Effects of therapeutic ultrasound on the nucleus and genomic DNA

Ultrasonics Sonochemistry xxx (2014) xxx–xxx

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Effects of therapeutic ultrasound on the nucleus and genomic DNA Yukihiro Furusawa a,1, Mariame A. Hassan a,b,⇑, Qing-Li Zhao a, Ryohei Ogawa a, Yoshiaki Tabuchi c, Takashi Kondo a a

Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr Al-Aini str., Cairo 11562, Egypt c Division of Molecular Genetics Research, Life Science Research Center, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan b

a r t i c l e

i n f o

Article history: Received 29 November 2013 Received in revised form 21 February 2014 Accepted 25 February 2014 Available online xxxx Keywords: Cavitation DNA strand breaks DNA damage repair response Free radicals Mechanical effects Therapeutic ultrasound

a b s t r a c t In recent years, data have been accumulating on the ability of ultrasound to affect at a distance inside the cell. Previous conceptions about therapeutic ultrasound were mainly based on compromising membrane permeability and triggering some biochemical reactions. However, it was shown that ultrasound can access deep to the nuclear territory resulting in enhanced macromolecular localization as well as alterations in gene and protein expression. Recently, we have reported on the occurrence of DNA double-strand breaks in different human cell lines exposed to ultrasound in vitro with some insight into the subsequent DNA damage response and repair pathways. The impact of these observed effects again sways between extremes. It could be advantageous if employed in gene therapy, wound and bone fracture-accelerated healing to promote cellular proliferation, or in cancer eradication if the DNA lesions would culminate in cell death. However, it could be a worrying sign if they were penultimate to further cellular adaptations to stresses and thus shaking the safety of ultrasound application in diagnosis and therapy. In this review, an overview of the rationale of therapeutic ultrasound and the salient knowledge on ultrasound-induced effects on the nucleus and genomic DNA will be presented. The implications of the findings will be discussed hopefully to provide guidance to future ultrasound research. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Ultrasound is cyclic sound pressure travelling at frequencies greater than the upper limit of human hearing (>20 kHz in healthy, young adults). Most of the medical ultrasound equipment operate at frequencies in the range from 1 to 15 MHz whereas therapeutic applications are usually restricted to the lower frequencies of this range (usually around 1 MHz) [1]. Despite being beyond human cognition, ultrasound is well perceived by cells. The relationship between cells and ultrasound is mutual where the way in which cells affect ultrasound waves provide the basis for medical sonography, while the way in which ultrasound affects cells – i.e. the bioeffects – provide the basis for therapeutic ultrasound. The

⇑ Corresponding author at: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. Tel.: +81 76 434 7265; fax: +81 76 434 5190. E-mail address: [email protected] (M.A. Hassan). 1 Current address: RIKEN Center for Integrative Medical Sciences (IMS-RCAI), Kanagawa 230-0045, Japan. The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

identification of ultrasound-induced bioeffects and understanding their underlying molecular mechanisms is important not only for uncovering the potential of ultrasound in therapeutic purposes, but also for avoiding adverse effects during diagnostic ultrasound imaging. In spite of the long decades of ultrasound use in medical practice and the wide strides taken in optimizing sonography, the various possibilities of bioeffects, their mediating acoustic effectors and biological pathways, and their dependence on operating ultrasound parameters still cannot be delimited or described properly. There are a wide spectrum of effects observed including differentiation and proliferation [2,3], transient membrane poration [4–7], necrosis, apoptosis [8–11], and cell lysis [12]. Retrospective analysis of sonicated cells shows that ultrasound impact on cells varies from cell deformation to cell lysis and from stimulating proliferation to cellular engagement in programmed cell death. In fact, there are many factors that render the complete understanding of ultrasound bioeffects a difficult task. The multiplicity of ultrasound effectors (thermal, mechanical, and chemical), and the various intracellular targets (cell membrane, cytoplasmic constituents, and nucleus) as well as the dynamic nature of cellular processes reflect the wide range of possibilities to arise. In addition,

http://dx.doi.org/10.1016/j.ultsonch.2014.02.028 1350-4177/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Y. Furusawa et al., Effects of therapeutic ultrasound on the nucleus and genomic DNA, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.02.028

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ultrasound studies employ different experimental setups and parameters, cells (origin: primate cells, human cells, etc.; orientation: suspended, adherent, 3d cultures, in-vivo, etc.). Thus, it is demanding not to spare effort to review the literature and scrutinize data among studies to come up with a provisional summary that would provide guiding signs for future ultrasound research to translate in clinical practice. In this review, we will try to present the salient knowledge on the bioeffects that are specifically observed in the nucleus upon exposure to therapeutic levels of ultrasound and discuss on the mediating ultrasound effectors and molecular pathways. 2. Rationale for therapeutic ultrasound In this section, a short overview of the mechanisms by which ultrasound can alter the biology of cells with relevance to their applications will be provided. 2.1. Thermal effects Unlike electromagnetic waves, ultrasound arises from energy transmission from a source to molecules which undergo vibration in consecutive compression (at high pressure) and expansion (at low pressure) cycles usually along the direction of propagation, and thus translating energy from location to another. This energy transmission can cause a relative rise in medium temperature. The extent of temperature rise depends on the initial energy of the acoustic beam. Thus, tuning ultrasound parameters (such as ultrasound frequency, intensity, irradiation time, etc.), as well as the sonicated area, can maximize or minimize the thermal effect on any object falling in the wave propagation field. Ultrasoundinduced hyperthermia contributes to some of the bioeffects of ultrasound and promotes for certain medical applications. For example, equipment emitting High Intensity Focused Ultrasound (HIFU) are now intended for cancer ablation therapy in some clinics all over the world. A focused beam of high intensity ultrasound (100–10,000 W/cm2) falls on a tumor mass to raise its temperature up to more than 60 °C in a short time [13]. This ‘‘flash’’ heating causes irreversible damage and cell killing through coagulative necrosis. Another application of thermal ultrasound includes physiotherapy in which a continuous beam of ultrasound is employed to heat up soft tissues (usually not to more than 10 °C) in order to relieve pain and swelling. By applying heat to the affected tissues, blood flow, elasticity and metabolic processes are increased [14]. In addition, ultrasound-induced hyperthermia has been claimed to enhance drug uptake in cells either through modulating the drug release from heat-sensitive dosage forms [15] or through modulating membrane permeability, especially in cells displaying multidrug resistance [16]. Extrapolating from the issue of acoustic energy transmission to surrounding particles, it is important to denote that the acoustic energy absorbed may not essentially manifest as increase in temperature. This issue has been explained by the ‘‘Frequency resonance hypothesis’’. It says that if enough acoustic energy transmits to certain moieties with critical structural conformations e.g. proteins, a modification in their resonant activity or a flip from one form to another may occur resulting in a modulation of their effector functions [17]. 2.2. Non-thermal/mechanical effects Although the temperature rise induced by ultrasound can be completely suppressed by altering the operating parameters (e.g. using pulsed waves, decreasing exposure time, decreasing ultrasound intensity), it is impossible to similarly silence the

pressure fluctuations in the acoustic field. Ultrasound waves are physical in their nature and the molecules of the transmitting medium must undergo alternative compression and expansion cycles in order to sustain ultrasound propagation. Any damping of this particulate translational movement attenuates or halts the transmission of ultrasound. Thus, in cases where ultrasound thermal effects are claimed the major player in the observed effects, it is still impossible to disregard the mechanical effects on nearby targets and their possible contribution to the observed outcomes. The mechanical (non-thermal) effects are in part due to the transmission of some of the beam momentum to the surrounding medium resulting in convection motion known as ‘‘acoustic streaming’’. In real biological environments, acoustic streaming is not a significant effector though, because of the efficiency of blood circulation in vasculature and the lack of sufficient fluid reservoirs in tissues [18]. However, the main ultrasound-induced mechanical stress arises when the acoustic beam transmits in a fluid containing either dissolved gases that can form (micro)-cavities, or artificial (stabilized) microbubbles (known as ultrasound contrast agents injected into the patients to enhance tissue echogenicity during sonography). These gaseous inclusions respond to the acoustic cyclic pressure alterations by oscillation to create convective motion (micro-streaming) and shear stresses in their vicinities. This phenomenon is recognized as ‘‘non-inertial’’/‘‘stable’’ cavitation. With continued oscillation, the shape and size of the microbubbles change, that, under certain conditions, they implode (‘‘Inertial’’/ ‘‘Transient’’ cavitation) releasing shear waves and liquid jets. After collapsing, fragments of the microbubbles can form smaller microbubbles that might eventually start another cycle of growth, oscillation, and collapse. The cavitational activity of microbubbles affects nearby cells and/or particles depending on the separating distance. The closer the targets to oscillating/collapsing microbubbles, the more rigorous the impact will be [19]. This relation also justifies for the heterogeneous nature of ultrasound bioeffects [20,21]. The mechanical effects of ultrasound are primarily observed on the cell membrane as mild membrane disruptions, cellular deformation [7], (transient) membrane poration [22], or lysis [23]. Moreover, because of the intricate network regulating mechanotransduction that can transduce extracellular physical forces deep into cells [24,25], ultrasound has been shown to result in a number of intracellular events that include triggering intracellular biochemical reactions and changes in gene expression [26,27] or inducing DNA damage [21,28]. Collectively, these bioeffects may culminate in increased cell proliferation [29,30] or recruitment in cell self-killing programs (apoptosis) [8,10] suggesting a role for ultrasound in wound and bone fracture-accelerated healing, and cancer eradication.

2.3. Sonochemical effects The oscillating microbubbles prior to their collapse encounter high internal pressure and high temperatures as well. Despite being highly localized and fleeting that they hardly contribute to any thermal effect, these high temperatures at the core can lead to water sonolysis and free radical formation. Hydrogen (H) atoms and hydroxyl (OH) radicals have been confirmed as the primary products of water sonolysis by electron paramagnetic resonance (EPR) and spin-trapping experiments [31,32]. These radicals can either combine to form H2, H2O2, and water, or attack solute molecules which are reduced or oxidized. Sonolysis in the presence of oxygen leads to the formation of oxygen atoms. Hydrogen atoms can react with oxygen to form HO2 radicals, the acid form of superoxide anion radicals [33]. The sonolytic formation of superoxide anion radicals was demonstrated in 1972 by use of the superoxide dismutase (SOD) inhibitable reduction of cytochrome C [34].

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As to their involvement in ultrasound-induced bioeffects, the literature has conflicting reports between support and denial. For example, the presence of free radical scavengers was shown to decrease the selective enhancement of caveolar-dependent permeability and the oxidative stress in endothelial cells treated with ultrasound [35,36]. In other case, transient permealization in cells exposed to ultrasound in presence of microbubbles was related to H2O2 formation [37]. This is in complete contrast to Mortimer and Dyson’s showing that Ca2+ uptake (as an indicator of cell permealization) was increased in sonicated fibroblasts in absence of transient cavitation and consequently the absence of free radicals [38]. In addition, Feril et al. has shown that ultrasound-induced apoptosis is triggered solely by a sonomechanical mechanism [8]. At this point, it has to be emphasized that the free radicals observed in ultrasound studies are of two types depending on where they are initially formed. One type is the free radicals that originate extracellularly as a result of inertial cavitation and can traverse freely into the cells. These free radicals are short-lived and thus their contribution to bioeffects is doubted [39]. However, if they can react with medium solutes and undergo a chain reaction that can extend their presence, some bioeffects may arise [35]. On the other hand, cells may produce free radicals intracellularly in response to mechanosensing or as a scheduled event within a triggered biochemical pathway (e.g. ultrasound – induced apoptosis) [11]. 3. Intracellular bioeffects of therapeutic ultrasound Since the early days of discovering the bioeffects of ultrasounds, the cell membrane has gained most of the attention being the first target to interact with acoustic waves and being the first defense line of the cell. As mentioned, cell membrane effects were readily observed after ultrasound irradiation with a wide range of lesions identified. They were shown to (momentarily) deform, liquefy or leak under acoustic stresses even in absence of transient cavitation [7,40,41]. Sustained effects occur under more vigorous exposure conditions that result in cell membrane tearing (cell lysis) [12,23]. In cases of extensive damage to the membrane, it is comprehensible that ultrasound gains access into deeper intracellular targets [42]. However, at milder exposures where (apparent) viability is conserved, the reported changes in gene expression and cellular functions are more difficult to explain. The knowledge that all the cell organelles are wired together in an intricate complex network sensing extracellular stresses through the cell membrane may provide a major clue for intracellular effects of ultrasound and again places the cell membrane on the top of the hierarchy of signal transduction. But is it possible that ultrasound can travel deeper and input directly into the intracellular targets? To resolve this ambiguity, studies on cell micro-rheology in an ultrasound field should be considered [43,44]. In the following sections, we will discuss the current knowledge on the bioeffects induced by therapeutic level ultrasound in the nuclear milieu. 4. Ultrasound- mediated nuclear delivery In 2005, opportunity was afforded to question the possibility that ultrasound can localize extracellular macromolecules directly into the nucleus. In these studies by Duvshani-Eshet et al. group, they reported on the successful nuclear delivery depending on the ultrasonic parameters provided that sonication time exceeds 10 min [45,46]. A further study then confirmed that the observed nuclear delivery was purely intracellular and is not related to sonoporation and increased/sustained membrane permeability [47]. It is suggested that part of this intracellular effects could be due to nuclear pore complex (NPC) opening [48]. Ultrasound results in

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an immediate elevation of intracellular calcium ions (Ca2+) due to Ca2+ influx when membrane permeability is compromised by the exposure [49]. This free cytosolic Ca2+ protects against the drainage of the intracellular Ca2+ stores including the cisternae of the nuclear membrane and the NPCs will remain open to macromolecules to traverse [50,51]. In fact, the study of the changes in the nuclear envelope is somewhat difficult and tedious because of the complex dynamic nature of NPCs and their uneven distribution among cells. However, recent efforts have been made to depict the morphological changes in the nuclear envelope in a time-dependent manner following exposure to therapeutic ultrasound using transmission electron microscopy (TEM). The study evaluated three criteria; namely, the membrane per pore area, the number of NPCs per unit area and the diameter of NPCs. With the assumption that the membrane area does not change after exposure, the study suggested that some NPCs temporarily disintegrated while the remaining portion had wider diameters [52]. All the observed morphological changes were reversed after 2 h incubation at 37 °C. Interestingly, this study sonicated cells for 10 min with continuous 1 W/cm2 at 1 MHz central frequency, making the conditions quite comparable to those employed for nuclear localization. The authors here did not provide data to whether similar changes can be detected following short exposure periods common to most delivery and cancer eradication-directed studies (usually 1 min) nor did they provide information on survival rates in their work. Nonetheless, the reversal of nuclear membrane changes might be taken as conserved viability. Whether these changes are relevant to ultrasound-enhanced nuclear delivery remains to be explored in future studies. Again, it is still unclear whether these morphological changes are caused by ultrasound mechanical effects (direct or transduced) or in response to oxidative stress (data not available).

5. Effects of therapeutic ultrasound on DNA DNA, the cell information bank, encounters different chemical and physical insults that can affect its integrity and result in structural alterations and mutations. DNA damage has been associated with many human disorders such as cancer, immunodeficiency and neurodegeneration. DNA damage can occur due to extrinsic factors (e.g. exposure to ionizing radiation or DNA-affinic moieties which include base analogues, intercalating agents, and alkylation compounds) or intrinsic factors (e.g. replication errors, exposure to intracellular reactive oxygen species (ROS) and/or oxidant metabolites of the body). DNA damage can occur through different approaches depending on the damaging agent. For instance, DNA can undergo oxidative or hydrolytic cleavage. The best known examples include the oxidation of guanine to 7,8-dihydro-8oxoguanine (oxoG) which readily pairs with adenine as well as cytosine, and the deamination of cytosine to uracil. The introduction of defects in one DNA strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs) can also occur through a direct damage or as a consequence of nucleotide excision repair where a mismatched base is excluded enzymatically from the sequence. Since DNA damage is a sort of daily incidence in each cell, cells had to be equipped with sophisticated mechanisms for repair that is either global or in-process during replication. DNA repair proceeds through replacing the erratically installed bases (base excision repair; BER) or the defective portions (nucleotide excision repair; NER) of the damaged DNA. In some cases, cells fail to reverse the damage and thus engage in self-killing programs with further DNA fragmentation. The interaction of ultrasound and purified DNA in aqueous solutions has been detected and identified decades ago. It is believed that both the mechanical and chemical effects of ultrasound are

Please cite this article in press as: Y. Furusawa et al., Effects of therapeutic ultrasound on the nucleus and genomic DNA, Ultrason. Sonochem. (2014), http://dx.doi.org/10.1016/j.ultsonch.2014.02.028

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responsible for the detected oxidative damage and the single- and double-strand raptures in the DNA helix [53,54]. This effect has made of ultrasound a useful laboratory tool for the preparation of DNA samples in-vitro. However, in a biological environment, it is uncertain how applied ultrasound can impact genomic DNA. Interesting research work claimed that ultrasound can image DNA loss of integrity and suggested that ultrasound can be used in monitoring cell death in response to therapeutics in cancer patients [55]. The principle depends on the increased ultrasoundbackscatter in the scans of cells with condensed DNA (treated cells). Under the study conditions, control untreated cells seemed refractory to ultrasound exposure. However, this is not the case with other studies which suggest the ability of ultrasound itself to induce DNA lesions or alter expression even in clinical applications [56,57]. Here, a thorough discussion on ultrasound-induced DNA alterations, causes and possible consequences will be presented. 5.1. Changes in gene expression Ultrasound’s potential to induce changes in gene and protein expression has been reported on several occasions. The roots for these observations lie in the responsiveness of the genetic machinery to mechanical stress [44]. There are two levels to discuss ultrasound-induced changes in gene expression. The first level is concerned with the changes in genomic DNA by ultrasound exposure in absence or in presence of cavitational activity. It was shown that low-intensity pulsed ultrasound (LIPUS) can modify the expression of some genes that stimulate cell growth [58–60]. On the other hand, a number of studies reported that after sonication the up-regulated genes served in the accomplishment of apoptosis [9,61]. Gene expression analysis following HIFU treatment showed different pattern of changes depending on the HIFU mode applied [62]. The major difference between all these studies that may justify for the different patterns of change in gene expression is the acoustic parameters applied. It might be that each ultrasound condition has a fingerprint change on the genomic processes. This derives from a broader concept that each physical stress owns a specific fingerprint effect on gene expression that reflects the integrated effector mechanisms of the respective treatment even though they may all finally converge into one biological response e.g. apoptosis. In our laboratory, we have found that ionizing radiation-, hyperthermia- and ultrasound- induced apoptosis in human leukemia (U937) cells underlay networks of interest that despite sharing up-regulated genes in common, they contain differentially up-regulated genes illustrating the intricate pathways responsive to each treatment (Fig. 1). To this end, we would recommend to construct a study-based database for differentially expressed genes with relevance to the acoustic parameters used hoping that this might end up in mapping the acoustic effects on gene expression. The second level of ultrasound effects on gene processes discusses its ability to promote the expression of exogenously introduced genes. This possibility has been suggested in several studies, however, the validity has not been confirmed yet [63,64]. Although the role of ultrasound in permealizing cells can be suppressed; however, the evaluation of the role of ultrasound on promoter activity still requires an elaborate experimental procedure to discriminate it from other possible intracellular effects [47]. 5.2. Evidence for single-strand breaks SSBs are the commonest lesions to occur to genomic DNA that can arise from ROS directly attacking the deoxyriboses or indirectly through enzymatic cleavage of the phosphodiester backbone during DNA BER.; but their repair is readily achievable once detected

based on the other intact template strand. In 1985, Kondo et al. showed that the irradiation of aqueous DNA solution with continuous 1.2 MHz ultrasound beam resulted in strand breaks. The group concluded that SSBs were induced mainly by free radicals from water sonolysis [65]. The induction of SSBs within the cellular context was then confirmed using human leukocytes in vitro [66]. Debates have arisen as to whether the detected SSBs occurred in cells surviving exposure or in non-viable cells. Miller et al. carried out extensive series of studies proving that cells directly irradiated with ultrasound were not to survive with their SSBs, but when incubated with ultrasound-induced free radical-laden phosphate buffer saline, they could tolerate SSBs induced by residual peroxide and could elicit a successful repair response in a time-dependent manner [67–69]. Later, Milowska et al. was able to detect reparable SSBs in viable fish erythrocytes exposed directly to ultrasound with repair peak at 1 h post exposure [39]. These observations were not eliminated when the exposure was performed in presence of catalase indicating that peroxide was not responsible for the induced SSBs. Similarly, the observed SSBs in the study of Ashush et al. could not be attributed to generated free radicals [70]. The discrepant results on ultrasound-induced SSBs could be explained by the difference in exposure conditions among studies. In the experiments carried out by Miller group the exposure of cells were performed at 2 °C using more vigorous ultrasound parameters. The sonication of cells under hypothermic conditions raise skepticism regarding the membrane rigidity and re-sealing efficiency following exposure [71]. Therefore, it seems that in near physiologic conditions, the short-lived free radicals generated from inertial cavitation may not access genomic DNA, however, intracellular oxidative stress in response to ultrasound-induced chemical and/ or mechanical stresses might be responsible for the observed lesions [72]. Despite the controversy on the mechanism of damage, ultrasound-induced SSBs are confirmed, and the extent of SSBs occurrence as well as repair efficiency depends on the acoustic parameters. 5.3. Evidence for double-strand breaks DSBs are considered the most dangerous of DNA lesions because their repair occurs at random in absence of an intact template copy. If the repair gene is incorrect, genomic rearrangements (deletions, translocations, and fusions in the DNA, etc.) can occur and all these consequences are strongly related to cancer development. DSBs were previously confirmed in a sonicated solution of pure DNA [73]. Recently, we have confirmed the occurrence of DSBs in different leukemia cell lines in vitro upon exposure to ultrasound using neutral comet assay (Fig. 2). The DNA damage was independent of apoptosis and was mainly induced by the cavitational (mechanical) activity. The use of the tri-atomic gas N2O to suppress cavitation was able to diminish DSBs whereas the addition of the radical scavengers DMSO and N-acetyl cysteine did not affect ultrasound-induced cH2AX. The extent of damage was comparable to a 10 Gy-dose of ionizing radiation, however, DNA damage response (DDR) and repair processes were shown to proceed differently [21,74]. In general, the histone H2AX is phosphorylated upon sensing DNA DSBs, and acts as platform for spatiotemporal assemblies of numerous DDR proteins to repair DNA. The activation of H2AX is downstream to the phosphoinositol-3-kinase kinase (PIKK) family proteins, namely, ataxia-talengiectasia-mutated (ATM), DNAdependent protein kinase (DNA-PKcs), and ataxia telangiectasia and Rad3-related protein (ATR). The detection of the phosphorylated H2AX provides a very sensitive method for the detection of DSBs (the presence of 1DSBs in cell nucleus can be detected using anti-cH2AX antibody). Although the phosphorylation of H2AX was confirmed in ultrasound-treated cells (Fig. 3), ultrasound appears to activate DNA-PKcs in preference to ATM as proven by chemical

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Fig. 1. Venn-diagram showing the up-regulated genes in human leukemia (U937) cells treated with isoeffective doses of ultrasound, hyperthermia, and ionizing-radiation. The different treatments induced 15–25% DNA fragmentation (apoptosis) based on DNA fragmentation assay described in [81]. Total RNA was extracted 3 h after treatment. Gene expression analysis and data extraction was carried out as described in [82]. ATF3: Cyclic ATP dependent transcription factor, BIRC3: Survivin, DDIT3: DNA damage induced transcript, DUSP1: Dual specificity phosphatase 1, HMOX1: Heme oxygenase-1, PPP1R15A: Protein phosphatase 1, regulatory (inhibitor) subunit 15A, SOD2: Superoxide dismutase 2.

Fig. 2. Detection of DNA DSBs by neutral comet assay in U937 cells immediately after exposure to ionizing radiation (IR, 10 Gy), or ultrasound at intensities of 0.3 or 0.4 W/cm2 (1 MHz central frequency, 10% duty cycle, 100 Hz pulse repetition frequency, 1 min). The relative tail moment (mean ± SD) is given by the ratio of comet tail moments of treated cells to those of control.

inhibition analysis using the pharmacological kinase inhibitors KU55993 and NU7026, respectively. Fluorescence imaging of ATM, DNA-PKcs and cH2AX in the nuclei of sonicated cells revealed unique distribution patterns (Illustration Fig. 4) in which the activation of H2AX and ATM was pan-nuclear and homogenous with discrete co-localized foci. It could be that the chromatin remodeling in

response to ultrasound mechanical stress resulted in global activation of ATM and H2AX. DNA-PKcs stained at T2609 phosphorylation cluster revealed that its intranuclear activation was independent from cH2AX foci, an observation similar to the case with ionizing radiation, whereas staining at S2056 phosphorylation cluster showed that DNA-PKcs-pS2056 was present at the periphery with overlapping regions of co-localization with cH2AX. Thus DNAPKcs-pS2056 may mediate non-homologous end joining (NHEJ) repair in bulk nuclear ultrasound-induced DSBs, and signal to cH2AX presence at the nuclear periphery. Still, this unique perinuclear distribution of DNA-PKcs-pS2056 cannot be explained in terms of the current knowledge. However, it is motivating to bring a mention of professor Foaini’s recent uncovering of the mechanosensing properties of ATR. Professor Foaini (Department of Biosciences, University of Milan, Italy) has spoken on several scientific events that ATR is a part of an integrated mechanical response coupling plasma membrane induced signaling with the changes within the nucleus. Whether this can hold true for the sonically induced-perinuclear DNA-PKcs in mammalian cells is left to future investigations. Unique to Furusawa’s et al. work that the group sets clear limits between ultrasound-induced DNA damage and ultrasoundinduced bioeffects leading to DNA damage by using apoptosis inhibitors (c.f. Ref. [21] versus [9]). However, it was not clear whether the cells with damaged DNA ended up in the non-viable fraction or not. It is important to discriminate the presence of DNA lesions with respect to viable fraction of cells because novel evidences show that DNA damage can be penultimate to other cellular responses and modifications that can result in cellular adaptation to applied stresses (discussed later). In summary, it has been confirmed that DSBs occur under certain conditions of sonication. They are also reported to occur in clinical applications of HIFU. They are accounted for by the mechanical stresses imposed by ultrasound. However, it remains unclear whether these mechanical stresses are laid directly on the nucleus or are indirectly transduced.

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5.4. Therapeutic ultrasound multidrug resistance Medical practitioners would have been fortunate enough if the claim that Ultrasound-induced DNA damage was observed mainly in dead cells held true. However, the rebuttal of this claim by subsequent studies added a new question about the possible consequences of such an effect. It is believed that faulty repair of DNA damage is where roads diverge to cell death or abnormal viability. It was notable in some reports that cells treated with ultrasound initially displayed features of cell death, but finally they could catch up with the control on the long term [75–77]. It was not until the early years of this century that a novel pathway for escaping genomic instability was discovered in which cells revert to an evolutionary preserved unusual type of division defined as neosis. Neosis is characterized by emergence of a progeny of small cells (>10) termed ‘‘Raju cells’’ with stem-cell like characters from a single mother cell through budding. Being favorable in p53-nonfunctional cells, a case encountered in most cancers, they not only can extend the life span of cancer cells, but also can develop resistance under chemical stresses [78]. In a pioneer study, signs of noetic division were observed post exposure to DNA-damaging ultrasound in vitro [28]. This phenomenon could shake the concept of therapeutic ultrasound if proven to occur under clinical conditions. It is worth mentioning here that in most of ultrasound studies, cells proved to revert to a pre-sonication state with respect to their cell cycle after 24 h post sonication [79]. It was not until recently that acoustic effects on cell cycle were probed to occur early after exposure and almost peak at 12 h [28,80]. Although this transient arrest in cell cycle may simply indicate that the cell had to halt its proliferation for a while to scan for and repair DNA lesions, it could also imply that cellular decisions to ultrasound-induced DNA damage are very early and rapid. 6. Conclusions

Fig. 3. Phosphorylation of H2AX induced by ultrasound treatment. Human leukemia U937 cells were treated with 1 MHz ultrasound at intensity of 0.3 W/cm2, 10% duty cycle for 1 min. After 30 min incubation period, cells were collected and immune staining of phosphorylated H2AX was performed as described in [21]. Cells were then analyzed flow cytometrically or microscopically (panel a). (b) Shows the dependence of H2AX phosphorylation on ultrasound intensity. US: ultrasound.

In this review, we have briefly overviewed the rationale for therapeutic ultrasound and focused on the bioeffects seen on the nucleus and the genomic DNA. As far as we know, this is the first review to focus on the biological effects of therapeutic level ultrasound observed in the nuclear territory. Evidence has been shown that ultrasound can induce DNA lesions (SSBs and DSBs) which may not always end in cell killing, it is important to consider and identify the possible consequences of these findings in future studies. This subject is of interest because it relates to the possible

Fig. 4. Illustrative diagram showing the distribution pattern of cH2AX, ATM pS1981, DNA-PKcs pT2609, and DNA-PKcs pS2056 in the nuclei of sonicated cells in response to DSBs sensing. The question mark indicates that it is unknown whether the mechanical effects of ultrasound to which the DNA damage is attributed access directly to the nucleus or transduced through the cytoskeleton.

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deleterious effects of ultrasonic diagnostic devices and the therapeutic applications especially for cancer eradication.

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