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Objectives This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Methods Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. Results The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P Conclusions Ultrasound sonication in the presence of methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells.

Summary Objectives Breast cancer is a common disease which threatens the life of women. To explore an alternative modality for combating breast cancer, a light-emitting diode (LED) that activates hypocrellin B was used in the present study to investigate apoptosis induction in breast cancer MDA-MB-231 cells. Materials and methods Photocytotoxicity was investigated 24 h after photodynamic treatment of hypocrellin B using MTT reduction assay and light microscopy. Apoptosis was observed 6 h after photodynamic treatment using flow cytometry with Annexin V/PI staining as well as fluorescent microscopy with Hoechst33258 staining. The ultrastructure of the treated cells was observed using transmission electron microscopy (TEM). Results Hypocrellin B-induced photocytotoxicity in MDA-MB-231 cells exhibited a dose-dependent manner. The amount of MDA-MB-231 cells attached to the bottom of well decreased significantly after photodynamic treatment of hypocrellin B. Flow cytometry showed that the early and late apoptotic rate of MDA-MB-231 cells increased remarkably up to 17.46% and 32.80%, respectively, after treatment of LED-activated hypocrellin B. In addition, nuclear condensation, fragmentation and chromatin margination, and topical apoptotic body in the treated cells were observed by nuclear staining and TEM. Conclusion Photodynamic action of hypocrellin B irradiated by light-emitting diodes could significantly kill breast cancer cells and induce apoptotic cell death, which suggests LED-activated hypocrellin B is a promising strategy for combating breast cancer.

In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hypporellin B concentration was kept at 2.5 μM and light doses from 0.5–4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

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 H(22) cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm(2) for 8 s. Cytotoxicity was investigated 24h 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 H(22) 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 H(22) 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 H(22) cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.

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.

Objectives: Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an effi-cient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruc-tion of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.Methods: Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 l m curcumin and thenwere treated by ultrasound for 8 s at the intensity of 0.46 W/cm 2 . Cytotoxicity was evaluated usingMTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanningmicrocopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electronmicroscopy (TEM).Results: MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treat-ment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combinedtreatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed thatmicrovillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, andmitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together.The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2cells after the combined treatment of curcumin and ultrasound.Conclusions: Our findings demonstrated that ultrasound sonication in the presence of curcumin signifi-cantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria,suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carci-noma cells in the presence of curcumin. 2010 Elsevier B.V. All rights reserved.

Ultrasound therapy is a new modality in the control of malignant cancers. The aim of the present study was to investigate the effect of 5-aminolaevulinic acid on the ultrasonic killing action in the cancer cells.The K562 cells as a cancer cell model were subjected to investigate the effect of 5-aminolaevulinic acid (5-ALA) on the ultrasonic killing action, in which the 5-ALA concentration was 2mM and the ultrasound exposure was 15 s at the intensity of 0.46 W/cm(2) and the frequency of 1.7MHz. Cytotoxicity was investigated 24h after ultrasound exposure using the trypan blue exclusion test. Ultrastructural cell morphology and mitochondrial changes were observed using transmission electron microscopy (TEM). Mitochondrial membrane potential (DeltaPsim) was evaluated using Rhodamine 123 assay.The death rates of the K562 cells in the controls including sham radiation and 5-ALA treatment alone were 1.81+/-0.13%, 1.27+/-0.20%, respectively. Those in ultrasound radiation alone and 5-ALA-ultrasound treatment were 12.61+/-2.63%, 46.87+/-4.09%, respectively. There were significant differences between 5-ALA-ultrasound treatment, ultrasound radiation alone and the controls (P0.05). TEM showed that the mitochondria expanding and some vacuoles were found in the ultrasound-treated cells. After the treatment of ultrasound and 5-ALA together some cells presented typical characteristics of apoptotic cells, such as nuclear condensation and crescent formation. Mitochondria of the cells were damaged more seriously than those treated by ultrasound alone, there were obvious swollen mitochondria and mitochondria in which cristae were almost perfectly disappeared, and more vacuolar mitochondria were founded. Mitochondrial membrane potential (DeltaPsim) was more significantly collapsed when the K562 cells were exposed to 2mM 5-ALA for 4h and then 0.46 W/cm(2) irradiation of ultrasound than ultrasound radiation alone.5-ALA pretreatment significantly enhanced the cytotoxicity of ultrasound radiation in K562 cells. The damage of mitochondria structure and function might be an important cause of cell death in K562 cells induced by the treatment of ultrasound radiation and 5-ALA together.

Objective. Hypocrellin is a natural photosensitizer from a traditional Chinese herb. In the present study, our aim is to investigate the effect of LED light-activated hypocrellin B on mitochondria of ovarian cancer cells.Material and Methods. Ovarian cancer HO-8910 cells were incubated with hypocrellin B at the concentration of 2.5 μM for 5 h and then irradiated by blue light from a novel LED source. Cell survival rate of HO-8910 cells was measured using MTT assay 24 h after photodynamic treatment of hypocrellin B. Mitochondrial morphology was observed using transmission electron microscopy (TEM). Mitochondrial membrane potential was measured using flow cytometry with JC-1 staining.Results. MTT assay showed that cell survival rate of HO-8910 cells in the photodynamic treatment group has significantly decreased down to27.22±1.26% (P<0.01). Light irradiation alone or hypocrellin B alone showed no significant impact. In our TEM mitochondria of the cells after photodynamic treatment of hypocrellin B showed severe damage with swollen mitochondria that had nearly nonexistant cristae. Mitochondrial membrane potential remarkably decreased after photodynamic action of hypocrellin B.Conclusion. The findings demonstrated that photodynamic action of hypocrellin B significantly decreased cell proliferation of ovarian cancer HO-8910 cells, caused severe damage to mitochondrial structure, and induced mitochondrial membrane collapse.

Objectives. Curcumin, a natural pigment from a traditional Chinese herb, has been attracting extensive attention. The present study aims to investigate cell death of nasopharyngeal carcinoma (NPC) cells induced by ultrasound sonication in the presence of curcumin in vitro. Methods. The NPC cell line CNE2 was chosen as a tumor model, and curcumin concentration was kept constant at 10 µM while the cells were subjected to ultrasound exposure for 8 s at an intensity of 0.46 W/cm2. Cell death was evaluated using flow cytometry with annexinV-FITC and propidium iodine staining, and nuclear staining with Hoechst 33258. Mitochondrial membrane potential and intracellular reactive oxygen species (ROS) were analyzed using flow cytometry with rhodamine 123 and dichlorodihydrofluorecein diacetate staining. Results. Flow cytometry showed that the combination of ultrasound and curcumin significantly increased the necrotic or late apoptotic rate by up to 31.37% compared with the controls. Nuclear condensation was observed in the nuclear staining, and collapse of ΔΨm and ROS increase were found in the CNE2 cells after the treatment with curcumin and ultrasound. Conclusions. The findings demonstrate that the presence of curcumin significantly enhances the ultrasound-induced cell death and ROS level, and induces the collapse of ΔΨm, suggesting that ultrasound sonication can increase the cell death of NPC cells in the presence of curcumin and that the treatment using curcumin and ultrasound together is a potential therapeutic modality in the management of malignant tumors.

Objective: The present study aims to investigate the possible mechanisms of hematoporphyrin monomethyl ether (HMME) enhancing the cytotoxicity of ultrasound in osteosarcoma cells. Methods: Osteosarcoma cell line UMR-106 was treated by HMME and ultrasound radiation, with the HMME concentration kept at 20 μg/mL and ultrasound radiation for 10 seconds at the intensity of 0.5 W/cm2. Cell proliferation was investigated at 12, 24, 36, and 48 hours using MTT assay after ultrasound and HMME treatment. Ultrastructural morphology was observed using transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) was measured using a flow cytometry with DCFH-DA staining and intracellular free calcium ion (Ca2+) with Fluo-3-AM staining. Results: The UMR-106 cells proliferated rapidly in the sham radiation and HMME treatment alone group, but ultrasound-treated cells and HMME-ultrasound-treated cells proliferated slowly. There was a significant difference between HMME-ultrasound treatment and the controls, including ultrasound radiation, HMME treatment alone, and sham radiation ( P < .05). TEM showed endoplasmic reticulum and mitochondrial swelling in the ultrasound-treated cells, and more cells presented apoptosis and necrosis after treatment with ultrasound and HMME together. Intracellular ROS and Ca2+ in the cells increased more significantly after both ultrasound and HMME treatment than after ultrasound treatment alone. Conclusions: HMME could effectively enhance the inhibition effect of ultrasound on osteosarcoma cells. Intracellular ROS and Ca2+ in the UMR-106 cells increased more significantly after the treatment of HMME and ultrasound together, indicating that the enhancement of HMME on ultrasound cytotoxicity to osteosarcoma cells possibly involves both intracellular ROS and Ca2+ elevation.

OBJECTIVE This study aimed to investigate the sonodynamic action of pyropheophorbide-a methyl ester (MPPa) in liver cancer cells to explore a novel therapeutic modality. METHODS H22 cells were chosen as model cells to investigate the sonodynamic action of MPPa on liver cancer. The MPPa concentration was kept constant at 2 micromol/L, and the cells were subjected to ultrasound exposure at an intensity of 0.97 W/cm(2). Cytotoxicity was investigated 24 hours after ultrasound exposure. Apoptosis was evaluated using flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodine staining and nuclear staining with Hoechst 33258. Reactive oxygen species (ROS) were analyzed using flow cytometry with 2,7-dichlorodihydrofluorescein diacetate staining. RESULTS No significant dark cytotoxicity of MPPa was shown in the H22 cells at the concentration of 2 micromol/L. The cell death rate induced by ultrasound treatment was significantly higher in the presence of MPPa than in the absence of it (P < .05). Flow cytometry showed that the sonodynamic action of MPPa significantly increased the early and late apoptotic rates of the H22 cells. Nuclear condensation and an ROS increase were found after sonodynamic treatment. CONCLUSIONS Our findings showed that MPPa-mediated sonodynamic action significantly enhanced death of H22 cells and the ROS level, suggesting that MPPa is a novel sonosensitizer and the sonodynamic action of MPPa might be a potential therapeutic modality in the management of liver cancer.

Objective. The mitochondrion is an important target of ultrasound-induced cell death. This study aimed to investigate the mitochondrial damage in nasopharyngeal carcinoma (NPC) cells induced by ultrasound radiation in the presence of hypocrellin B (HB). Methods. The NPC cell line CNE2 was used to investigate the effect of HB on ultrasonic action with an HB concentration of 2.5 μmol/L and ultrasound exposure for 15 seconds at an intensity of 0.65 W/cm2. Cytotoxicity was investigated 24 hours after ultrasound exposure. Mitochondrial structure changes were observed by transmission electron microscopy. The mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. Results. The mean death rates of the CNE2 cells ± SD were 25.14% ± 1.50% after ultrasound radiation alone and 76.72% ± 1.13% after ultrasound radiation in the presence of HB. Transmission electron microscopy showed that slightly enlarging mitochondria were found in the ultrasound-treated cells. After treatment with ultrasound and HB together, some cells had seriously damaged mitochondria, namely, obvious swollen mitochondria and mitochondria in which cristae had almost completely disappeared. The mitochondrial membrane potential was more significantly collapsed when the CNE2 cells were exposed to HB for 5 hours and then ultrasound at 0.65 mW/cm2 than with ultrasound radiation alone (P < .05). Conclusions. Hypocrellin B significantly enhanced the cytotoxicity of ultrasound radiation in the CNE2 cells. The damage to the mitochondrial structure and function might be an important cause of death in the CNE2 cells induced by treatment with ultrasound radiation and HB together.