It has been shown that 1 and 3 MHz low-intensity ultrasound was able to affect the fragile and leaky angiogenic blood vessels in a
tumor. However, the
biological effects of 21 kHz low-intensity ultrasound on
tumors remain unclear. The aim of the present study was to explore the effects of 21 kHz ultrasound with
microbubbles on the regulation of
vascular endothelial growth factor (
VEGF),
cyclooxygenase-2 (COX-2) and apoptosis in subcutaneous prostate
tumors in nude mice. The study included three parts, each with 20
tumor-bearing nude mice. Twenty nude mice were divided into four groups: control (
sham treatment),
microbubble ultrasound
contrast agent (UCA), low-frequency ultrasound (US) and US+UCA groups. The UCA used was a
microbubble contrast agent (
SonoVue). The parameter of ultrasound: 21 kHz, an intensity of 26 mW/cm2, 40% duty cycle (on 2 sec, off 3 sec), 3 min, once every other day for 2 weeks. In the first study, all subcutaneous
tumors were examined by contrast-enhanced ultrasonography (CEUS) at the initiation and completion of the experiments. Peak intensity (PI), time to peak intensity (
TTP) and area under the curve (AUC) on the time intensity curve (
TIC) were analyzed. In the second study, the intensity of
VEGF and COX-2
protein expression in the vascular endothelium and cytoplasm was evaluated using immunohistochemistry and
laser confocal microscopy. In the third study,
terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used for the evaluation of cell apoptosis in
tumor tissues. The
tumor cells and vasculature were examined by transmission electron microscopy (TEM). Only in the US+UCA group, PI and AUC decreased. The intensity of COX-2 and
VEGF in the US+UCA group in immunohistochemical staining and
laser confocal microscopy was lower compared to that of the other three groups. More cell apoptosis was found in the US+UCA group compared to the other 3 groups. In the control, UCA and US groups, the
tumors had intact vascular endothelium and vessel lumens in TEM. However, lumen occlusion of vessels was observed in the US+UCA group. Twenty-one kHz low-intensity ultrasound with
microbubbles may have anti-angiogenic effects on subcutaneous
tumors in nude mice.