Treatment of transplanted adriamycin-resistant ovarian cancers in mice by combination of adriamycin and ultrasound exposure

Ovarian cancer models were established in cyclophosphamide immunosuppressed mice by subrenal capsular cell fibrin clot transplantation. SKOV3 cancers were treated by adriamycin alone, or adriamycin combined with ultrasound exposure. SKOV3/ADR cancers were treated with adriamycin, as well as verapamil and insonation were administrated alone or concurrently. The results were: (1) Insonation alone could not suppress growth of tumours. (2) In SKOV3 cancers, ultrasound exposure potentiated the efficiency of adriamycin. (3) In SKOV3/ADR cancers, insonation reversed adriamycin resistance, but verapamil was not effective and no synergism existed between it and ultrasound. These findings revealed that ultrasound exposure enhanced the efficiency of adriamycin to both chemosensitive and chemoresistant ovarian cancers in vivo. Mechanisms were discussed.     

The use of models in "target" theory to evaluate the survival curves of human ovarian carcinoma cell line exposure to adriamycin combined with ultrasound

The models of "target" theory in radiation biology were used to evaluate the survival curves of human ovarian carcinoma cell line after exposure to adriamycin combined with ultrasound. 3AO cells were exposed to adriamycin in group ADR, to adriamycin after ultrasound exposure in group US+ADR, and to adriamycin prior to insonation in group ADR+US. The cell survival was determined by a clonogenic assay. The dose-response curves were fitted to two models, (1) single-hit, single-target model, (2) single-hit, multi-target model. The single-hit, multi-target model could fit the survival curve in group ADR, but it could only describe the survivals in groups US+ADR and ADR+US while the concentration of adriamycin was less than 0.05 mug/ml. These suggested that the single-hit, multi-target model could be conditionally used to describe the survival while cells were exposed to the combination of adriamycin and acoustic exposure. The models also were tools to understand the potentiation attributable to insonation.     

Potentiation of cytotoxicity of adriamycin on human ovarian carcinoma cell line 3AO by low-level ultrasound.

The aim of this study was to determine whether the ultrasound, with a dosage that did not lead to acute and delayed inhibition, could potentiate the cytotoxicity of adriamycin to human ovarian carcinoma cell line 3AO in vitro. Drug sensitivity was analyzed by clonogenic assay, cells were treated by adriamycin singly in group ADM (control), adriamycin prior to ultrasound exposure in group ADM + US, and ultrasound irradiation prior to adriamycin administration in group US + ADM. The intracellular drug accumulation in each group was determined by fluorometry. The results were: (1) the values of IC50 were 0.0083, <0.001 and 0.0065 microg/ml in group ADM, ADM + US and US + ADM respectively; the clone surviving rate in group ADM + US and in group US + ADM were decreased (P < 0.001, P < 0.01), compared with control; the surviving rate in group ADM + US was lower than that in group US + ADM (P < 0.01). (2) The intracellular drug accumulations in group ADM + US were promoted (P < 0.01) and not increased significantly in group US + ADM (P > 0.05). These suggested that the low-level ultrasound could enhance the cytotoxicity of adriamycin to human ovarian carcinoma cells and promoted intracellular drug contents played the leading role.     

The effect of free radical scavenger and antioxidant on the increase in intracellular adriamycin accumulation induced by ultrasound

Ultrasound could potentiate cytotoxicity of adriamycin on cancer cell line as a result of increased intracellular accumulation ascribed to cavitation. In order to determine which free radical led to increase of drug content, effects of the free radical scavenger and antioxidant on increased intracellular adriamycin accumulation by ultrasound were investigated. The intracellular drug content of adriamycin was lower in the group where histidine was administrated before ultrasound exposure and in the group where mannitol was added after sonication. Drug accumulation was also decreased in groups in which vitamin C administrated either before or after ultrasonic exposure. These results suggested that hydroxyl radical play the leading role in synergism between ultrasound and adriamycin.     

Biological effects of ultrasound exposure on adriamycin-resistant and cisplatin-resistant human ovarian carcinoma cell lines in vitro

Human ovarian cancer cell lines, SKOV3 and its adriamycin-resistant substrain SKOV3/ADR and COC1 and its cisplatin-resistant substrain COC1/DDP, were subjected to acoustic exposure. The critical levels (LC), which resulted in no immediate cell killing, were determined in four cell lines, respectively. LC were the same in four cell lines. After being insonated by LC, cell proliferation and clone forming of SKOV3/ADR were suppressed but those of SKOV3 were not affected (1); cell reproduction of COC1 was triggered but that of COC1/DDP was not influenced (2); flow cytometry detected sub-G1 peaks in SKOV3/ADR and COC1/DDP (3). These findings suggested that there were differences in the responses to ultrasound exposure between chemosensitive and chemoresistant human ovarian cancer cells.     

Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells

The effects of low-power ultrasound, the anti-cancer drug cisplatin, and their combined application were studied in two lines of human ovarian carcinoma cells, A2780 and A2780cis. Four modes of treatment were used: exposure to ultrasonic field, application of cisplatin, exposure to ultrasound followed by cisplatin, and presence of cisplatin followed by exposure to application ultrasound. Ultrasound was used at intensities of 0.5 W/cm² and 1.0 W/cm² for 10 min, cisplatin was applied at concentrations of 1 μM and 6 μM per cell suspension treated in A2780 and cisplatin-resistant A2780cis cells, respectively. The results of each experimental treatment were assessed by the resultant cell viability related to the viability of control cells, using a standard MTT test. It was shown that a combined effect of ultrasound and cisplatin was more effective than that of ultrasound or cisplatin alone. It also appeared that the order of application played a role, with the cisplatin-ultrasound treatment lowering cell viability more than the ultrasound-cisplatin treatment. It can be assumed that the exposure of cells to a low-power ultrasonic field has an immediate effect on the structure of cell surfaces and, consequently, on entry of cisplatin into the cell.The study also included observations on changes in the cell cycle associated with the treatments used in both cell lines and their evaluation by flow cytometry.     

Stability of alteplase in presence of cavitation

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.     

Ultrasound-induced cytolysis of cancer cells is enhanced in the presence of micron-sized alumina particles

Micron-sized alumina particles have been shown to enhance sonochemical free radical formation in aqueous solutions and simultaneously increase the solution temperature and acoustic (white) noise, effects attributable to enhanced inertial cavitation [T. Tuziuti, J. Phys. Chem. A 109 (2005) 4869–4872]. In the current study, the same ultrasound exposure system was applied to in vitro cancer cells as a model system to determine the effect of alumina particles on the long-term survival of cells and on the major pathways of cell death, i.e., either apoptosis or necrosis. Following 6 h of incubation after ultrasound treatment, it was found that the cells died mainly through necrosis, irrespective of whether the exposure was conducted in the presence of alumina particles or not. Alumina particles were non-toxic to cells alone, but were found to decrease the long-term survivability of cells that survived the initial exposure. This effect depended on the size and concentration of particles. These results correlated well with the effect of alumina particles on the sonochemical oxidation of KI under the same exposure conditions. Spin-trapping with 5,5-dimethyl-pyroline N-oxide (DMPO) and electron spin resonance spectroscopy indicated that the sonochemical formation of OH radicals increased in the presence of alumina particles. The current study is consistent with the well known observation that micron-sized particles enhance the acoustic cavitation process.     

Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.     

Basic study on apoptosis induction into cancer cells U-937 and EL-4 by ultrasound exposure

Recently, the low invasive cancer treatments with small aftereffects have been considered. We are studying on the suppression methods of cancer cell proliferation with ultrasound. Cancer cells of mouse T lymphoma (EL-4) have been used in our study. The human histitocytic lymphoma cells (U-937) was used in this time. The cancer cells were cultured in a culture medium of RPMI1640. The standing wave acoustic field was formed in a water tank of our ultrasound exposure system by a vibrating plate driven with a Langevine type transducer. The U-937 and EL-4 were exposed to ultrasound in the acoustic field with spatial average acoustic intensity of 350 mW/cm(2) at 150 kHz. The viable rate of EL-4 decreased with the lapse of culture time after ultrasound exposure. U-937 did not show the remarkable decrease tendency. The proliferation of U-937 which exposed to ultrasound with 700 mW/cm(2) was suppressed. It can be thought that apoptosis was induced in the cancer cells in this condition. We observed the morphological change on the U-937 exposed to ultrasound with this condition. The morphological changes by apoptosis like the shrink of cells, formation of apoptotic bodies etc. can be observed with an optical microscope and a phase contrast microscope.     

Cytotoxic effects and in vitro reversal of multidrug resistance by therapeutic ultrasound in human hepatocarcinoma cell line (HepG2)

Multidrug resistance (MDR) is one of the major obstacles to successful chemotherapy of human malignancies. Although many strategies have been explored to overcome MDR, none of them have been proven to be clinically useful until now. The aim of this study was to investigate whether a novel therapeutic ultrasound (US) approach would have useful effects on the reversal of MDR in cancer cells. Wild-type and MDR phenotype (HepG2/ADM) cells of the human hepatocarcinoma cell line HepG2 were exposed to 0.8 MHz US at an intensity of 0.43 W/cm² for a 9 s exposure (total energy density: 3.87 J/cm²). After US exposure, cell number and viability were counted immediately, and flow cytometry was performed to measure retention of rhodamine 123 and adriamycin in HepG2 and HepG2/MDR cells. Both cell lines were then incubated in suspension with adriamycin, vincristine, etoposide, cisplatin and 5-fluorouracil, respectively, and the MTT assay was used to determine cytotoxicity. The results showed that US exposure could significantly increase the uptake of Rh123 and ADM by HepG2/ADM tumor cells. The resistant index for the chemotherapeutic drugs was significantly lower in the US-exposed HepG2/ADM cells than in those not exposed to US. It was therefore concluded that US exposure could enhance the sensitivity of HepG2/ADM tumor cells to these chemotherapeutic agents, and the functional and structural changes induced by previous US exposure in MDR tumor cells may be responsible for it. However, further study is needed to investigate the mechanism behind US-mediated reversal of MDR.     

Effects of nonlinear propagation, cavitation, and boiling in lesion formation by high intensity focused ultrasound in a gel phantom

The importance of nonlinear acoustic wave propagation and ultrasound-induced cavitation in the acceleration of thermal lesion production by high intensity focused ultrasound was investigated experimentally and theoretically in a transparent protein-containing gel. A numerical model that accounted for nonlinear acoustic propagation was used to simulate experimental conditions. Various exposure regimes with equal total ultrasound energy but variable peak acoustic pressure were studied for single lesions and lesion stripes obtained by moving the transducer. Static overpressure was applied to suppress cavitation. Strong enhancement of lesion production was observed for high amplitude waves and was supported by modeling. Through overpressure experiments it was shown that both nonlinear propagation and cavitation mechanisms participate in accelerating lesion inception and growth. Using B-mode ultrasound, cavitation was observed at normal ambient pressure as weakly enhanced echogenicity in the focal region, but was not detected with overpressure. Formation of tadpole-shaped lesions, shifted toward the transducer, was always observed to be due to boiling. Boiling bubbles were visible in the gel and were evident as strongly echogenic regions in B-mode images. These experiments indicate that nonlinear propagation and cavitation accelerate heating, but no lesion displacement or distortion was observed in the absence of boiling.     

Quantification of optison bubble size and lifetime during sonication dominant role of secondary cavitation bubbles causing acoustic bioeffects

Acoustic cavitation has been shown to deliver molecules into viable cells, which is of interest for drug and gene delivery applications. To address mechanisms of these acoustic bioeffects, this work measured the lifetime of albumin-stabilized cavitation bubbles (Optison) and correlated it with desirable (intracellular uptake of molecules) and undesirable (loss of cell viability) bioeffects. Optison was exposed to 500 kHz ultrasound (acoustic pressures of 0.6-3.0 MPa and energy exposures of 0.2-200 J/cm2) either with or without the presence of DU145 prostate cancer cells (10(6) cells/ml) bathed in calcein, a cell-impermeant tracer molecule. Bubble lifetime was determined using a Coulter counter and flow cytometer, while bioeffects were evaluated by flow cytometry. The lifetime of Optison cavitation nuclei was found to decrease and bioeffects (molecular uptake and loss of cell viability) were found to increase with increasing acoustic energy exposure. These bioeffects correlated well with the disappearance of bubbles, suggesting that contrast agent destruction either directly or indirectly affected cells, probably involving unstabilized cavitation nuclei created upon the destruction of Optison. Because Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects, the indirect mechanism involving secondary cavitation bubbles is more likely.     

Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection

The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated. HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene. Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium. When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively. When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases. These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases.     

超声增强脂质体与细胞相互作用的研究

理论及实验研究了超声增强脂质体与细胞的相互作用.实验制作了包裹荧光素的脂质体,利用1 MHz聚焦超声增强脂质体与乳癌细胞的相互作用,采用荧光显微镜观察与荧光素结合前后细胞的变化,流式细胞仪定量检测细胞中包含的荧光素.结果表明,在声压幅度为0.24 MPa超声作用40 s后,细胞吸收荧光素能力比对照组（无超声作用）有8.78％的提高.理论讨论了超声增强脂质体与细胞相互作用的可能物理机制,指出超声诱发的脂质体运动及脂质体的粒径变化是超声增强脂质体与细胞相互作用的原因. 关键词： 脂质体 药物传递 超声辐射力     

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity I_SATA (spatial averaged, temporal averaged) varied from 0.33 W/cm² to 8 W/cm² (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm².     

Ultrasound-mediated disruption of cell membranes. I. Quantification of molecular uptake and cell viability

Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells. Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation. This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased. Varying pulse length produced no significant effect. All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure. This suggests that acoustic energy exposure may be predictive of ultrasound's nonthermal bioeffects.     

Biological effect of paramecium in diffused ultrasonic fields

Since the cell structure or biophysical reaction includes in the interaction between ultrasound and living matter. When multi-cell creature is exposed to ultrasound, this reaction will lead the biological effect becomes complex. Therefore, in this paper, a single cell creature is chosen to study the biological effects induced by ultrasound exposure. The paramecium, which possesses many features typical of higher-order animal cells, was considered an appropriate choice for this study. The ability shown by ultrasound in promoting and/or accelerating many reactions has been shown to be a useful field. The growth phase of paramecium by using the ultrasound irradiation is an important parameter in this study. In our experiment, the exponential and stationary phases were employed. Three important factors must be taken into account when an ultrasonic biological reaction is investigated: the medium system, the bubbles' field, and the acoustic field. The medium system involves the physical parameters of the medium field. The bubbles' field includes the reacting bubble size of the liquid and the resonance frequency of the reaction bubbles. The oscillation of the cells in response to the ultrasound radiation is simulated using Rayleigh-Plesset's bubble activation theory. The resonance frequency of the unicellular creature is then calculated. The acoustic field is about using the diffuse field theory of Sabine to create a uniform sound field for the radiation experiment. The resonance frequency of the paramecium vacuole is among 0.54-1.09 MHz. When the 0.25 and 0.5 MHz frequencies of ultrasound was irradiated in the stationary phase of the paramecium, the relative growth rate was about 20% lower than that of unexposed sample. Therefore, the phenomenon of inhibition and destruction appeared during irradiation. The exponential phase of the paramecium samples appear to be different when irradiated with 1 MHz ultrasound. As can be seen in the results, the maximum relative growth rate was increasing 18% with 1 MHz ultrasound exposure.     

Increased accumulation of paclitaxel and doxorubicin in proliferating capillary cells and prostate cancer cells following ultrasound exposure

Introduction

We have previously reported enhanced cytotoxic effects of both doxorubicin and antisense oligonucleotides using an optimized ultrasound regime of a single 10 s exposure in burst-mode (4 MHz, 32 W/cm²(SaTa), 50 ms burst period) in both PC3 (prostate cancer) cells and angiogenic Huvec (human umbilical cord endothelial cells). The objective of this study was to investigate the effect of ultrasound on the cellular uptake of both hydrophilic agents (rhodamine R123, doxorubicin hydrochloride and mannitol) and hydrophobic agents (rhodamine R6G and paclitaxel) using the same 4 MHz ultrasound exposure system.

Methods

PC3 cells and Huvec were incubated with solutions of radioactive or fluorescent compounds for 1 h and ultrasound was then applied to cells. Following washing and lysis of cells, the degree of drug uptake was measured using liquid scintillation counting or fluorescence spectroscopy.

Results

Ultrasound exposure resulted in the enhanced uptake of both hydrophilic and hydrophobic compounds into cells. For paclitaxel, approximately 100% increased uptake was observed when the drug was encapsulated in a nanoparticulate micellar formulation compared to approximately 50% for free drug.

Conclusions

The 4 MHz, 32 W/cm² ultrasound exposure regime (using burst-mode with 50 ms burst period) allows for the enhanced uptake of both water soluble and insoluble compounds into proliferating cancer and angiogenic cells.     

Improving dispersion of nanometer-size diamond particles by acoustic cavitation

A novel acoustic-dispersion method for fine diamond particles was developed. Two samples of nanometer-sized diamond particles were used. They had primary particle sizes of 5 nm (ND5) and 150 nm (ND150). Disaggregation of agglomerated particles using ultrasound and surface modification of ND5 and ND150 were investigated. The ND5 and ND150 particles aggregated to secondary particles, having sizes on the order of micrometers. The surfaces of ND5 and ND150 particle were modified due to chemical reactions and the particles were disaggregated by acoustic cavitation. The ND5 particles were disaggregated to give an average particle size of about 100 nm by ultrasound exposure with average acoustic intensities higher than 800 W/m(2). The agglomerated ND150 particles with size of 15 microm were disaggregated to reach an average particle size of about 300 nm by ultrasound exposure with an average acoustic intensity higher than 2000 W/m(2). The surfaces of ND5 and ND150 particles were found to be modified with hydroxyl groups resulting from acoustic cavitation. This could lead to a well dispersed solution of nanometer-sized diamond particles in water.