Ultrasound-induced Apoptosis Research Articles

280 - Differential Role of Small Sized Gold Nano-Particles on X-Irradiation and Ultrasound-Induced Cell Death

X-irradiation (X-rays) and Ultrasound have been shown to induce apoptosis in cancer cells. Gold nanoparticles (Au-NPs) are used as promising sensitizers due to the high atomic number and greater biocompatibility than other metals. However, effects of Au-NPs (2 nm size) with physical modalities remain to be elusive. Therefore, this study is intended to investigate the effects of Au-NPs, on X-rays and ultrasound induced apoptosis in human lymphoma U937 cells. Effects of Au-NPs on X-rays and ultrasound-induced apoptosis were determined by observing the changes in intracellular reactive oxygen species (ROS) formation and apoptotic signaling pathways. Radiation-induced apoptosis was significantly inhibited in the cells pre-treated with Au-NPs. In contrast, Au-NPs showed enhancement of apoptosis in combination with ultrasound. ROS generation was increased in the combined treated cells with ultrasound and Au-NPs, which ultimately enhanced the DNA damage, while no such changes were observed following combination of radiation and Au-NPs. Our findings indicate the potential use of Au-NPs in combination treatment and would further clarify the role of Au-NPs in radiation and ultrasound-aided therapies.

Suppression of human 8-oxoguanine DNA glycosylase (OGG1) augments ultrasound-induced apoptosis in cervical cancer cells

PurposeHuman 8-oxoguanine DNA glycosylase (OGG1) is a major base excision repair enzyme, and it was reported to suppress the activation of intrinsic apoptotic signaling pathway in response to oxidative stress. In this study, our aim was to investigate the effects of OGG1 downregulation on ultrasound-induced apoptosis in cervical cancer cells. MethodsOGG1 expression was silenced by shRNA in the cervical cancer SW756 and CaSki cells. Cell viability was evaluated by MTT assay after OGG1 knockdown following ultrasound treatment. Ultrasound-induced apoptosis was measured by Annexin V-FITC/propidium iodide. Intracellular reactive oxygen species (ROS) production and Ca2+ concentration were detected using a fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and a green fluorescent dye fluo-4AM, respectively. Western blotting was used to analyze the expression of Bcl-2, Bax, cleaved caspase-3, and nuclear factor-κB p65 (NF-κB p65). ResultsThe results indicated that OGG1 knockdown did not suppress cell proliferation, but significantly augmented ultrasound-induced inhibitory effects on the cell viability, and increased ultrasound-induced early apoptosis and late apoptosis and necrosis in the SW756 and CaSki cells when exposure to ultrasound (1MHz) at 1.5W/cm2 for 30 and 60s. OGG1 knockdown significantly increased intracellular ROS production and Ca2+ concentration after incubation of 6, 24, and 48h post-ultrasound treatment. The downregulation of Bcl-2 protein and the upregulation of Bax, cleaved caspase-3, and NF-κB p65 protein levels were observed in the shRNA-OGG1 cells and mock-shRNA cells, but no significant change of these protein levels was found between of them. ConclusionsThese results indicate that downregulation of OGG1 expression can augment ultrasound-induced apoptosis in cervical cancer cells, which suggests that OGG1 suppression might provide a new insight for ultrasound-induced therapeutic effects on cervical cancer treatment.

Caveolin-1 Mediates Low-Intensity Ultrasound-Induced Apoptosis via Downregulation of Signal Transducer and Activator of Transcription 3 Phosphorylation in Laryngeal Carcinoma Cells

Low-intensity ultrasound therapy has been found to be a potential tool in the management of malignant tumors in recent years. However, the molecular mechanism underlying low-intensity ultrasound-induced apoptosis is still not clear. In this study, we investigated the effects of low-intensity ultrasound-induced apoptosis in HEp-2 cells. We found that low-intensity ultrasound significantly induced apoptosis, and the expression level of caveolin-1 (Cav-1) was dramatically increased after ultrasound treatment of HEp-2 cells. After inhibiting the expression level of Cav-1 using siRNA transfection, we found that the cellular apoptosis induced by low-intensity ultrasound was significantly suppressed. In addition, inhibition of Cav-1 expression promoted phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that the STAT3 signaling pathway was involved in low-intensity ultrasound-induced apoptosis via Cav-1 regulation. Our results indicate that Cav-1/STAT3 signaling pathway may mediate low-intensity ultrasound-induced apoptosis, and this technology could potentially be used clinically for the treatment of cancers.

Potential mechanism of apoptosis induced by ultrasound in human hepatocarcinoma cells via comparative proteomic analysis.

To analyze the potential molecular mechanism of ultrasound induced apoptosis in cancer cells, comparative proteomic methods were introduced in the study. After ultrasound exposure at the intensity of 1.2 W/cm2, the human SMMC-7721 hepatocarcinoma cells were stained by trypan blue to detect the morphologic changes, and then the flow cytometry was used to examine the percentage of early apoptosis via double staining of FITC-labelled Annexin V and Propidium iodide. The proteins were separated by two-dimensional (2D) SDS polyacrylamide gel electrophoresis (PAGE). Among them, the differently expressed proteins were identified by MALDI-TOF mass spectrometry to reveal the key proteins response to ultrasound exposure. It's proved early apoptosis of cells were induced by focused ultrasound. After ultrasound exposure, the expressing characteristics of several proteins changed, in which some proteins in HSP family are associated with apoptosis initiation. It is suggested that the focused ultrasound could be applied in the assistant cancer therapy. Moreover, it is proved the comparative proteomic methods could supply information about the protein expression to analyze the metabolic processes related to bio-effects of biomedical ultrasound.

Low intensity-pulsed ultrasound induced apoptosis of human hepatocellular carcinoma cells in vitro

The present study was conducted to determine whether low intensity-pulsed ultrasound (LIPUS) could induce apoptosis of human hepatocellular carcinoma cells, SMMC-7721, and to define the mechanism of ultrasound-induced apoptosis, in vitro. MTT assay was used to measure cell proliferation. Apoptosis was investigated by multiple methods such as flow cytometry, DNA fragmentation, Ca2+ mobilizations, pro- and anti-apoptotic protein expression, and light as well as ultramicroscopic morphology. The results provide evidence that LIPUS induced a dose-dependent effect on cell viability and apoptosis of SMMC-7721 cells. Specifically, exposure of cells to >0.5W/cm2 intensity significantly increased cell apoptosis, caused shifts in cell cycle phase, and induced structural changes. Ultrasound significantly increased intracellular Ca2+ concentrations and modulated expression of caspase-3, Bcl-2 and Bax. The findings suggest that this novel technology can be used to induce SMMC-7721 apoptosis via the Ca2+/mitochondrial pathway and could potentially be of clinical use for the treatment of hepatocellular carcinoma (SMMC-7721 cell line) and other cancers.

Contrast-enhanced harmonic endoscopic ultrasound imaging: basic principles, present situation and future perspectives.

Over the last decade, the development of stabilised microbubble contrast agents and improvements in available ultrasonic equipment, such as harmonic imaging, have enabled us to display microbubble enhancements on a greyscale with optimal contrast and spatial resolution. Recent technological advances made contrast harmonic technology available for endoscopic ultrasound (EUS) for the first time in 2008. Thus, the evaluation of microcirculation is now feasible with EUS, prompting the evolution of contrast-enhanced EUS from vascular imaging to images of the perfused tissue. Although the relevant experience is still preliminary, several reports have highlighted contrast-enhanced harmonic EUS (CH-EUS) as a promising noninvasive method to visualise and characterise lesions and to differentiate benign from malignant focal lesions. Even if histology remains the gold standard, the combination of CH-EUS and EUS fine needle aspiration (EUS-FNA) can not only render EUS more accurate but may also assist physicians in making decisions when EUS-FNA is inconclusive, increasing the yield of EUS-FNA by guiding the puncture with simultaneous imaging of the vascularity. The development of CH-EUS has also opened up exciting possibilities in other research areas, including monitoring responses to anticancer chemotherapy or to ethanol-induced pancreatic tissue ablation, anticancer therapies based on ultrasound-triggered drug and gene delivery, and therapeutic adjuvants by contrast ultrasound-induced apoptosis. Contrast harmonic imaging is gaining popularity because of its efficacy, simplicity and non-invasive nature, and many expectations are currently resting on this technique. If its potential is confirmed in the near future, contrast harmonic imaging will become a standard practice in EUS.

Cytotoxicity enhancement of DOX-liposome-containing microbubbles combined with ultrasound to the multi-drug resistant cell

Multidrug resistance (MDR) in cancer cells is a significant obstacle to successful cancer therapy. Doxorubicin (DOX) is very active chemotherapeutic agent for the treatment of breast cancer and the efficacy of DOX is also restricted by multidrug resistance.Ultrasound (US) targeted destruction of drug loaded microbubbles (MBs) is gaining more and more attention as a promising strategy for locally drug delivery. In this article, through avidin–biotin binding of DOX-containing liposomes to the microbubbles, we developed DOX-liposome-containing microbubbles in order to investigate its effect of enhancing cellular uptake and cytotoxicity of DOX against drug-resistant cancer cells. The results demonstrated that treatment of cells with ultrasound and DOX-liposome-containing microbubbles caused a significant higher drug uptake in DOX-resistant MCF-7 cells, compared with control (DOX-liposome). More importantly, a significant enhancement of tumor growth inhibition against DOX-resistant MCF-7 cells was found when using the drug-liposome-containing microbubbles combined with ultrasound.Compared with DOX-liposome treatment, the cytotoxicity effect was greatly enhanced from 21% to 60%. By further mechanism study, DOX-loaded microbubbles plus ultrasound induced significant apoptosis in MDR line of MCF-7 cells.

Bioeffects of Low-Intensity Ultrasound In Vitro

The purpose of this study was to evaluate the potential molecular mechanism of low-intensity ultrasound-induced apoptosis by analyzing protein profile alteration in response to ultrasound exposure. Human hepatocarcinoma SMMC-7721 cells were used in this study. Cell viability was measured by a trypan blue dye exclusion test. Morphologic changes were examined by light microscopy. Apoptosis was assessed by phosphatidylserine externalization and DNA fragmentation. The pattern of the mitochondrial membrane potential decrease was determined by flow cytometry. Protein profile alteration was analyzed by comparative proteomics based on 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Low-intensity ultrasound (3.0 W/cm(2), 1 minute, cells incubated for 6 hours after ultrasound exposure) induced early apoptosis (mean +/- SD, 26.5% +/- 6.2%) significantly (P < .05) with minimal lysis in human hepatocarcinoma cells in vitro. On a molecular level, several proteins, eg, cellular tumor antigen protein 53, BH3-interacting domain death agonist, apoptosis regulator Bcl-2, and heme oxygenase 1 were identified as responding to ultrasound irradiation, suggesting that mitochondrial dysfunction and oxidative stresses were involved in ultrasound-induced apoptosis. It was also assumed that mitofilin-regulated crista remodeling may be a potential channel of mitochondrial membrane permeabilization pore formation involved in low-intensity ultrasound-induced apoptosis. This study suggests that 2 potential molecular signaling pathways are involved in ultrasound-induced apoptosis. It is a first step toward low-intensity ultrasound-induced apoptotic cancer therapy via understanding its relevant molecular signaling and key proteins.

Saving Cells from Ultrasound-Induced Apoptosis: Quantification of Cell Death and Uptake Following Sonication and Effects of Targeted Calcium Chelation

Applications of ultrasound for noninvasive drug and gene delivery have been limited by associated cell death as a result of sonication. In this study, we sought to quantify the distribution of cellular bioeffects caused by low-frequency ultrasound (24 kHz) and test the hypothesis that Ca 2+ chelation after sonication can shift this distribution by saving cells from death by apoptosis. Using flow cytometry, we quantitatively categorized sonicated cells among four populations: (i) cells that appear largely unaffected, (ii) cells reversibly permeabilized, (iii) cells rendered nonviable during sonication and (iv) cells that appear to be viable shortly after sonication, but later undergo apoptosis and die. By monitoring cells for 6 h after ultrasound exposure, we found that up to 15% of intact cells fell into this final category. Those apoptotic cells initially had the highest levels of uptake of a marker compound, calcein; also had highly elevated levels of intracellular Ca 2+; and contained an estimated plasma membrane wound radius of 100–300 nm. Finally, we showed that chelation of intracellular Ca 2+ after sonication reduced apoptosis by up to 44%, thereby providing a strategy to save cells. We conclude that cells can be saved from ultrasound-induced death by appropriate selection of ultrasound conditions and Ca 2+ chelation after sonication. (E-mail: prausnitz@gatech.edu)

High-intensity focused ultrasound induced apoptosis with caspase3, 8, and 9/6 activation in rat hepatoma.

The purpose of the present study is to investigate anticancer efficacy and apoptosis confirmed by caspase under several exposure conditions of high-intensity focused ultrasound (HIFU). Twenty-five rats with KDH-8 hepatoma were treated by HIFU at several acoustic energies to evaluate treatment efficacy. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst 33258 staining, and caspase3, 8, and 9/6 activity was respectively assayed. The KDH-8 subcutaneous tumors were reduced by HIFU, and these rats survived longer than the nontreatment rats (P<0.01). The minimal threshold of HIFU energy was 30W×1.0s for tumor control and long-term survival. The tumors exposed to HIFU exhibited marked apoptotic features under conditions of less than 10W×1.0s. In cultured KDH-8 cells, apoptosis was caused at less than 30W×1.0s (P<0.01), and more was induced as the energy went down. Caspase3, 8, and 9/6 were more activated at low energy under 10W×1.0s (P<0.01), and caspase8, which is death receptor dependent, was significantly more activated than caspase9/6, which is mitochondria dependent (P<0.01). HIFU-induced apoptosis invivo and invitro is one of the mechanisms for tumor control and is mediated by caspase3, 8, and 9/6. The significantly greater activation of caspase8 than of caspase9/6 suggests that the apoptosis pathway induced by HIFU might be more mitochondria dependent than death receptor dependent. However, further examination will be needed.

Ultrasound-induced apoptosis in the presence of Sonazoid and associated alterations in gene expression levels: A possible therapeutic application

Ultrasound (US) has been shown to induce apoptosis and cell lysis in cancer cells. In this study, we report on the potential of using Sonazoid, a new echo-contrast agent, that is chemically more stable and US-resistant in combination with US in cancer therapy. The biological effects and their mechanisms in the presence or absence of ultrasonic exposure in vitro were investigated. In addition, the impact of the agent on the expression levels of genes responsive to US was studied using global-scale microarrays and computational gene expression analysis tools. Our results show that the combination led to enhanced cell killing in the presence of 1 MHz acoustic field. The apoptosis induction was shown to be mediated by the mitochondrial pathway. The occurrence of US-induced DNA damage was also observed. Despite these findings, the agent at concentrations similar to those clinically used can be considered as well tolerated. Furthermore, Sonazoid enhanced expression of genes that related to apoptosis and are responsive to US, although it alone had almost no effect. These results indicate the potential of Sonazoid for US contrast enhancement as well as the possibility of its use in US-aided therapies.

Potential mechanism in sonodynamic therapy and focused ultrasound induced apoptosis in sarcoma 180 cells in vitro

Sonodynamic therapy employs a combination of ultrasound and a sonosensitizer to enhance the cytotoxic effect of ultrasound and promote apoptosis. However, the mechanism underlying the synergistic effect of ultrasound and hematoporphyrin is still unclear. In this study, we investigated mechanism of the induction of apoptosis by sonodynamic therapy in Sarcoma 180 cells. The cell suspension was treated by 1.75-MHz focused continuous ultrasound at an acoustic power ( I SATA) of 1.4 ± 0.07 W/cm 2 for 3 min in the absence or presence of 20 μg/ml hematoporphyrin. The proportion of apoptotic cells was determined by flow cytometry. We then analyzed the reactive oxygen species generation and localization by confocal microscopy. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the expression of caspase-8, caspase-9, poly(ADP)-ribose polymerase, and nuclear factor-κB. The findings of our study indicate that ultrasound treatment induced the activation of nuclear factor-κB as an early stress response. When cells were pretreated with hematoporphyrin, the initial response to the therapy was the formation of 1O 2 in the mitochondria. Our results primarily demonstrate that the mechanisms of induction of apoptosis by ultrasound and hematoporphyrin-sonodynamic therapies are very different. Our findings can provide a basis for explaining the synergistic effect of ultrasound and hematoporphyrin.

Bioeffects of Low-Energy Continuous Ultrasound on Isolated Sarcoma 180 Cells

Background: The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. Methods: After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Results: Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. Conclusion: ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis.

Growth inhibition of neurofibroma by ultrasound-mediated interferon γ transfection.

We have previously shown that ultrasound-mediated transfection (sonotransfection) can be optimized using a concept based on the ultrasound-induced apoptosis produced in our in vitro experiments. At optimized conditions, we have shown, using five cancer cell lines, that sonotransfection is superior to other conventional nonviral methods. Interferon gamma (IFN-γ) transfection using lipofection has been found to markedly inhibit the proliferation of neurofibroma cell lines. In this study, we investigated whether sonotransfection of IFN-γ to neurofibroma cell lines can suppress cell proliferation. The ultrasound device used was the SonoPore KTAC-4000, which is capable of various acoustic settings. Ultrasound transducers at an oscillation frequency of 1.011 MHz were used; the potential ideal conditions were an intensity of 0.17 W/cm(2) at a burst frequency of 0.5 Hz, 25% duty factor, and 30-s sonication duration. Cells were assayed at 3 and 5 days after sonication. The transfection efficiency was found to be 12%. The ultrasound-treated cells were successfully transfected with IFN-γ genes as detected by enzyme-linked immunosorbent assay, and the cell growth ratio in the IFN-γ sonotransfection group tended to be lower than that in the other experimental groups. These results suggested that IFN-γ sonotransfection could potentially become a nonsurgical method for treating skin lesions such as neurofibromas.

Low intensity ultrasound-induced apoptosis in human gastric carcinoma cells

To investigate the low intensity ultrasound (US)-induced apoptosis in human gastric carcinoma cells and its potential mechanism and to suggest a new therapeutic approach to gastric carcinoma. Human SGC-7901 gastric carcinoma cells were cultured in vitro and irradiated by low intensity US for 10 min at different intensities with different incubation times after irradiation. Morphologic changes were examined under microscope with trypan blue staining and then the percentage of early apoptotic cells was detected by flow cytometry (FCM) with double staining of fluorescein isothiocyanate (FITC)-Annexin V/propidium iodide (PI). Two-dimensional electrophoresis (2DE) was used to get the protein profile and some proteins differently expressed after US irradiation were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Functional analysis was performed to investigate the mechanism of US-induced cell apoptosis. The percentage of apoptotic cells increased about 10% after US irradiation (12.0 W/cm(2), 12 h culture). The percentage of early apoptosis and secondary necrosis in the US-irradiated cells increased with the increased US intensity. Moreover, apoptotic cells increased with the increased culture time after US irradiation and reached its maximum at about 12 h. Several new proteins appeared after US irradiation and were up or down regulated more than 2 times. Some heat shock proteins (HSPs) were found to be associated with the signal process simulating the apoptosis of cells. Low intensity US could induce apoptosis in human gastric carcinoma cells. US-induced apoptosis is related to US intensity/culture time. US-induced apoptosis may be caspases-dependent and endoplasmic reticulum (ER) stress-triggered apoptosis may also contribute to it. Proteomic experimental system is useful in finding the protein alteration in carcinoma cells after US irradiation, helping to develop a new cancer therapy.

Activation of Bak in ultrasound-induced, JNK- and p38-independent apoptosis and its inhibition by Bcl-2

The molecular mechanisms underlying ultrasound-induced apoptosis remain poorly understood. We have demonstrated that in Jurkat cells, the over-expression of the anti-apoptotic protein Bcl-2 inhibited ultrasound-induced apoptosis, but not necrosis. Inhibition of caspase activity also protected the cells from apoptosis, but not from necrosis, showing the involvement of different mechanisms in ultrasound-induced apoptosis and necrosis. Bak, a pro-apoptotic member of the Bcl-2 family proteins, was activated by ultrasound and its activation was completely inhibited by Bcl-2 over-expression, but not by caspase inhibition. Antioxidant N-acetyl cysteine did not protect the cells from ultrasound-induced apoptosis or necrosis, nor did the inhibition of either c-Jun N-terminal kinase or p38, key factors in the radical oxygen species (ROS)-mediated cell stress response, suggesting that ROS do not play a crucial role in ultrasound-induced apoptosis. Our results confirm that ultrasound induces apoptosis via a pathway that involves Bak, Bcl-2, and caspases, but not ROS.

An echo-contrast agent, Levovist, lowers the ultrasound intensity required to induce apoptosis of human leukemia cells

To verify the effect of echo-contrast agent (ECA) on apoptosis induced by ultrasound, leukemia cell lines (Jurkat, Molt-4 and U937) were sonicated at intensities previously shown to induce optimal apoptosis with or without Levovist, an ECA. The results showed that loss of viability and apoptosis can be induced in all three cell lines, apoptosis highest with Molt-4, based on viability and DNA fragmentation assay. Such finding was supported by corresponding increase of cells with low mitochondrial membrane potential, high superoxide production, increased intracellular calcium concentration, and phosphorylation of histone H2AX after sonication. Optimal ultrasound condition was 0.3W/cm2, 1MHz, 10% duty factor pulsed at 100Hz; but in the presence of Levovist, an apparent shift of cell killing induction was observed at 0.2W/cm2. While these results further confirmed previous findings on ultrasound-induced apoptosis, they also suggest that use of an enhancing factor, such as addition of ECA, may be useful in cancer therapy when a much lower intensity is desired.

Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents

To determine the effects of echo-contrast agents (ECAs) on ultrasound (US)-induced apoptosis and cell lysis, human myelomonocytic lymphoma U937 cells in suspension were exposed to 1 MHz continuous waves US for 1 min at an intensity of 0.5, 1.0, 2.0 or 4.0 W/cm 2 with or without non-shell type ECA, Levovist™ (2 mg/ml), and shell type, Optison™ (1 μl/ml) or YM454 (1 μl/ml). Levovist™ minimally enhanced the US-induced apoptosis at 1.0 W/cm 2 while Optison™ and YM454 did at 2.0 and 4.0 W/cm 2, as detected by flow cytometry. Cell lysis was also augmented when Levovist™ was combined with US at 2.0 W/cm 2, and when Optison TM was combined with US at 2.0 and 4.0 W/cm 2. YM454 showed the highest rate of enhanced cell lysis at 1.0, 2.0 and 4.0 W/cm 2. Therefore, this study shows that Optison™ and YM454 are effective in augmenting the US-induced cell killing, but not Levovist™. Another result indicates that cavitation plays a role in the augmented effects and that inertial cavitation appears necessary for Optison TM and YM454 to effect their actions. In addition, results show that the rate of apoptosis is lower in the presence of ECAs with higher free radical scavenging activity, suggesting a possible role for free radicals in apoptosis. These findings suggest that some ECAs have potential to be adjuncts in cases wherein augmented US-induced cell killing is needed, such as in cancer therapy with US. (E-mail:kondot@ms.toyama-mpu.ac.jp)