5-Aminolevulinic Acid Enhances Ultrasound-mediated Antitumor Activity via Mitochondrial Oxidative Damage in Breast Cancer

Materials and Methods

Chemicals. 5-ALA hydrochloride was obtained from SBI Pharmaceuticals Co. Ltd. (Tokyo, Japan). Doxorubicin hydrochloride was from Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan); aminophenyl fluorescein (APF) from Sekisui medical Co. Ltd. (Tokyo, Japan); 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and JC-1 were purchased from Funakoshi Co. Ltd. (Tokyo, Japan); PpIX, tert-butyl hydroperoxide (tBHP) and 1,3-diphenylisobenzofuran (DPBF) were all from Tokyo chemical industry Co. Ltd. (Tokyo, Japan); MitoSOX Red from Thermo Fisher Scientific Inc. (Waltham, MA, USA); and WST-1 from Dojindo laboratories Co. Ltd. (Kumamoto, Japan).

Cell culture. Mouse mammary EMT6 tumor cells (supplied by Dr. Shin-ichiro Masunaga, Kyoto University, Kyoto, Japan) were maintained in Eagle's minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (JR Scientific, Inc., Woodland, CA, USA). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C.

Cytotoxicity and intracellular kinetics of 5-ALA. To evaluate the cytotoxicity of 5-ALA, the cells (2×10² cells/well) were seeded into a 96-well plate and incubated with various concentrations (0-10 mM) of 5-ALA for 5 h, washed with PBS and, then, incubated for 72 h. The survival rate of the cells was determined using the WST-1 assay. To evaluate the intracellular kinetics of PpIX synthesized from 5-ALA, 3×10⁴ cells/dish were incubated for 24 h and, then, 100 μM of 5-ALA was added. The cells were washed with PBS and collected using 2% Triton X-100 solution at every measurement time point. Fluorescence intensity of PpIX (excitation: 410 nm, emission: 630 nm) was measured using a microplate reader, Infinite M200 (Tecan Group Ltd. Männedorf, Switzerland).

5-ALA-SDT for EMT6 cells. EMT6 cells (1.0×10³ cells/well) were seeded into a 24-well plate and incubated for 24 h. 5-ALA was added to the growth medium at a final concentration of 1 mM and the cells were incubated for 5 h in the dark. Subsequently, the cells were exposed to ultrasound (1 MHz, 20% duty cycle, 2.15 W/cm², 2 min) using the ultrasonic generator UST-770 (ITO Co. Ltd., Tokyo, Japan). After 72 h, the survival rate of the cells was measured using the WST-1 assay.

5-ALA-SDT for a tumor-bearing mouse model. Eight-week-old female BALB/c mice were purchased from the Japan SLC Inc. (Shizuoka, Japan) and housed in dedicated pathogen-free barrier facilities. EMT6 cell suspensions (1×10⁶ cells/50 μl) were subcutaneously injected into the back of the BALB/c mice. When the tumors reached about 100 mm³ (six days after inoculation), 5-ALA (300 mg/kg) was administrated orally. After 3 h, they were exposed to ultrasound (1 MHz, 50% duty cycle, 3.0 W/cm², 10 min, 3 times/week) through a 1-cm gel by using the ultrasonic generator UST-770 under general anesthesia. The mice were administered doxorubicin hydrochloride (1 mg/kg) intraperitoneally twice a week as a positive control. All animal protocols were approved by the Animal Care and Use Committee of Tokushima University (No. 14081).

ROS detection. ROS production, by the reaction between PpIX and ultrasound (1 MHz, 20% duty cycle, 2.15 W/cm², 2 min) or between PpIX and tBHP (20 mM), was determined by measuring the absorbance of DPBF at 410 nm for singlet oxygen or fluorescence of APF (490 nm excitation and 515 nm emission wavelengths) and DCFH for hydroxyl radicals (488 nm excitation and 522 nm emission wavelengths). Intracellular ROS content was determined by measuring the fluorescence of DCFH-DA for cytoplasmic ROS and MitoSOX Red for mitochondrial ROS. The cells (4×10⁴ cells/well for DCFH-DA or 2×10⁴ cells/well for MitoSOX Red) were seeded in a 24-well plate and 1 mM 5-ALA was added for incubation for 5 h. DCFH-DA (50 μM) or MitoSOX Red (5 μM) was added and then exposed to ultrasound (1 MHz, 20% duty cycle, 2.15 W/cm², 2 min). The fluorescence intensity of DCFH was measured at 488 nm excitation and 530 nm emission wavelengths by using a microplate reader at every measurement time point. After 3 h, the fluorescence intensity of MitoSOX Red was measured at 550 nm excitation and 600 nm emission wavelengths using a fluorescence microscope BZ-X700 (KEYENCE Co. Ltd., Osaka, Japan).

Mitochondrial membrane potential (MMP) detection. The decrease in MMP was determined using a fluorescence microscope with JC-1 staining. The cells (2×10⁴ cells/well) were seeded and 1 mM 5-ALA was added for incubation for 4 h. JC-1 (10 μg/ml) was added and then exposed to ultrasound (1 MHz, 20% duty cycle, 2.15 W/cm², 2 min). Fluorescence intensity was measured at 485 nm excitation and 535 nm emission wavelengths for a monomer type of JC-1 and 550 nm excitation and 600 nm emission wavelengths for an aggregate type of JC-1 at every measurement time point.

Statistical analysis. Data are expressed as mean and standard deviation values. The statistical significance of the differences between the results of the independent experiments was analyzed using the Student's t-test. A p-value of <0.05 was considered statistically significant.