5-Aminolevulinic Acid-Based Sonodynamic Therapy Induces the Apoptosis of Osteosarcoma in Mice

Abstract

Objective: Sonodynamic therapy (SDT) is promising for treatment of cancer, but its effect on osteosarcoma is unclear. This study examined the effect of 5-Aminolevulinic Acid (5-ALA)-based SDT on the growth of implanted osteosarcoma and their potential mechanisms in vivo and in vitro.

Methods: The dose and metabolism of 5-ALA and ultrasound periods were optimized in a mouse model of induced osteosarcoma and in UMR-106 cells. The effects of ALA-SDT on the proliferation and apoptosis of UMR-106 cells and the growth of implanted osteosarcoma were examined. The levels of mitochondrial membrane potential (ΔψM), ROS production, BcL-2, Bax, p53 and caspase 3 expression in UMR-106 cells were determined.

Results: Treatment with 5-ALA for eight hours was optimal for ALA-SDT in the mouse tumor model and treatment with 2 mM 5-ALA for 6 hours and ultrasound (1.0 MHz 2.0 W/cm2) for 7 min were optimal for UMR-106 cells. SDT, but not 5-ALA, alone inhibited the growth of implanted osteosarcoma in mice (P<0.01) and reduced the viability of UMR-106 cells (p<0.05). ALA-SDT further reduced the tumor volumes and viability of UMR-106 cells (p<0.01 for both). Pre-treatment with 5-ALA significantly enhanced the SDT-mediated apoptosis (p<0.01) and morphological changes. Furthermore, ALA-SDT significantly reduced the levels of ΔψM, but increased levels of ROS in UMR-106 cells (p<0.05 or p<0.01 vs. the Control or the Ultrasound). Moreover, ALA-SDT inhibited the proliferation of osteosarcoma cells and BcL-2 expression, but increased levels of Bax, p53 and caspase 3 expression in the implanted osteosarcoma tissues (p<0.05 or p<0.01 vs. the Control or the Ultrasound).

Conclusions: The ALA-SDT significantly inhibited osteosarcoma growth in vivo and reduced UMR-106 cell survival by inducing osteosarcoma cell apoptosis through the ROS-related mitochondrial pathway.

Fig 1. Schematic diagram of the sonication device.

Fig 2. The dynamics of 5-ALA metabolism in tumors and ALA-SDT mediated inhibition on the growth implanted osteosarcoma in mice.

BALB/c nude mice were inoculated with UMR-106 cells to induce solid tumor and the tumor-bearing mice were treated with 250 mg/kg 5-ALA. The contents of generated PpIX in the tumors or surrounding tissues of individual mice were evaluated longitudinally at the indicated time points. In addition, the tumor-bearing mice were treated with vehicle alone as the Control, with 5-ALA (ALA), ultrasound or both as the ALA-SDT daily for 10 consecutive days. The volumes of tumor were monitored daily. Data are expressed as the mean ± SD of individual groups (n = 3 per group for the measurement of PpIX, n = 10 per group for measurement of in vivo tumor growth). A. The dynamics of PpIX generation in the tumor. B. The growth of implanted tumors in mice. *P<0.05, **p<0.01 vs. the Control.

Fig 3. Optimization of ALA-SDT for UMR-106 cells in vitro.

UMR-106 cells were treated with 0–10 mM 5-ALA for 2–12 house and during the last 2-h culture, the cells were exposed to MTT for determine the optimal concentration of 5-ALA by the survival of cells. Furthermore, the cells were treated with 0–4 mM and exposed to ultrasound at 2.0 W/cm² for 5 minutes, followed by determining the survival of cells. In addition, the cells were treated with 2 mM 5-ALA for 2–12 hours and the intracellular contents of PpIX in individual groups of cells were determined longitudinally using a fluorescent microscope and spectrophotometer. Moreover, the cells were treated with 2 mM 5-ALA and then with ultrasound for 1–15 minutes, followed by determining the survival rates of cells. Finally, the cells were treated with vehicle as the Control, 2 mM 5-ALA or/and ultrasound for 7 minutes and the viability of individual groups of cells was determined by MTT. Data are representative fluorescent images or expressed as the mean ± SD of individual groups of cells from three separate experiments. A. The dose effects of 5-ALA on the survival of cells. B. The dose effect of ALA_SDT on the survival of cells. C. The fluorescent images (magnification x 400) of intracellular PpIX. D. The quantitative analysis of intracellular PpIX. E. The time effect of ALA-SDT on the survival of cells. F. The effect of ALA-SDT on the viability of cells. *P<0.05, **p<0.01 vs. the Control.

Fig 4. ALA-SDT induces osteosarcoma cell apotosis in vitro.

UMR-106 cells were treated with vehicle (Control), 2 mM 5-ALA or/and ultrasound for 7 minutes, cultured for 6 hours and the percentages of apoptotic cells were determined by flow cytometry. In addition, the cells damages of individual groups of cells were characterized by TEM. Data are representative images of individual groups of cells from three separate experiments. A. Flow cytometry analysis of apoptotic cells. B. TEM analysis of cell damages (Magnification×20000). The white arrows indicate apoptotic bodies.

Fig 5. ALA-SDT significantly reduces the mitochondrial membrane potential (ΔψM) and promotes high levels of ROS production in UMR-106 cells.

UMR-106 cells were treated, as described above, and stained with JC-1 or DCFH-DA. Subsequently, the changes in the ΔψM and ROS production in individual groups of cells were determined by fluorescent imaging and spectrophotometer. Data are representative images (magnification x400) or expressed as the means ± SD of individual groups of cells from three separate experiments. A. Fluorescent images of JC-1 staining. B. Quantitative analysis of ΔψM. C. Fluorescent images of ROS production. D. Quantitative analysis of ROS production. *P<0.05, **p<0.01 vs. the Control. #p<0.05 vs. the ultrasound alone.

Fig 6. ALA-SDT inhibits the proliferation and promotes the apoptosis of implanted osteosarcoma cells in vivo.

The PCNA expression and the frequency of apoptotic cells in tumor sections from the different groups of mice were characterized by immunohistochemistry and TUNEL assays, respectively. Data are representative images (magnification x 400) or expressed as the mean % ± SD of in individual groups of tumors (n = 10 per group). A. Immunohisochemsitry for PCNA expression. B. Quantitative analysis of the frequency of PCNA+ tumor cells. C> The TUNEL analysis of apoptotic cells in the tumors. D. The Quantitative analysis of apoptotic tumor cells. *P<0.05, **p<0.01 vs. the Control. #p<0.05 vs. the ultrasound alone.

Fig 7. Immunohistochemsitry analysis of the levels of apoptosis-related regulators in the tumors.

The levels of BcL-2, Bax, p53 and caspase 3 expression in the tumor sections from the different groups of mice were characterized by immunohistochemistry. Data are representative images (magnification x 200) or expressed as the mean ± SD of the IOD from the different groups of mice (n = 10 per group). A. Immunohisochemsitry analysis. B. Quantitative analysis of the IOD values. *P<0.05, **p<0.01 vs. the Control. #p<0.05 vs. the ultrasound alone.