Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved.
OBJECTIVE
In this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally, the acoustic mechanisms involved were investigated.


METHODS
The effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects.


RESULTS
The combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect.


CONCLUSIONS
This study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic conditions.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan. kondot@med.u-toyama.ac.jp.
Takashi
T
Kondo
Second Department of Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Toru
T
Yoshida
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Ryohei
R
Ogawa
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Mariame A
MA
Hassan
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Yukihiro
Y
Furusawa
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Qing-Li
QL
Zhao
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Akihiko
A
Watanabe
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Akihiro
A
Morii
Department of Anatomy, Fukuoka University School of Medicine, Fukuoka, Japan.
Loreto B
LB
Feril
Department of Anatomy, Fukuoka University School of Medicine, Fukuoka, Japan.
Katsuro
K
Tachibana
Laboratory of Animal Histophysiology, Division of Animal Science, Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
Hiroshi
H
Kitagawa
Life Science Research Center, University of Toyama, Toyama, Japan.
Yoshiaki
Y
Tabuchi
Life Science Research Center, University of Toyama, Toyama, Japan.
Ichiro
I
Takasaki
Department of Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Mohammad H
MH
Shehata
Laboratory of Biomedical Instrumentation and Measurements, Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan.
Nobuki
N
Kudo
Second Department of Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Kazuhiro
K
Tsukada
apoptosis; doxorubicin; low-intensity ultrasound