasound in vitro.
]. The effect of a direct irradiation (e.g., ultrasound, laser, light) on certain molecules (porphyrins, psoralenes, and anthracyclines) is to generate highly active oxygen species such as singlet oxygen, superoxyde radicals, or fatty acid radicals, which can play an important role in cancer treatment, acting selectively on malignant cells [
- Kessel D
- Jeffers R
- Fowlkes J
- Cain C
Porphyrin-induced enhancement of ultrasound cytotoxicity.
- Briviba K
- Klotz L.O
- Sies H
Toxic and signaling effects of photochemically or chemically generated singlet oxygen in biological systems.
According to the origin of the photons, the therapy is called PDT (photodynamic therapy) or, if by sonoluminescence, SDT (sonodynamic therapy). The effects generated by SDT and PDT are different in terms of cell viability; both SDT (specifically related to the ultrasonic cavitational activity) and PDT generate active oxygenated species and lead to a diminution of the intracellular thiol levels [
- Kessel D
- Jeffers R
- Fowlkes J
- Cain C
Effects of sonodynamic and photodynamic treatment on cellular thiol levels.
]. In the case of PDT by ultraviolet-A (UVA), apoptosis of T helper cells is induced by the generation of singlet oxygen, but this effect depends essentially on the initial concentration in photosensitizers (PS) and on the local oxygen concentration [
- Morita A
- Werfel T
- Stege H
- et al.
Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy.
]. For SDT, as a result of the high energies involved, the cell lysis is the major phenomenon, probably masking other effects on the surviving cells [
- Miller D.L
- Thomas R.M
- Frazier M.E
Single strand DNA breaks in CHO cells after exposure to ultrasound in vitro.
]. Classical SDT leading to apoptosis involves specific sensitizing molecules, and requires an electrical power of about 5 W/cm2
and irradiation time of several minutes. In the process described here, we used ultrasound at low energy to induce apoptosis specifically in leukemic cells, in the absence of any chemical agent synergy. Since singlet oxygen and hydroxyl radicals seem implicated in the induction of apoptosis, we have named this technique SLDT: Sonochemical Low-energy Dynamic Therapy.
Materials and methods
Human leukemia cell lines (K562, Nalm-6, KG1a, and HL-60) obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in RPMI-1640 (BioWhittaker, Walkersville, MD, USA) supplemented with 10% fetal calf serum (Gibco, Grand Island, NY, USA) and 1% L-glutamine (Gibco). Leukemic cells were harvested, resuspended in phosphate-buffered saline (PBS, pH = 7.2, Gibco), and immediately used for the experiment at a concentration of 106 total. Heparinized venous blood was obtained from healthy volunteers and leukemic patients after informed consent was obtained. Mononuclear cells were separated by Ficoll-Hypaque gradient density centrifugation (International Medical Products, Brussels, Belgium).
The ultrasonic treatment and its specific results are part of patent PCT/BE97/00078 (pending) under the name of Eric Cordemans de Meulenaer et al.
A total of 2.5 mL of cell suspension in 13 × 100-mm disposable plastic tubes (Greiner, Frickenhausen, Germany) was treated in our system with a frequency of 1.8 MHz during various times of exposure. Unless indicated otherwise, the power supplied by the generator to the ceramic disk is 0.22 W/cm2, which represents an acoustical power transmitted to the interior of the test cell of 0.007 W/mL. This calorimetric measurement was made at the University of Coventry, England, under Professor Tim Mason. The irradiation (ON/OFF) cycles are 5.5 ms/3 ms.
The cell viability was assessed by the trypan blue exclusion test immediately after ultrasonic treatment and after 18 hours culture in the incubator (37°C and 5% CO2).
Cytocentrifuge preparations were made from the cell suspension and after air drying, cells were stained with May Grünwald Giemsa and analyzed by light microscopy.
Annexin V binding assay
Flow cytometric analysis of annexin-V–fluorescein isothiocyanate (FITC)- and propidium iodide (PI)-stained cells was performed using the kit purchased from Biosource International (Camarillo, CA, USA) as recommended by the manufacturer. Data are presented as dot plots showing the change in mean fluorescence intensity of annexin-V–FITC/propidium iodide.
The level of DNA fragmentation of apoptotic cells was determined using the Apotarget Quick DNA Ladder Detection Kit (Biosource). Cell pellets (106 cells) were resuspended in 20 μL of lysis buffer and DNA was extracted according to manufacturer's instructions. DNA was analyzed after separation by gel electrophoresis (1% agarose). As positive control, cells were irradiated with UV light by placing a plate directly under a UV transilluminator for 10 minutes (intensity of 5 mW/cm2). Cells were then incubated at 37°C for 5 and 18 hours before apoptosis was assessed.
Quantification of cells with degraded DNA was also performed using a method described by Nicoletti et al. [
- Nicoletti I
- Migliorati G
- Pagliacci M.C
- Grignani F
- Riccardi C
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
]. After permeabilization, cells were incubated with solution containing PI and RNAse (Coulter DNA-prep Reagent). The tubes were placed at 4°C in the dark overnight before analysis by flow cytometry to identify the sub-G0 peak corresponding to apoptosis.
Mitochondrial potential was estimated by incorporation of the cationic fluorochrome DiOC6 immediately after cell treatment according to a published protocol [
- Macho A
- Castedo M
- Marchetti P
- et al.
Mitochondrial dysfunctions in circulating T lymphocytes from human immunodefiency virus-1 carriers.
]. Briefly, K562 cells (106
/mL) were incubated with 2.5 nmol/L 3,3′-dihexyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, OR) for 15 minutes at 37°C, followed by flow cytometric analysis.
Cell Tracker green CMFDA (5-chloromethyl fluorescein diacetate; Molecular Probes) was used for determining levels of intracellular glutathione as previously described [
Evaluation of methods for measuring cellular glutathione content using flow cytometry.
]. Glutathione level was evaluated in viable cells (PI−
Analysis of caspase-3 activity
Caspase-3 was detected by flow cytometric analysis using the phycoerythrin (PE)-conjugated polyclonal rabbit antibody anti-active caspase-3 monoclonal antibody (BD-Pharmingen, San Diego, CA, USA). Cells were fixed and permeabilized using Fix and Perm kit (Caltag, Burlingame, CA) for 15 minutes at room temperature. Cells were then stained with anti-caspase-3 Ab and incubated for 15 minutes. Cells were washed and analyzed by flow cytometry. The enzymatic activity of caspase-3 was determined using the Apotarget caspase-3/cpp32/colorimetric protease assay kit (Biosource), as suggested by the manufacturer. Caspase-3 activation was also indirectly evaluated by PARP cleavage using a rabbit anti-PARP cleavage site AB, FITC conjugate (Biosource).
bcl-2 and bax expression
After permeabilization, cells were incubated with isotype-matched negative control, FITC-labeled mouse anti-human bcl-2 (Dako, Glostrup, Denmark), and polyclonal rabbit antibodies to bax. Subsequently, a FITC-labeled secondary antibody (Dako) was added to bax. To quantify bcl-2 and bax expression, the cytometer was calibrated using a mixture of beads labeled with known amounts of fluorochrome (Dako). The values of mean fluorescent intensity (MFI) were then converted to molecules of equivalent soluble fluorochrome (MESF) using a calibration curve.
Clonogenic assay for K562 cell line
Colony-forming unit-leukemic (CFU-L) cells were assayed as previously described [
- Lagneaux L
- Marie J.P
- Delforge A
- et al.
Comparison of in vitro inhibition of etoposide (VP16) on leukemic and normal myeloid, erythroid clonogenic cells.
Briefly, the culture medium consisted of IMDM supplemented with 20% FCS and methylcellulose at a final concentration of 4%. Cultures were incubated at 37°C in 5% CO2 air, and colonies (> 20 cells) were scored after 5 days. The clonogenic efficiency of K562 cell line was 16%.
Assay for hematopoietic colony-forming cells
Hematopoietic colony-forming cells (CFU-GEMM, CFU-GM, and BFU-E) were assayed using fetal bovine serum (FBS)-free methylcellulose medium supplemented by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, GCSF, and 3 U/mL EPO (erythropoietin; Methocult H4536, StemCell Technologies, Vancouver, BC, Canada). Cultures were plated in 0.25-mL volumes in 4-well tissue culture plates (Nunclon, Life Technologies, Merelbeke, Belgium). Mononuclear cells were plated at 104 cells/well. After 14 days at 37°C in 5% CO2 in air and 100% humidity, the colonies were counted.
The Wilcoxon Signed Ranks test was used to analyze the statistical significance of experimental results.
Apoptosis ensures the homeostasis of tissues during development, host defense, and aging and occurs in response to a large variety of signals including γ-irradiation and ultraviolet exposure [
Molecular mechanisms of UV-induced apoptosis.
- Belyaev I.Y
- Czene S
- Harms-Ringdahl M
Changes in chromatin conformation during radiation-induced apoptosis in human lymphocytes.
- Carloni M
- Meschini R
- Ovidi L
- Palitti F
PHA-induced cell proliferation rescues human peripheral blood lymphocytes from X-ray-induced apoptosis.
]. Apoptotic cell death is characterized by early changes in the nuclear membrane and by chromatin condensation, followed by DNA fragmentation [
- Arends M.J
- Morris R.G
- Wyllie A.H
Apoptosis the role of the endonuclease.
]. Ultrasound at high power also induces structural and functional changes in sonicated cells [
- Alter A
- Roznszajn L.A
- Miller H.I
- Rosenschein U
Ultrasound inhibits the adhesion and migration of smooth muscle cells in vitro.
]. Moreover, ultrasound beams have the potential to treat malignant tumors when combined with sensitizers such as porphyrins [
- Yumita N
- Nishigaki R
- Umemura K
- et al.
Sonochemical activation of hematoporphyrin An ESR study.
]. Recently, in vitro studies demonstrated therapeutic ultrasound-induced apoptosis in cultured cells via the process of cavitation [
- Ashush H
- Leon A
- et al.
Apoptosis induction of human myeloid leukemic cells by ultrasound exposure.
]. This therapeutic ultrasound (750 kHz), characterized by a “high-intensity” delivery to generate cavitation leading to apoptotic cell death, was observed in the surviving cells besides a large amount of necrosis (more than 40%). In contrast to the mechanism reported for γ-irradiation, apoptosis induced by ultrasound seems independent of cell-cycle modifications [
- Yu Y.Q
- Giocanti N
- Averbeck D
- Megnin-Chanet F
- Favaudon V
Radiation-induced arrest of cells in G2 phase elicits hypersensitivity to DNA double-strand break inducers and an altered pattern of DNA cleavage upon re-irradiation.
In this study, the induction of apoptosis in leukemic cells by a “low-energy” ultrasonic treatment is demonstrated. This treatment leads to a sequence of events recognized as hallmarks of apoptosis: a drop in mitochondrial potential, loss of phosphatidylserine asymmetry, morphological variations, DNA fragmentation, and, finally, loss of plasma membrane. This “low-energy” ultrasound-induced apoptosis involves activation of caspase-3 and is accompanied by the proteolytic degradation of the caspase substrate PARP and by the modulation of bcl
-2/bax ratio, demonstrated using flow cytometric analysis. A comparable mechanism has been demonstrated after photodynamic therapy [
Photodynamic therapy a mitochondrial inducer of apoptosis.
- He J
- Whitacre C.M
- Xue L.Y
- Berger N.A
- Oleinick N.L
Protease activation and cleavage of poly(ADP-ribose) polymerase an integral part of apoptosis in response to photodynamic treatment.
Soon after ultrasonic treatment, an important decrease of intracellular glutathione level is observed, suggesting that oxidative stress may play a role in ultrasound-induced apoptosis. Loss of glutathione and oxidative damage have been suggested to constitute early signaling events in apoptotic cell death [
- Macho A
- Hirsch T
- Marzo I
- et al.
Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis.
A mechanism coherent with all our observations involves ultrasound-induced sonochemical luminescence triggering photosensitized singlet oxygen production to initiate apoptosis as previously described for direct photoirradiation [
- Sharman W.M
- Allen C.M
- Van Lier J.E
Role of activated oxygen species in photodynamic therapy.
]. In classic ultrasonic irradiation conditions, the direct destructive cavitation effects dominate the sonoluminescence, which is weak in the absence of an air/liquid interface injected into the medium. The fact that there are no ultrasound effects in the absence of bubbles suggests the major role played by 1
in our system.
Data obtained in the presence of histidine, a quencher of 1O2, suggest the importance of singlet oxygen in the induction of apoptosis by ultrasound under the “low energy” conditions. However, the fact that mannitol, an inhibitor of hydroxyl radicals, protects against ultrasound-induced apoptosis also implies the intervention of these radicals in our system.
Evidence against singlet oxygen formation during sonodynamic therapy has been presented by Miyoshi et al. [
- Miyoshi N
- Igarashi T
- Riesz P
Evidence against singlet oxygen formation by sonolysis of aqueous oxygen-saturated solutions of Hematoporphyrin and Rose Bengal. The mechanism of sonodynamic therapy.
], but these data are only consistent with a long and “high-energy” ultrasound exposure, leading to an accumulation of sensitizer-derived free radicals either by direct pyrolysis or due to reactions with Ho
radicals formed by pyrolysis of the water solvent. In the present study, the supernatant of cells submitted to one or several ultrasonic treatments is unable to induce apoptosis in K562 cells, demonstrating that the effects generated do not originate from chemical species produced by the solvent. At the chosen frequencies, the “low-energy” ultrasound does not directly generate free radicals such as those originating in the sonolysis of the solvent or those from the addition of molecules such as dimsethylsulfoxide (DMSO) or N, N-dimethylformamide (DMF), which generate a sonodynamic action on leukemia cells [
Peroxyl radical formation in aqueous solutions of N,N-dimethylformamide and N-methylformamide and dimethylsulfoxide by ultrasound implications for sonosensitized cell killing.
- Jeffers R
- Feng R
- Fowlkes J
- Hunt J
- Kessel D
- Cain C
Dimethylformamide as enhancer of cavitation-induced cell lysis in vitro.
Sonoluminescence being in our case the only possible inducing physical phenomenon due to the presence of topical cellular PS, it is normal that these results can be compared to photolytical damages suffered after an exposure to classic ultrasound. The effects obtained with our technique are achieved without the necessity of classical PS. The physiological effects obtained with techniques such as phototherapy depend at the same time on the radiation dose, on the nature of the PS used, on their concentration, and on their localization [
- Kessel D
- Luo Y
- Deng Y
- Chang C.K
The role of subcellular localization in initiation of apoptosis by photodynamic therapy.
]. The major targets of PDT are cell membranes but, significantly, the technique discussed here is the only one to act directly within the interior of the cells. Within the cells, it is the endogenous photoabsorbing molecules such as the porphyrins and flavoproteins that play the photosensitizing role [
Photosensitization of organisms with special reference to natural photosensitizers.
]. Indeed, an implication of endogenous porphyrins in photodynamic DNA damage has been proposed [
Photodynamic DNA damage mediated by δ-aminolevulinic acid-induced porphyrins.
In our technique, the net effects of the ultrasonic action suggest that endogenous PS may be implicated in the structure where their local concentration is high. Endogenous PS are localized mainly in the membrane structures such as lysosomes, mitochondria, nuclear membranes, and the microsomes of the endoplasmic reticulum, of which the relative surface represents nearly 50% of the cell membrane surface [
- Moan J
- Berg K
- Kvam E
- et al.
Intracellular localization of photosensitizers.
Under these soft conditions to which they are subjected, healthy cells are much less sensitive to ultrasound than leukemic cells. This difference in behavior between the healthy and leukemic cells cannot be related to a difference in the localization of the endogenous PS but is probably due to a modification of the fundamental cell mechanisms (e.g., p53 status, signaling pathways, resistance to oxidative stress).
In conclusion, this “low-energy” ultrasonic treatment (SLDT) induces a similar sequence of events as that induced by PDT: a rapid formation of 1O2, having on the one hand an effect on mitochondrial membranes (drop of mitochondrial potential) and provoking on the other hand a lipidic oxidation of the membrane (decrease of cellular GSH level). Specific tests have confirmed the very rapid induction of apoptosis in the absence of necrosis. This ultrasonic treatment could be a promising tool for the ex vivo elimination of leukemic cells by means of apoptosis.