In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells

Sonodynamic therapy (SDT) has been shown to mediate apoptosis in many experimental systems, but the detailed mechanism of this process is unclear. In this study, we aim to investigate the potential participation of the mitochondria-caspase signaling pathway in the SDT-induced apoptosis in isolated sarcoma 180 (S180) cells. The cell suspension was treated with 1.75 MHz continuous ultrasound (US) at an acoustic intensity (I_SATA) of 1.4 W for 3 min in the absence or presence of 20 μg/ml hematoporphyrin (Hp). At different times after the SDT-treatment, the apoptotic cells were identified under a scanning electron microscope, and the apoptosis index (AI) was determined by flow cytometry. In addition, the mitochondrial membrane potential, permeabilization of the inner mitochondrial membrane, and translocation of apoptosis-related proteins were assessed by confocal microscopy. Simultaneously, the activation of some special apoptosis-associated proteins [caspase-9, caspase-3, polypeptide poly (ADP-ribose) polymerase (PARP), and Bax] was evaluated by western blotting. Our results indicate that the ultrasonically activated Hp can cause obvious cell apoptosis (AI, 57.66%) at 3 h after treatment, and this effect can be significantly reduced by caspase-9 inhibitor (AI, 20.76%) and the oxygen scavenger NaN₃ (20.11%). However, the apoptosis induced by ultrasound alone was relatively lower (28.33%) and was not reduced by NaN₃. Further, SDT caused an 82.1% reduction in the mitochondrial membrane potential and a 70.7% reduction in the permeabilization of the inner mitochondrial membrane immediately after treatment, and these two effects were obviously prevented by NaN₃. In comparison with the control cells, the SDT-treated cells showed obvious cytochrome-c and Bax translocations, caspase activation, Bax expression, and PARP cleavage at 1 h after SDT-treatment. However, in the cells treated with ultrasound alone, these phenomena partially and weakly occurred 3 h after exposure. These results primarily showed that the mitochondria-caspase signaling pathway in S180 cells was activated in the US- and SDT-induced apoptosis. Moreover, Hp significantly accelerates the process of apoptosis and enhances the cytotoxic effect of ultrasonic treatment. Singlet oxygen may be responsible for the mitochondrial damage and the activation of the apoptotic signaling pathway.     

Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells

Objectives

The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT).

Methods

The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (4′-6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis.

Results

The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 μg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase-3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes.

Conclusions

These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis.     

The role of p53 in the response of tumor cells to sonodynamic therapy in vitro

p53 plays a pivotal role in apoptosis. In addition, p53 is currently extensively investigated as a promising strategy for highly specific anticancer therapy in chemotherapeutics and photodynamic therapy. However, the role of p53 in the response of tumor cells to sonodynamic therapy treatment is still unclear. In this study, we aim to investigate the activation of p53 in sonodynamic therapy. Three murine tumor models with distinct aggressiveness (S180, H-22 and EAC) were treated with 1.75 MHz continuous ultrasound at an acoustic intensity (I_SATA) of 1.4 W for 3 min in the presence of 20 μg/ml hematoporphyrin. The DNA fragment and nuclear damage were observed by TUNEL and single cell gel electrophoresis. Western blotting and RT-PCR were used to analyze the expression of p53, PUMA, Bax and Fas. Then we checked the translocation of p53 by confocal microscopy. DNA sequencing was used to determine the status of p53 gene in three tumor cell lines. Our results indicated that the level of p53 protein and mRNA increased significantly, and p53 activated the expression of its downstream pro-apoptosis gene PUMA, Bax and Fas in the S180 and H-22 cells. Meanwhile, p53 protein translocated onto mitochondria. In the EAC cells, expression and translocation of p53 was not found; the level of PUMA, Bax and Fas remained unaltered. The S180 cells showed most serious DNA fragment and nuclear damage with 77.43% TDNA; H-22 cells in the middle with 58.85% TDNA; whereas EAC cells appeared less nuclear material lost with just 15.82% TDNA. The results of DNA sequencing showed that the sequences of exons 5-8 of the p53 gene of S180, H-22 and EAC cells were the same with the sequences of wild-type p53 provided by NCBI. These results primarily demonstrated that: (1) p53 was activated to promote SDT-induced apoptosis through extrinsic and intrinsic signaling pathways in the S180 and H-22 cells; (2) cellular responses of different cells to SDT were distinct, the aggressive S180 cells were much more sensitive than H-22, whereas EAC cells were relatively less sensitive. The discrepancy among the cell lines may be due to different activation time of p53 protein.     

Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

Background  

Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O₂) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-κB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells.     

Insights into the novel application of Fe-MOFs in ultrasound-assisted heterogeneous Fenton system: Efficiency,kinetics and mechanism

In this work, as a new strategy, ultrasound/H₂O₂/MOF system was firstly applied by environmental-benign Fe-MOFs (MIL-53, MIL-88B and MIL-101) for tetracycline hydrochloride removal. The synthetic Fe-MOFs were characterized by XRD, FTIR, SEM, XPS, N₂ sorption-desorption isotherms and CO-FTIR. MIL-88B demonstrated the best catalytic performance because of its highest amount of Lewis acid sites. Influencing factors, contrast experiment, and corresponding dynamics were carried out to obtain the best experimental conditions and reaction system. Under optimal conditions ([Tetracycline hydrochloride] = 10 mg/L, [MIL-88B] = 0.3 g/L, [H₂O₂] = 44 mM, [ultrasound power] = 60 W, and pH = 5.0), the-first-order kinetic rate constant k was calculated to be 0.226 min⁻¹, higher than the simple combination of the ultrasound system (0.004) and MIL-88B/H₂O₂ system (0.163), indicating the importance of synergistic effect between ultrasound and Fenton reaction. EPR test and quenching experiment proved that ·OH is mainly responsible for tetracycline hydrochloride removal. The major reaction path is the adsorption and decomposition of H₂O₂ by coordinative unsaturated iron sites on Fe-MOF, but it is not the only path. The direct decomposition of H₂O₂ and the cavitation effect caused by ultrasound also contribute to the generation of OH.     

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

Sonocatalytic injury of cancer cells attached on the surface of a nickel–titanium dioxide alloy plate

The present study demonstrates ultrasound-induced cell injury using a nickel–titanium dioxide (Ni–TiO₂) alloy plate as a sonocatalyst and a cell culture surface. Ultrasound irradiation of cell-free Ni–TiO₂ alloy plates with 1 MHz ultrasound at 0.5 W/cm² for 30 s led to an increased generation of hydroxyl (OH) radicals compared to nickel–titanium (Ni–Ti) control alloy plates with and without ultrasound irradiation. When human breast cancer cells (MCF-7 cells) cultured on the Ni–TiO₂ alloy plates were irradiated with 1 MHz ultrasound at 0.5 W/cm² for 30 s and then incubated for 48 h, cell density on the alloy plate was reduced to approximately 50% of the controls on the Ni–Ti alloy plates with and without ultrasound irradiation. These results indicate the injury of MCF-7 cells following sonocatalytic OH radical generation by Ni–TiO₂. Further experiments demonstrated cell shrinkage and chromatin condensation after ultrasound irradiation of MCF-7 cells attached on the Ni–TiO₂ alloy plates, indicating induction of apoptosis.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Experimental study on killing tumor cells by activation of hematoporphyrin derivatives by bi-frequency focal ultrasound in vitro

Tumor cells K562 were killed by the hematoporphyrin derivatives (HpD) activated by bi-frequency focal ultrasound in which a choice of irradiating parameter of ultrasound was tested by experiment. The effect of killing tumor cells was investigated by MTT method and compared with the contrast group. The results showed that the bi-frequency ultrasound exhibited an improved effect of killing tumor cells than single frequency ultrasound. It was 2-3 times higher than the single frequency ultrasound for the killing effect of tumor cells. After the irradiation of ultrasound, the cell-killing effect of hatching 16 h is better than that of hatching 4 h in the hatching tank. This might be due to delay of cell apoptosis.     

Targeted sonodynamic therapy using protein-modified TiO2 nanoparticles

Our previous study suggested new sonodynamic therapy for cancer cells based on the delivery of titanium dioxide (TiO₂) nanoparticles (NPs) modified with a protein specifically recognizing target cells and subsequent generation of hydroxyl radicals from TiO₂ NPs activated by external ultrasound irradiation (called TiO₂/US treatment). The present study first examined the uptake behavior of TiO₂ NPs modified with pre-S1/S2 (model protein-recognizing hepatocytes) by HepG2 cells for 24 h. It took 6 h for sufficient uptake of the TiO₂ NPs by the cells. Next, the effect of the TiO₂/US treatment on HepG2 cell growth was examined for 96 h after the 1 MHz ultrasound was irradiated (0.1 W/cm², 30 s) to the cells which incorporated the TiO₂ NPs. Apoptosis was observed at 6 h after the TiO₂/US treatment. Although no apparent cell-injury was observed until 24 h after the treatment, the viable cell concentration had deteriorated to 46% of the control at 96 h. Finally, the TiO₂/US treatment was applied to a mouse xenograft model. The pre-S1/S2-immobilized TiO₂ (0.1 mg) was directly injected into tumors, followed by 1 MHz ultrasound irradiation at 1.0 W/cm² for 60 s. As a result of the treatment repeated five times within 13 days, tumor growth could be hampered up to 28 days compared with the control conditions.     

ROS-generating alginate-coated gold nanorods as biocompatible nanosonosensitisers for effective sonodynamic therapy of cancer

Sonodynamic therapy (SDT) emerges as a promising non-invasive alternative for eradicating malignant tumours. However, its therapeutic efficacy remains limited due to the lack of sonosensitisers with high potency and biosafety. Previously, gold nanorods (AuNRs) have been extensively studied for their applications in photodynamic or photothermal cancer therapy, but their sonosensitising properties are largely unexplored. Here, we reported the applicability of alginate-coated AuNRs (AuNRs^ALG) with improved biocompatibility profiles as promising nanosonosensitisers for SDT for the first time. AuNRs^ALG were found stable under ultrasound irradiation (1.0 W/cm², 5 min) and maintained structural integrity for 3 cycles of irradiation. The exposure of the AuNRs^ALG to ultrasound irradiation (1.0 W/cm², 5 min) was shown to enhance the cavitation effect significantly and generate a 3 to 8-fold higher amount of singlet oxygen (¹O₂) than other reported commercial titanium dioxide nanosonosensitisers. AuNRs^ALG exerted dose-dependent sonotoxicity on human MDA-MB-231 breast cancer cells in vitro, with ∼ 81% cancer cell killing efficacy at a sub-nanomolar level (IC₅₀ was 0.68 nM) predominantly through apoptosis. The protein expression analysis showed significant DNA damage and downregulation of anti-apoptotic Bcl-2, suggesting AuNRs^ALG induced cell death through the mitochondrial pathway. The addition of mannitol, a reactive oxygen species (ROS) scavenger, inhibited cancer-killing effect of AuNRs^ALG-mediated SDT, further verifying that the sonotoxicity of AuNRs^ALG is driven by the production of ROS. Overall, these results highlight the potential application of AuNRs^ALG as an effective nanosonosensitising agent in clinical settings.     

Modeling the elimination of mature biofilms formed by Staphylococcus aureus and Salmonella spp. Using combined ultrasound and disinfectants

Biofilm formation by foodborne pathogens on food processing surfaces has contributed to numerous disease outbreaks and food recalls. We evaluated the following strategies for elimination of mature biofilm formed by Staphylococcus aureus and Salmonella spp. on stainless steel surfaces: acidic electrolyzed water (AEW), ozone water (OW), or ultrasound (40 kHz) alone, and combinations of ultrasound and disinfectants. The dynamics of elimination by combinations were determined using the Weibull and biphasic models. Treatment with AEW alone reduced the number of biofilm cells by approximately 3.0 log cfu/cm², whereas less than 0.8 log cfu/cm² of cells reduction was observed in biofilm exposed to OW or ultrasound alone, even with treatment for 20 min. The combination of AEW and ultrasound produced an obvious synergistic effect on biofilm reduction, achieving approximately 4.8 log cfu/cm² reduction in Salmonella spp. biofilm. Interestingly, the biphasic model was a better fit than the Weibull model for the elimination process of mature biofilm formed by both pathogens and subjected to a combination of ultrasound and AEW, as determined by smaller values of the statistical parameters RMSE and AIC, although both models could evaluate the dynamic processes. Our findings indicated that a combination of ultrasound and AEW could effectively reduce the biofilm formed by pathogens on food contact surfaces, and that the biphasic model could predict the number of residual cells after biofilm exposure to this intervention approach.     

Enhanced sonodynamic therapy by carbon dots-shelled microbubbles with focused ultrasound

Sonodynamic therapy involving the non-invasive and local generation of lethal reactive oxygen species (ROS) via ultrasound (US) with sonosensitizers has been proposed as an emerging tumor therapy strategy. However, such therapy is usually associated with inertial cavitation and unnecessary damage to healthy tissue because current sonosensitizers have insufficient sensitivity to US. Here, we report the use of a new proposed sonosensitizer, carbon dots (C-dots), to assemble microbubbles with a gas core (C-dots MBs). As the C-dots were directly integrated into the MB shell, they could effectively absorb the energy of inertial cavitation and transfer it to ROS. Our results revealed the appearance of ¹O₂, •OH, and H₂O₂ after US irradiation of C-dots MBs. In in vitro experiments, treatment with C-dots MBs plus US induced lipid peroxidation, elevation of intracellular ROS, and apoptosis in 32.5%, 45.3%, and 50.1% of cells respectively. In an animal solid tumor model, treatment with C-dots MBs plus US resulted in a 3-fold and 2.5-fold increase in the proportion of ROS-damaged cells and apoptotic cells, respectively, compared to C-dots MBs alone. These results will pave the way for the design of novel multifunctional sonosensitizers for SDT tumor therapy.     

Ultrasound-mediated destruction of oxygen and paclitaxel loaded lipid microbubbles for combination therapy in hypoxic ovarian cancer cells

We synthesized oxygen and paclitaxel (PTX) loaded lipid microbubbles (OPLMBs) for ultrasound mediated combination therapy in hypoxic ovarian cancer cells. Our experiments successfully demonstrated that ultrasound induced OPLMBs destruction significantly enhanced the local oxygen release. We also demonstrated that OPLMBs in combination with ultrasound (300 kHz, 0.5 W/cm², 15 s) yielded anti-proliferative activities of 52.8 ± 2.75% and cell apoptosis ratio of 35.25 ± 0.17% in hypoxic cells at 24 h after the treatment, superior to other treatment groups such as PTX only and PTX-loaded MBs (PLMBs) with or without ultrasound mediation. RT-PCR and Western blot tests further confirmed the reduced expression of HIF-1α and MDR-1/P-gp after ultrasound mediation of OPLMBs. Our experiment suggests that ultrasound mediation of oxygen and drug-loaded MBs may be a useful method to overcome chemoresistance in the hypoxic ovarian cancer cells.     

Targeted sonocatalytic cancer cell injury using avidin-conjugated titanium dioxide nanoparticles

In this study, we applied sonodynamic therapy to cancer cells based on the delivery of titanium dioxide (TiO₂) nanoparticles (NPs) modified with avidin protein, which preferentially discriminated cancerous cells from healthy cells. Subsequently, hydroxyl radicals were generated from the TiO₂ NPs after activation by external ultrasound irradiation (TiO₂/US treatment). Although 30% of the normal breast cells (human mammary epithelial cells) exhibited the uptake of avidin-modified TiO₂ NPs, over 80% of the breast cancer cells (MCF-7) exhibited the uptake of avidin-TiO₂ NPs. Next the effect of the TiO₂/US treatment on MCF-7 cell growth was examined for up to 96 h after 1-MHz ultrasound was applied (0.1 W/cm², 30 s) to cells that incorporated the TiO₂ NPs. No apparent cell injury was observed until 24 h after the treatment, but the viable cell concentration declined to 68% compared with the control at 96 h.     

Synergistic antibacterial effects of ultrasound and thyme essential oils nanoemulsion against Escherichia coli O157:H7

Essential oil nanoemulsions have been proven to have stronger antimicrobial effects compared to the essential oil alone or coarse emulsion. Sonoporation could be the promising candidate to trigger a synergistic effect with thyme essential oil nanoemulsion (TEON) and produce a more effective antibacterial efficacy. Therefore, in this study, the bactericidal effects of ultrasound (US) in combination with TEON treatments against Escherichia coli (E. coli) O157:H7 were investigated. The remarkable synergistic effects of US (20 kHz, 255 W/cm², 9 min) and TEON (0.375 mg/mL) treatments at 22 °C reduced E. coli O157:H7 populations by 7.42 ± 0.27 log CFU/mL.The morphological changes of cells exposed to different treatments were observed by scanning electron microscopy and transmission electron microscopy. The results showed that the synergistic effects of the ultrasound and TEON treatments altered the morphology and interior microstructure of organism cells. Laser scanning confocal microscopy (LSCM) images revealed that the combination treatments of ultrasound and TEON altered the permeability of cell membranes, and this affected the integrity of E. coli O157:H7 cells. This was further indicated by the high amounts of nucleic acids and proteins released from these cells following treatment.The results from this study illustrated the mechanisms of the synergistic effects of sonoporation and TEON treatments and provided valuable information for their potential in food pasteurization.     

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.     

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

Objective

The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT).

Methods

The MB concentration was kept constant at 100 μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5 s with an intensity of 0.46 W/cm². The cytotoxicity was investigated 24 h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT.

Conclusions

Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.     

Low-intensity ultrasound enhances the antimicrobial activity of neutral peptide TGH2 against Escherichia coli

In recent years, foodborne diseases caused by Escherichia coli are a major threat to the food industry and consumers. Antimicrobial peptides (AMPs) and ultrasound both have good inhibitory effects on E. coli. In this work, the mechanism of action and synergistic effect of an in silico predicted AMP, designated as TGH2 (AEFLREKLGDKCTDRHV), from the C-terminal sequence of Tegillarca granosa hemoglobin, combined with low-intensity ultrasound was explored. The minimal inhibitory concentration (MIC) of TGH2 on E. coli decreased by 4-fold to 31.25 μg/mL under 0.3 W/cm² ultrasound treatment, while the time kill curve analysis showed that low-intensity ultrasound combined with peptide TGH2 had an enhanced synergistic bactericidal effect after 0.5 h. The permeability on E. coli cell membrane increased progressively during combined treatment with peptide TGH2 and low-intensity ultrasound, resulting in the leakage of intracellular solutes, as shown by transmission electron microscopy (TEM). Structural analysis using circular dichroism (CD) revealed that peptide TGH2 has an α-helical structure, showing a slight untwisting effect under 0.3 W/cm² ultrasound treatment for 0.5 h. The findings here provide new insight into the potential application of ultrasound and AMPs combination in food preservation.     

The tumor affinity of chlorin e6 and its sonodynamic effects on non-small cell lung cancer

ObjectiveSonodynamic therapy (SDT) is a promising new approach for cancer therapy. The aim of this study was to investigate the tumor affinity of chlorin e6, a photosensitizer, and its sonodynamic effects on NSCLC.MethodsHuman lung adenocarcinoma cells SPCA-1 and mice bearing SPCA-1 tumor xenograft were exposed to ultrasound in the presence or absence of chlorin e6. Chlorin e6 distribution was detected by laser scan confocal microscope. Cell apoptosis and necrosis were studied by flow cytometry analysis. Tumor size and weight were measured after different treatments.ResultsThe concentration of chlorin e6 in tumor tissue was remarkably higher than that in normal muscle near tumor, and the difference was greatest at 18 h (the fluorescence intensity was 5.38-fold higher in tumor than in muscle, P < 0.05). In vivo, ultrasound (0.4–1.6 W/cm²) or chlorin e6 (10–40 mg/kg) alone had no remarkable anti-tumor effects, but the combination of ultrasound (1.6 W/cm²) with chlorin e6 (SDT) hampered tumor growth significantly (P < 0.05). Intraperitoneal injection of 40 mg/kg chlorin e6 exerted no notable side effect on blood, liver and kidney function. Flow cytometry analysis showed that chlorin e6-mediated sonodynamic effect was mainly through the induction of cell necrosis.ConclusionChlorin e6 is a promising sonosensitizer and chlorin e6-mediated SDT may provide a new approach for NSCLC therapy.