Nagahiko Yumita

Sonochemical activation of hematoporphyrin: a potential modality for cancer treatment

In-vitro and in-vivo studies demonstrate the antitumor effect of hematoporphyrin sonochemically activated by ultrasound irradiation. Tumor cell suspensions with and without hematoporphyrin (Hp) are irradiated by continuous-wave ultrasound. Ultrasound cell damage with Hp is significantly greater than that without Hp, while no cell damage by Hp alone was detected. Growth of tumors implanted in mice is stopped by the ultrasound irradiation after Hp administration. Because the cell-damage enhancement by Hp is suppressed by adding histidine to the suspension but not by adding mannitol, the enhancement is most likely due to sonochemical generation of singlet oxygen.<<ETX>>

Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

Sonodynamic approach to tumor treatment

The localization of sonochemical reactions with focused ultrasound is investigated by developing a sonochemically active tissue mimicking phantom. It is demonstrated that the sonochemical effects can be localized within a region of size not larger than typical tumors to be treated. Sonochemically efficient methods of ultrasound irradiation, which may neither require standing wave situations nor extremely high ultrasound intensity in order to induce substantial sonochemical effects, are studied. The ultrasound intensity threshold for the sonochemical reaction is drastically decreased by the second harmonic superimposition. It is also demonstrated by in vitro experiments that a gallium-deuteroporphyrin complex, ATX-70, has three times higher sonochemical activity than hematoporphyrin (Hp) and induces cell damage at twice the rate of Hp in combination with ultrasound.<<ETX>>

Sonodynamically induced apoptosis with porfimer sodium in HL-60 cells.

Sonodynamically induced apoptosis with porfimer sodium in HL-60 cells was investigated. HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of porfimer sodium. After the exposure, sonodynamically induced apoptosis was assessed according to morphologic changes, DNA fragmentation and caspase-3 activation. The cells treated with 50 μg/ml porfimer sodium and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas no significant morphologic change was observed in the cells exposed to either ultrasound alone or porfimer sodium alone. DNA ladder formation was observed in the cells treated with ultrasound in the presence of porfimer sodium. Activation of caspase-3 was also observed after the treatment with ultrasound and porfimer sodium. Both sonodynamically induced apoptosis and caspase-3 activation were significantly suppressed by histidine. These results indicate that combination treatment with ultrasound and porfimer sodium induced apoptosis in HL-60 cells. Significant reduction by histidine in both sonodynamically induced apoptosis and caspase-3 activation suggests that some ultrasonically generated active species, deactivatable by histidine, are the major mediators to induce the observed apoptosis.

In vitro hemolysis using a switched spiral focal field

An efficient method of ultrasound irradiation to induce biologically effective cavitation is presented. A sector-vortex transducer, a phased array transducer with multiple sectors and a geometric focus, is used to produce ultrasound focal fields with spirally shaped wavefronts. Clockwise and counterclockwise spiral focal fields with basically the same ultrasound power distribution but having different wavefront angles are produced by using the same array transducer. The in vitro bioeffects from the periodic switching between such a pair of spiral focal fields are investigated. Suspended rat erythrocytes were insonated for 1 min at an ultrasound frequency of 750 kHz. A significantly high hemolysis rate is observed at a switching period of 10 ms. The rate is about ten times higher than those at switching periods of 0.1 ms or 1 s. Similar switching period dependence is also seen in the enhancement of hemolysis by adding sonochemical sensitizers.<<ETX>>

Sonodynamically-induced cytotoxicity by rose bengal derivative and microbubbles in isolated sarcoma 180 cells

It is known that the combination of ultrasound and sonodynamic sensitizer (SDS) is effective in noninvasive tumor treatment, referred to as sonodynamic therapy (SDT). Microbubbles have been used in ultrasound therapy as well. The purpose of this paper is to clarify the effect of microbubbles on SDT. Sarcoma 180 cells were suspended in air-saturated phosphate-buffered saline and exposed to ultrasound with the SDS rose bengal derivative (RBD) in standing wave mode in the presence and absence of microbubbles [sonazoid (SZ)]. The ultrasonically induced cytotoxicity with RBD and SZ was about 20 times higher than without either, and about 80% of the SZ microbubbles were destructed by ultrasonic exposure in as short as five seconds. Since microbubbles induce significant cytotoxicity even with short duration, low intensity ultrasound, the application of microbubbles in SDT shows promise in anti-tumor treatment.

Anticancer Effects of Functionalized Carbon Nanotubes Combined with Ultrasound Irradiation through Reactive Oxygen Species Generation

The sonodynamically induced antitumor effect of functionalized carbon nanotubes (CNT) combined with ultrasound through reactive oxygen species generation was investigated. Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. The rate of ultrasonically induced damage on isolated sarcoma 180 cells was enhanced by twice with 80 mM CNT. This enhancement was significantly inhibited by histidine, which may suggest it was mediated by ultrasonically induced oxidation. The coadministraion of 25 mg/kg and ultrasonic exposure suppressed the growth of implanted colon 26 tomors at an intensity with which ultrasound alone showed only a slight antitumor effect. In addition, the combination of CNT and ultrasound substantially enhanced singlet oxygen generation. Sonodynamically induced cell damage and singlet oxygen generation were significantly suppressed by histidine and NaN3. These results indicate singlet oxygen plays an important role in sonodynamically induced antitumor effect. In conclusion, the presented results suggest that CNT is a potential sensitizer for sonodynamic tumor treatment.

Antitumor effect of sonodynamically activated pyrrolidine tris-acid fullerene

In this study, the sonodynamically induced antitumor effect of pyrrolidine tris-acid fullerene (PTF) was investigated. Sonodynamically induced antitumor effects of PTF by focused ultrasound were investigated using isolated sarcoma-180 cells and mice bearing ectopically-implanted colon 26 carcinoma. Cell damage induced by ultrasonic exposure was enhanced by 5-fold in the presence of 80 µM PTF. The combined treatment of ultrasound and PTF suppressed the growth of the implanted colon 26 carcinoma. Ultrasonically induced 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (4oxoTEMPO) production in the presence and absence of PTF was assessed, and it was shown that 80 µM PTF enhanced 4oxoTEMPO production as measured by ESR spectroscopy. Histidine, a reactive oxygen scavenger, significantly reduced cell damage and 4oxoTEMPO generation caused by ultrasonic exposure in the presence of PTF. These results suggest that singlet oxygen is likely to be involved in the ultrasonically induced cell damage enhanced by PTF.

Apoptosis induction by aluminum phthalocyanine tetrasulfonate-based sonodynamic therapy in HL-60 cells

The present study aims to investigate sonodynamically-induced apoptosis using the phthalocyanine, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS). HL-60 cells were exposed to ultrasound for up to 3 min in the absence and presence of AlPcTS. Apoptosis was analyzed by cell morphology, DNA fragmentation, and caspase-3 activity. Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells showing membrane blebbing and cell shrinkage after combined treatment (ultrasound and AlPcTS) was significantly higher than following other treatments, including ultrasound alone and AlPcTS alone. Furthermore, DNA ladder formation, caspase-3 activation and enhanced nitroxide generation were observed in cells treated with ultrasound and AlPcTS. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. The significant reduction by histidine indicated that ultrasonically generated reactive oxygen species, such as singlet oxygen, is an important mediator of sonodynamically-induced apoptosis.

Involvement of Reactive Oxygen Species in Sonodynamically Induced Apoptosis by 4-Formyloximeetylidene-3-Hydroxyl-2-Vinyl-euterio-Porphynyl (IX)-6-7-Diaspartic Acid (ATX-S10)

Background: In this study, we investigated the induction of apoptosis by ultrasound in the presence of the photochemically active chlorine, 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl (IX)-6-7-diaspartic acid (ATX-S10). Methods: HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of ATX-S10, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Results: Cells treated with 80μM ATX-S10 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or ATX-S10 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and ATX-S10 but not in cells treated with ultrasound or ATX-S10 alone. In addition, the combination of ATX-S10 and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. Conclusions: These results indicate that the combination of ultrasound and ATX-S10 induces apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. General significance: The results reported in this paper are experimental, but they significantly support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.