The present invention is directed to using high-frequency,
low-energy ultrasound to treat liquid mediums. In specific embodiments the devices
and methods herein can induce significant apoptosis in cells suspended in a physiological
Cells can be damaged by exposure to ultrasound. For example,
ultrasound can cause irreversible cell damage and induce destructive cell membrane
modifications. Several reports have suggested that cavitation resulting from the
collapse of gas bubbles generated by acoustic pressure fields may be the cause for
cell damage following ultrasonic irradiation. It has also been suggested that cavitation
induces single-strand breaks in DNA by the action of residual hydrogen peroxide.
The use of ultrasound in cancer therapy has become an important
issue. Ultrasound has been used in conjunction with hyperthermia, and photo-, radio-,
and chemotherapy. Malignant cells are known to be more susceptible to these combined
methods than their normal counterparts. The effect of direct irradiation (e.g.,
ultrasound, laser, light) on certain molecules (e.g., classical photosensitizers
and sonosensitizers) is the generation of highly active oxygen species such as singlet
oxygen, superoxide radicals, hydroperoxides, or fatty acid radicals, which can play
an important role in cancer treatment, acting selectively on malignant cells.
According to the origin of the radiation, the above-described
therapy is termed PDT (photodynamic therapy) or, if by ultrasound or sonoluminescence:
SDT (sonodynamic therapy). Addition of a photosensitizer is a pre-requisite for
both therapies. While the general effects induced 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. In the case of PDT by ultraviolet-A (UVA), apoptosis
of T helper cells can be 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. For SDT, as a result of the high energies involved,
the cell lysis is the major phenomenon, probably masking other effects on the surviving
U.S. Patent No. 4,971,991 to Umemura et al. discloses the
use of ultrasound to treat tumor cells, but relies on high ultrasound power levels,
and does not describe the use of microbubbles. Other patents describing ultrasound
and microbubbles, such as U.S. Patent No. 5,215,680 to D'Arrigo, rely on the use
of the cavitational and thermal effects of ultrasound to treat tumors, as opposed
to individual cancer cells, the extent of which is determined by duration and number
of treatments. This type of treatment uses high power and long irradiation times,
predominantly producing cell lysis and necrosis. See Kondo, Cancer Letters
178(1), 63-70, (2002).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a drawing showing one embodiment of an ultrasonic
treatment device described herein.
Figure 2 is a drawing showing three views of an apparatus
for treating hyperproliferative cells in a suspension with ultrasound and microbubbles.
The far left view is an upper view of the apparatus, the middle view is a front
view, and the far right view is a lateral view of the apparatus.
Figure 3 is a bar graph showing the effects of ultrasonic
treatment on cellular glutathione levels. Data are expressed in percentage of cells
displaying glutathione level comparable to untreated cells. Values are mean ±
SEM of 3 independent experiments.
Figure 4 is a bar graph representing the effects of high-frequency
ultrasound on cellular caspase-3 activity.
Figure 5 is a bar graph showing the effect of irradiation
on cloning efficiency of K562 cells. The results are expressed as mean ± SEM
of 3 independent experiments.
Figure 6 is a bar graph showing the percentage of apoptotic
K562 cells 5 hours after 1 or 3 ultrasonic treatments. The rate of apoptosis is
determined by flow cytometry after Annexin-V staining and the results are expressed
as mean ± SEM of 7 independent experiments.
Figure 7 is a point graph showing the changes of phosphatidylserine
distribution according to time and successive ultrasonic treatments. Results from
one representative experiment are expressed as percentage of cells stained with
Figure 8 is a bar graph showing the effect of ultrasonic
treatment on the apoptosis of normal mononuclear cells (MNC) and leukemic cells
(Nalm-6, KG1a, HL-60, and primary leukemic cells obtained from 5 patients). Results
are mean ± SEM of 5 independent experiments.
Apoptosis, or programmed cell death, is a normal component
of the development and health of multicellular organisms. Apoptosis ensures the
homeostasis of tissues during development, host defense, aging, and occurs in response
to a large variety of signals including &ggr;-irradiation and ultraviolet exposure.
Cells die in response to a variety of stimuli, typically during apoptosis they do
so in a controlled fashion. This makes apoptosis distinct from another form of cell
death called necrosis in which uncontrolled cell death leads to lysis of cells,
inflammatory responses and, potentially, to serious health problems. Apoptosis,
by contrast, is a process in which cells play an active role in their own death,
which is why apoptosis is often referred to as cell suicide.
Upon receiving specific signals instructing the cells to
undergo apoptosis, a number of distinctive biochemical and morphological changes
typically occur in the cell. For example, a family of proteins known as caspases
are typically activated in the early stages of apoptosis. These proteins breakdown
or cleave key cellular substrates that are required for normal cellular function,
including structural proteins in the cytoskeleton and nuclear proteins such as DNA
repair enzymes. Caspases can also activate other degradative enzymes such as DNases,
which cleave the DNA in the nucleus. In general, apoptotic cell death is characterized
by early changes in the nuclear membrane, chromatin condensation, and DNA fragmentation.
These biochemical changes result in morphological changes in the cell.
The teachings herein are directed towards devices and methods
which can neutralize, prevent the growth of, and remove hyperproliferative cells
(e.g., tumor cells) present in a liquid medium. In more specific embodiments,
the methods and devices provided herein induce apoptosis in hyperproliferative cells
present in a suspension, such as a physiological fluid. Treatable physiological
fluids include blood, plasma, serum, and cerebrospinal fluid which can be extracted
from and/or administered to animals, including mammals, humans, and the like.
Low-energy, high-frequency ultrasonic treatment according
to the teachings herein can induce apoptotic effects in hyperproliferative cells.
These effects include for example, having an effect on mitochondrial membranes (drop
of mitochondrial potential), loss of phosphatidylserine asymmetry, provoking a lipidic
oxidation of the membrane (decrease of cellular GSH level), morphological variations,
DNA fragmentation, loss of plasma membrane, and the like. Furthermore, low-energy
ultrasound-induced apoptosis can involve activation of caspase-3, the proteolytic
degradation of the caspase substrate PARP, and the modulation of bcl-2/bax
ratio in the cells.
Specific tests (see Examples) have confirmed the very rapid
induction of apoptosis with limited amounts of necrosis.
Devices and Methods
Embodiments of the devices that can be used to implement
the inventive methods can be found in U.S. Provisional Application 60/423,368, U.S.
Application No. 10/358445, and U.S. Patent No. 6,540,922 to Cordemans et al. Methods
of treating hyperproliferative cells can be performed with the devices disclosed
herein. One particular embodiment of a device that can be used for treating a liquid
medium such as an aqueous medium (e.g., physiological fluids) is illustrated in
FIGURE 1. In certain embodiments the fluids to be treated contain hyperproliferative
cells. In other embodiments, the fluids to be treated can be a physiological liquid
suspected of containing hyperproliferative cells, such as after diagnosis, for example.
Cells which are not completely differentiated such as stem cells, as well as solutions
containing viruses and/or virus infected cells can also be treated. Examples of
treatable viruses can include HIV, HCV, HBV, Herpes virus, hantavirus, influenza,
and Ebola, for example.
Referring to FIGURE 1, the devices described herein include
a compartment 2, preferably in the shape of a cylinder or a rectangular cross-section.
In certain embodiments the compartment 2 can be in communication with a reservoir
(not shown) which holds the liquid medium to be treated. In other embodiments (e.g.,
when a human or animal physiological fluid is treated), the devices provided herein
do not contain a reservoir that is directly connected to the human or animal body.
Such embodiments include those wherein the physiological fluid is extracted and/or
administered (e.g., reinjection) during extracorporeal treatment of the human
or other animal body. Accordingly, an animal such as a human can be substituted
for any reference herein to a "reservoir."
In other embodiments, a hyperproliferative cell suspension
can be treated in a device as shown in FIGURE 2. In this embodiment, an air inlet
tube 3 is used as a microbubble emitter 3 to emit microbubbles 5 into the hyperproliferative
cell suspension 22 contained in a compartment (or poach) 20. The compartment (or
poach) 20 ,containing the cell suspension 22 may be immersed in a water bath 24,
such as an incubator, for example.
In further embodiments, the compartment 2 contains (e.g.,
along its wall or adjacent to the bottom) one or more high-frequency ultrasound
emitters 1 that emit ultrasound 4 into the compartment 2 (advantageously toward
the center of this compartment 2). In other embodiments the container can also have
one or more microbubble emitters 3 for emitting gas microbubbles 5, which are arranged
so as to emit the gas microbubbles 5 into the ultrasound 4 field emitted in the
The term "microbubbles," as used herein is intended to
refer to gas bubbles with an average diameter of less than 1 mm. In some embodiments,
the diameter is less than or equal to 50 µm. Still in other embodiments, the
microbubbles have a diameter less than 30 µm. In certain embodiments the microbubbles
are selected from air, oxygen, and ozone microbubbles or a mixture thereof. To lower
operating costs, it can be advantageous to use microbubbles that are not ozone microbubbles,
such as air microbubbles. Advantageous embodiments of the invention do not rely
on the generation of a thermal effect to treat cells. While in certain embodiments
the use of stabilized microbubbles can be effective in treating cells, in preferred
embodiments, the use of stabilized microbubbles is unnecessary. Lipid boundary microbubbles
are an example of a stabilized microbubble.
The term "hyperproliferative cells" is intended to refer
to cells that divide, reproduce, or otherwise proliferate at a relatively high rate,
and can include cancer cells (e.g., leukemic cells), precancerous cells,
tumor cells, bone marrow cells, and totipotent cells.
In certain embodiments, the term "liquid medium" relates
to physiological liquids which may be administered to man or animals, and/or extracted
from man or animals. In specific embodiments, physiological fluids are reinjected
after treatment (e.g., an ex vivo treatment). In certain embodiments
the term "physiological liquids" can include, blood, serum, cephalorachidian, cerebrospinal
fluid, plasma, and the like. U.S. Patent No. 5,401,237, issued to Tachibana et al.,
describes a process of extracting and readministering physiological fluid.
In specific embodiments, the methods and devices herein
include low energy, high-frequency ultrasound to treat hyperproliferative cells.
The term "high frequency" is intended to refer to frequencies greater than 100 kHz
and up to several MHz. In certain embodiments, the high frequencies used are between
200 kHz and 20 MHz. In various embodiments, the ultrasound frequency can be selected
from between 200 kHz to 10 MHz. In a preferred embodiment, the frequency used is
between 200 kHz and 1.8 MHz.
In various embodiments of the devices described herein,
the microbubble emitter 3 for emitting gas microbubbles 5 is arranged at the base
11 of the compartment 2, (i.e., at the bottom of the compartment 2), such that the
microbubbles move by rising naturally or by entrainment of the gas in the flow of
In further embodiments, the devices and methods described
herein induce apoptosis in hyperproliferative cells. It has been discovered that
healthy cells are much less sensitive to high-frequency 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 photosensitizers
but is probably due to a modification of the fundamental cell mechanisms such as
p53 status, signaling pathways, and resistance to oxidative stress, for example.
Specifically, apoptosis can be induced in cancerous cells (e.g., leukemic),
precancerous cells, tumor cells, bone marrow cells, totipotent cells, and the like.
In more specific embodiments the devices and methods provided
herein can produce radicals such as ROS (reactive oxygen species), H.,
.OH and HOO. which can also form H2O2,
this molecule and/or these radicals being toxic to hyperproliferative cells and
thus bring about their inactivation and/or destruction. Lipid peroxydation products,
resulting from the oxidative stress created under the ultrasonic conditions are
also potential participants to this biomechanism.
While evidence against singlet oxygen formation during
sonodynamic therapy has been presented, 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 H.
or .OH radicals formed by pyrolysis of the water solvent.
The species created using the disclosed methods and devices
are thought to be derived from the reaction of high-frequency ultrasound on a water
molecule, most likely giving rise (in particular in the presence of oxygen) to the
H. + .OH
O2 → HOO.
+ HOO. → H2O2 + O2,
.OH → H2O2
Advantageously, the energy required to produce these toxic
species is reduced if the process is performed in the presence of microbubbles,
as described herein. In certain embodiments, a generator is configured to supply
power to the ultrasound emitter at less than 1 W/cm2. In preferred embodiments,
the power is supplied at about 0.5 W/cm2 or lower, or more in many advantageous
embodiments about .25 W/cm2 or lower. In advantageous embodiments, the
power dissipated in the volume of physiological fluid from this level of power applied
to the emitter is less than 30 mW/cm3. In some embodiments, the power
is dissipated at about 7mW/cm3.
While in certain embodiments, the ultrasound can be administered
continuously, in other embodiments, the ultrasound can be administered intermittently,
using ON/OFF cycles. Those with skill in the art can determine effective ON/OFF
cycle times depending on the volume of cells, type of cells, and other relevant
In further embodiments the teachings herein relate to treating
hyperproliferative cells in suspension, as opposed to a tumor or neoplastic mass.
In these embodiments, the teachings herein do not rely on microbubbles to concentrate
or pool at a particular tumor site. This allows for the treatment of unwanted hyperproliferative
cells that do not happen to be clumped together.
Another advantage of the methods and devices provided herein
is that the hyperproliferative cells can be effectively treated in short periods
of time. In specific embodiments, hyperproliferative cells can be treated in under
1 minute. In even more specific embodiments, the cells can be treated in under 30
seconds, including between 5-20 seconds, for example.
As known in the art, biophysical modes of ultrasonic action
are classified as having either thermal, cavitational, or non-thermal and non-cavitational
effects. It is important to note that using the above-described power ranges and
short treatment times, significant fluid and/or cell heating is avoided such that
little or no heat generated cell death occurs. As an example, treatments resulting
in a non-thermal effect include treatments conducted at temperatures below 40°C,
35°C, and 30°C. The power levels are also such that cavitation does not
occur to a significant extent, such that cell membrane injury due to the ultrasound
is substantially avoided.
Under higher ultrasound powers, it has been recently appreciated
that the injection of microbubbles into the ultrasound field gives rise to an increase
in the phenomenon of sonoluminescence, by superposition of the microbubbles onto
the cavitation bubbles induced by the ultrasound, the number of excited and toxic
species can be multiplied. This phenomenon is observed on a macroscopic level when
the ultrasound treatment is synergistically combined with the presence of microbubbles
of suitable size.
In additional embodiments, the devices and methods provided
herein have the advantage that there is no need to devote the ultrasound to specific
zones, since it is observed that the treatment system functions by diffusing the
products formed in situ (for example radicals and H2O2 formed)
towards the reservoir 6 of the aqueous medium to be treated.
In further embodiments, the one or more ultrasound 4 emitters
1 in the devices described herein are oriented so as not to give rise to any standing-wave
phenomena. For example, in certain embodiments, one or more ultrasound emitters
can be oriented obliquely relative to the axis 9 of the compartment 2 (acute angle
not perpendicular to this axis 9) and relative to the flow of liquid and to the
flow of microbubbles 5 (see FIGURE 1) This characteristic makes it possible for
all the microbubbles 5 in the compartment 2 to be treated in a statistically identical
manner, without creating stationary zones in the compartment 2.
The devices and methods herein can include emitting gas
microbubbles with an average diameter of less than 1 mm into a high-frequency ultrasound
field in the treated liquid medium. In some embodiments the diameter of the microbubbles
is less than or equal to 50 µm. Still in other embodiments the microbubbles
have a diameter less than 30 µm. In certain embodiments the microbubbles are
selected from air, oxygen, and ozone microbubbles. In other embodiments the microbubbles
are not ozone microbubbles.
According to other embodiments, the devices and methods
described herein can include a light emitter 12 (i.e. an electromagnetic radiation
emitter) which emits into the compartment 2 in the ultrasound 4 field, radiation,
with a frequency that is mostly in the visible range. However, for certain applications,
in order to remove certain specific hyperproliferative cells, it is advantageous
to emit electromagnetic radiation with a frequency that is mostly non-visible, as
ultraviolet radiation (e.g., UVA, UVB or UVC type), infrared, laser or microwaves.
It has recently been discovered, unexpectedly, that a treatment
comprising the emission of microbubbles into the fields combined with ultrasound
and optionally light radiation is particularly effective at inactivating and removing
hyperproliferative cells present in a liquid medium, such as a physiological fluid.
The phenomenon of luminescence can promote the production of extremely active oxygenated
species such as the superoxide radical or singlet oxygen, which can result in a
series of biochemical reactions that are extremely toxic for certain hyperproliferative
cells. In advantageous embodiments, the radiation is emitted intermittently in ON/OFF
cycles. In more specific embodiments the ON/OFF cycle can be about 5.5ms/3 ms.
It is known that luminescence can take place in the presence
of so-called sensitizing molecules (e.g., photosensitizers and sonosensitizers),
so as to give rise to an anti-tumor action on certain cancer cells. Such molecules
can include: porphyrins, chlorines, tetracyclines, methylene blue, fluorescein,
acridine, rhodamine, and the like. These active agents can be injected into the
organism or administered orally and subsequently activated by sonoluminescence.
After activation, these agents can produce singlet oxygens which in turn plays a
fundamental role, in particular in biochemical processes resulting from oxidative
stress. Specifically, a singlet oxygen can oxidize the various cell components,
such as the proteins, lipids, amino acids and nucleotides, for example.
In other embodiments, solid particles or solid surfaces
can be used to synergize the luminescence and/or emission of radiation. These solids
can include TIO2, clays, and ceramics, for example.
Various embodiments are directed towards devices and methods
which do not require additional chemical products such as photosensitizers and/or
sonosensitizers to neutralize, prevent the growth of, and/or remove hyperproliferative
cells from a physiological medium. It is not always necessary to add a photosensitizing
or sonosensitizing agent to the liquid medium to be treated, since it has been unexpectedly
observed that luminescence can be produced in situ on certain hyperproliferative
cells (e.g, leukemic cells) present in physiological fluids (e.g., blood)
already containing these photosensitizing molecules.
While the devices and methods provided herein can be used
in conjunction with other drugs such as photosensitizers, sonosensitizers, chemotherapeutic
agents, antibiotics, antiviral drugs, it is important to note that the effectiveness
of the provided methods and devices in treating hyperproliferative cells is not
dependent on the use of other chemicals, reagents, or drugs. Accordingly, the methods
and devices described herein can be used without additional substances, including
chemicals, reagents, hormones, peptides, proteins, nucleic acids, carbohydrates,
DNA vaccines, angiogenesis stimulators or drugs. In even more specific embodiments,
the teachings herein do not rely on the cellular absorption of these substances.
Specifically, the effects obtained with the teachings herein
can be achieved without the necessity of classical photosensitizers and sonosensitizers.
The physiological effects obtained with techniques such as PDT depend at the same
time on the radiation dose, on the nature of the photosensitizer used, on their
concentration, and on their localization. While being requirements for traditional
treatments, sensitizers are not needed for the teachings provided herein, thereby
considerably simplifying the methods and devices.
In certain embodiments, the net effects of the ultrasonic
action implicate endogenous photosensitizers in the structure where their local
concentration is high. For example, endogenous photosensitizers are localized mainly
in the membrane structures such as lysosomes, mitochondria, nuclear membranes, Golgi
apparatus, and the microsomes of the endoplasmic reticulum, of which the relative
surface represents nearly 50% of the cell membrane surface.
In some embodiments, the devices and methods described
herein can include a pump for circulating the liquid medium, as well as one or more
apparatuses for recovering, preferably by filtration, centrifugation or precipitation
(such as cyclones, etc.), hyperproliferative cells present in the liquid medium.
In certain embodiments the pump and/or apparatus for recovery are arranged between
the reservoir (or animal) containing the liquid medium to be treated and the compartment
In certain embodiments the devices and methods herein can
be used to extract physiological fluid (e.g., blood) from a subject, suspected
of (e.g., diagnosed) having cancer (e.g., leukemia). After extraction,
the physiological fluid can be treated with high-frequency, low-energy ultrasound
and gas microbubbles with diameters less than 1 mm. In certain embodiments the methods
induce apoptosis on the cancerous cells (e.g., leukemia). After treating
the physiological fluid such that the hyperproliferative cells have been either
sufficiently neutralized, prevented from growing, or removed, the fluid can then
be administered back to the subject. These methods can be performed similar to other
ex vivo methods, such as hemodialysis, for example.
A blood treatment subject can be attached to one of the
devices described herein. According to certain embodiments, the bloodstream of the
subject can be connected to a ultrasound device described herein, through an internal
fistula in their arm. This involves having an artery and a vein connected surgically.
When they are joined, the stronger blood flow from the artery causes the vein to
become larger. Needles can be inserted in the enlarged vein to connect the subject
to the ultrasound device.
Another way to provide access to the bloodstream is to
insert an internal graft. In this procedure an artery is surgically connected to
a vein with a short piece of special tubing placed under the skin, which a needle
can be inserted into.
In other embodiments, when it is necessary to gain access
to the bloodstream quickly, or when the veins in the arms are too small to provide
enough blood for ultrasonic treatment for example, a central venous catheter can
be used. In this procedure, a soft tube is surgically inserted into a large vein
in the neck or near the collarbone. In some embodiments this method can be temporary
until a permanent access site is ready.
Subjects that can be treated according to the methods described
herein can include any animal, such as a mammal, including humans, mice, monkeys,
dogs or pigs.
In further embodiments, the devices and methods herein
utilize low-energy, high-frequency ultrasonic waves to prevent, treat, or neutralize
hyperproliferative cells by inducing apoptosis in the cells (e.g. leukemic cells).
Inducing apoptosis in hyperproliferative cells can lead to a sequence of characteristic
events including a drop in mitochondrial potential, loss of phosphatidylserine asymmetry,
morphological variations, DNA fragmentation, loss of plasma membrane, and the like.
Furthermore, low-energy ultrasound-induced apoptosis can involve activation of caspase-3,
the proteolytic degradation of the caspase substrate PARP, and the modulation of
bcl-2/bax ratio in the cells.
Additional methods involve initiating apoptosis using ultrasound-induced
sonochemical luminescence to trigger photosensitized singlet oxygen production from
direct photoirradiation. In classic ultrasonic irradiation conditions, the direct
destructive cavitation effects dominate the sonoluminescence, which is fairly weak
in the absence of an air/liquid interface injected into the medium. Accordingly,
it can be advantageous to utilize microbubbles in conjunction with ultrasound in
order to enhance luminescence over cavitational effects.
The following examples describe treating cells with high-frequency,
low-energy ultrasound and various assays to indicate the presence of apoptosis in
Cell preparation and high-frequency ultrasound treatment
Human leukemia cell lines (K562, Nalm-6, KGla, 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. Heparinized venous blood was obtained from
healthy volunteers and leukemic patients. Mononuclear cells were separated by Ficoll-Hypaque
gradient density centrifugation (International Medical Products, Brussels, Belgium).
The following describes the ultrasonic treatment on the
cells. Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic
cells, and normal mononuclear cells were treated by ultrasound at a frequency of
1.8 MHz during various exposure times at an acoustical power of 7 mW/mL and irradiation
(ON/OFF) cycles of 5.5ms/3 ms.
After 18 hours culture in the incubator (37°C and
5% CO2) the cells were successfully tested for cell viability by a trypan
blue exclusion assay. Additional tests were performed on the treated cells and are
described in more details in the following examples. Apoptosis was evaluated by
cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial
potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax
ratio were tested by flow cytometry. Cloning efficiency was evaluated by assays
Effect of high-frequency ultrasound on DNA fragmentation
DNA fragmentation has been associated with apoptosis. Quantification
of cells with degraded DNA was performed using a method described by Nicoletti,
I. et al. J Immunol Methods 139(2):271 (1991) and an Apotarget Quick DNA
Ladder Detection Kit (Biosource). Cell pellets (106 cells) were resuspended in 20
L of lysis buffer and DNA was extracted according to the manufacturer's instructions.
DNA was analyzed after separation by gel electrophoresis (1% agarose). As a 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.
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.
Classic nucleosomal DNA ladder patterns were observed in
DNA samples from cells treated by ultraviolet (positive control) and by ultrasound.
Internucleosomal DNA cleavage was barely noticeable 5 hours after the ultrasonic
treatment but became clearly evident 18 hours afterwards (results not shown). Furthermore,
an increase in the number of nuclei with fragmented DNA was observed with PI staining,
5 hours after treatment. Specifically, 15% of the treated cells had fragmented DNA
and 2% of the untreated cells had fragmented DNA (data not shown).
Effect of high-frequency ultrasound on mitochondrial transmembrane potential
The early disruption of mitochondrial transmembrane potential
(&Dgr;Ym), preceding advanced DNA fragmentation, has been observed in several
models of cell apoptosis. The following assay was performed to determine the effect
of high-frequency ultrasonic treatment on &Dgr;Ym.
Mitochondrial potential was estimated by incorporation
of the cationic fluorochrome DiOC6 immediately after cell treatment according to
the published protocol found in A. Macho, et al., Blood 86(7): 2481 (1995).
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.
Ultrasonic treatment was accompanied by an increase of
cell populations displaying a low &Dgr;Ym (results not provided). A population
of cells displaying a reduced DiOC6 incorporation was evidenced 30 minutes after
treatment, and the drop of mitochondrial potential was very clear 5 hours after
the ultrasonic treatment with more than 50% of the cells having a low &Dgr;Ym.
These results provide evidence of cellular apoptosis.
Effect of high-frequency ultrasound on cellular glutathione levels
It has been shown that there is a depletion of glutathione
during apoptosis. An assay was performed to determine cellular glutathione content
after ultrasonic treatment. Cell Tracker green CMFDA (5-chloromethyl fluorescein
diacetate; Molecular Probes) was used for determining levels of intracellular glutathione
as previously described in D.W. Hedley et al. Cytometry 15: 349 (1994).
Directly after ultrasonic treatment, a subpopulation appeared
with lower levels of GSH than that observed in untreated cells (>50% of cells
displaying a low level of GSH) as shown in FIGURE 3. The results of successive treatments
indicated a larger GSH depletion after 5 hours. The results, expressed as a percentage
of cells displaying a GSH level comparable to untreated cells, clearly demonstrate
that high-frequency ultrasonic treatment is associated with GSH depletion.
Effect of high-frequency ultrasound on cellular caspase-3 activity
Caspase-3 has been shown to play an important role in chemotherapy-induced
apoptosis. Specifically, activation of caspases leads to cell demise via cleavage
of cellular substrates such as actin, gelsolin, or PARP. To directly address the
involvement of caspase-3 in ultrasound-induced apoptosis, caspase activity was determined
using flow cytometry and colorimetric assay.
Specifically, 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).
As shown in FIGURE 4, high-frequency ultrasonic treatment
led to the activation of caspase-3. Moreover, this protease activity was maximal
at 1 hour post-treatment. Furthermore, cleavage of PARP was apparent 2 hours after
treatment, with 40% of cells stained by the rabbit anti-PARP FITC vs 5% for untreated
cells (results not shown).
Effect of high-frequency ultrasound on cellular BCL-2/BAX ratio
Different proteins of the bcl-2 family have been implicated
in triggering or preventing apoptosis. The following assay was performed to determine
whether bcl-2 and bax, the two major members of the bcl-2 family, were involved
in the induction of apoptosis by ultrasound. 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.
The results (data not provided) demonstrated that untreated
cells expressed high levels of the anti-apoptotic protein-bcl-2 protein (47 ±
4 × 103 MESF) and this expression appears as a unimodal peak of fluorescence.
One hour after ultrasonic treatment, the expression of bcl-2 protein was already
downregulated (respectively 40 ± 0.9 and 32 ± 0.9 × 103 MESF in K562
cells treated by 1 or 3 ultrasonic treatments). Two hours after treatment, bcl-2
expression appeared clearly bimodal, the cells displaying either a bcl-2high (comparable
to untreated cells) or bcl-2low phenotype (11 ± 2 × 103 MESF).
In contrast to bcl-2, levels of the pro-apoptotic protein,
bax, were higher in cells treated with ultrasound as compared with untreated cells
(respectively 85 ± 0.5 and 48 ± 5 × 103 MESF for treated and untreated
K562). The ratio of bcl-2/bax was thus significantly reduced during high-frequency
ultrasonic treatment, providing evidence of cellular apoptosis (0.98 for control
cells vs 0.38 for ultrasound-treated cells).
Effect of high-frequency ultrasound on levels of cellular phosphatidylserine
During apoptosis, phosphatidylserine residues flip from
the inside to the outside of the plasma membrane and this change can be detected
using Annexin-FITC, which binds to the PS residues. The following describes an Annexin
V binding assay that was performed. 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 were presented as dot plots showing the change in mean
fluorescence intensity of Annexin-V-FITC/propidium iodide (not shown).
The results indicated that the changes of phosphatidylserine
distribution varied according to time. Specifically, the results showed that ultrasonic
treatment provoked plasma membrane injury in a low percentage of cells, demonstrating
that the necrotic action of the tested ultrasound treatment is very weak. Interestingly,
an increase in apoptotic cells was observed two hours after treatment. Five hours
after the treatment, 35% of cells were Annexin-V-positive, demonstrating the ultrasonic
induction of apoptosis in K562 cells.
Effect of high-frequency ultrasound on colony formation
An important proof for an effect on cell viability is the
inability of a cell to multiply and form a colony. The following describes a clonogenic
assay that was performed on a K562 cell line to determine the effect of high-frequency
ultrasonic irradiation on cloning efficiency. 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%. As shown in FIGURE 5, a significant reduction in cloning efficiency
of K562 cells is observed after 1 and 3 treatments (respectively 25% and 42% of
inhibition), confirming the sensitivity of leukemic cells to high-frequency ultrasound.
Effect of oxygen scavengers on apoptosis
The following procedure was performed to determine the
effect of active oxygen scavengers on the induction of apoptosis by high-frequency
ultrasound. K562 cells were incubated with L-histidine (10 mM) and/or mannitol (100
mM). Some cells were treated by high-frequency ultrasound and others were not. Cell
apoptosis was detected by an Annexin-V/PI assay. The results, as provided in Table
1, demonstrate that the ultrasonically induced cell damage is significantly reduced
in the presence of histidine and mannitol (respectively 43% and 47% of inhibition
of apoptosis induced by 3 successive treatments).
of active oxygen scavengers on ultrasonically induced cell apoptosis
No US treatment
1 US treatment
3 US treatments
18 ± 6
42 ± 8
63 ± 5
15 ± 5
24 ± 8
36 ± 11
11 ± 4
21 ± 5
30 ± 3
Histidine + Mannitol
11 ± 2
16 ± 3
25 ± 5
Results are expressed
as percentage of cells displaying phosphatidylserine extemalization 5 hours post-treatment
(mean ± SEM from 4 independent experiments).
The association of mannitol and histidine led to more than
60% of inhibition of apoptosis. The effectiveness of these agents on reducing cellular
apoptosis induced by ultrasonic treatment provides evidence that ultrasonically
induced singlet oxygen and hydroxyl radicals are important mediators to induce apoptosis.
Effect of successive treatments of high-frequency ultrasound on cells
A flow cytometry follow-up was performed on K562 cells
cultured 0.5, 2, and 5 hours after ultrasonic treatment. After one treatment, the
level of apoptotic cells observed was three times that of the control (respectively
26% and 8% after 5 hours of culture for treated and untreated cells). A necrotic
effect of 5 to 10% was also observed, which is well below that found when using
drugs or photodynamic treatment (PDT) treatment. With successive irradiations, under
the same conditions (7 mW/mL, 20 seconds) and at different intervals, apoptosis
of K562 cells increased to 37 ± 3% (p < 0.02) and 49 ± 5% (p < 0.02)
after respectively 1 and 3 successive treatments (FIGURE 6). Morphological variations
(e.g., cell shrinkage, membrane blebbing, chromatin condensation) were also observed
after successive treatments (results not shown). FIGURE 7 demonstrates that the
amount of cellular phosphatidylserine increases after successive high-frequency
ultrasound treatments as detected by an Annexin-V assay.
Effect of high-frequency ultrasound on various cell lines
In addition to K562, the effect of ultrasonic treatment
was tested on other normal and malignant cell lines, including KG1a (immature minimally
differentiated acute myeloid leukemia blasts), HL-60 (promyelocytic leukemia), and
Nalm-6 (ALL cell line). The results presented in FIGURE 8 demonstrate that the sensitivity
to ultrasound depends on cell type, but successive treatments led to a significant
increase in the number of apoptotic cells for all cell lines evaluated.
Mononuclear cells from 5 patients (1 refractory anemia
with excess of blast cells [RAEB], 1 secondary acute myelogenous leukemia [AML],
and 3 cases of AML French-American-British [FAB] classification M3, M4, and M4Eo)
were also treated by ultrasound, and blast cells were discriminated from contaminating
normal cells on the basis of their CD45 expression as previously described in F.
Lacombe, F. et al. Leukemia 11:1878 (1997). These cells had also been labeled
for their phosphatidylserine exposure by FITC-annexin. This method makes it possible
to compare the respective apoptotic behavior of leukemic blast cells and normal
cells treated by ultrasound. The results presented in FIGURE 8 demonstrate that
primary leukemic cells are sensitive to ultrasonic treatment with more than 37 ±
18% of apoptotic cells observed 5 hours after 3 treatments.