Xinshu Xia

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 100μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5s with an intensity of 0.46W/cm(2). The cytotoxicity was investigated 24h 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.

Mitochondrial Damage in Nasopharyngeal Carcinoma Cells Induced by Ultrasound Radiation in the Presence of Hypocrellin B

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.

Hypocrellin B-encapsulated nanoparticle-mediated rev-caspase-3 gene transfection and photodynamic therapy on tumor cells.

Gene therapy and photodynamic therapy are two kinds of important therapeutic strategies for treating malignant tumors. In order to explore the combined effects of gene therapy and PDT on tumor cells, rev-caspase-3 gene was transfected into the tumor model CNE2 cells using hypocrellin B-encapsulated nanoparticle (nano-HB) as a carrier. The transfected CNE2 cells were then irradiated by light from a LED source and the survival rate was investigated 18 h after PDT. Apoptosis was analyzed by a flow cytometer with propidium iodine (PI) staining and the active caspase-3 expression was measured using flow cytometry with phycoerythrin (PE)-conjugated anti-active caspase-3 antibody. The result from the flow cytometer showed that the level of the activated caspase-3 significantly increased up to 63.10% in the transfected CNE2 cells. The survival rate 18 h after gene transfection alone and nano-HB-mediated PDT was 96.6±2.07%, 72.6±4.15%, respectively. However, the survival rate of the transfected CNE2 cells 18 h after LED exposure significantly decreased to 50.6±5.98% under the light energy of 4 J/cm(2). Apoptotic rate 18 h after the combination of gene transfection and PDT increased up to 24.65%. Our findings demonstrated that nano-HB could significantly enhance the transfection efficiency of rev-caspase-3 gene in the CNE2 cells. LED irradiation could effectively kill the treated CNE2 cells and induce apoptosis, suggesting hypocrellin B-encapsulated nanoparticle as an efficient gene carrier and a novel photosensitizer. The combination of gene therapy and PDT using nanoparticle as a mediator can be developed for treating nasopharyngeal carcinoma.

5-Aminolaevulinic acid enhances ultrasound-induced mitochondrial damage in K562 cells.

BACKGROUND 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. MATERIALS/METHODS 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. RESULTS 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 (P<0.05). 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. CONCLUSION 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.

Ultrasound-Induced Cell Death of Nasopharyngeal Carcinoma Cells in the Presence of Curcumin

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 ∆Ψm 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 ∆Ψm, 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.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/cm(2). 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.

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

OBJECTIVES Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro. METHODS Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10μm curcumin and then were treated by ultrasound for 8s at the intensity of 0.46W/cm(2). Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM). RESULTS MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16±2.37% and 24.93±8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67±7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial 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 CNE2 cells after the combined treatment of curcumin and ultrasound. CONCLUSIONS Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.

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 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.

Apoptosis of breast cancer cells induced by hypocrellin B under light-emitting diode irradiation

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 24h after photodynamic treatment of hypocrellin B using MTT reduction assay and light microscopy. Apoptosis was observed 6h 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.

Effect of blue light radiation on curcumin-induced cell death of breast cancer cells

In the present study, we have successfully set up a novel blue light source with the power density of 9 mW/cm2 and the wavelength of 435.8 nm and then the novel light source was used to investigate the effect of light radiation on curcumin-induced cell death. The cytotoxicity was investigated 24 h after the treatment of curcumin and blue light radiation together using MTT reduction assay. Nuclear chromatin was observed using a fluorescent microscopy with Hoechst33258 staining. The results showed blue light radiation could significantly enhance the cytotoxicity of curcumin on the MCF-7 cells and apoptosis induction. These findings demonstrated that blue light radiation could enhance curcumin-induced cell death of breast cancer cells, suggesting light radiation may be an efficient enhancer of curcumin in the management of breast cancer.

Sonodynamic Action of Pyropheophorbide-a Methyl Ester in Liver Cancer Cells

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 μmol/L, and the cells were subjected to ultrasound exposure at an intensity of 0.97 W/cm2. 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 μmol/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.