Inhibitory effects of low-intensity pulsed ultrasound sonication on the proliferation of osteosarcoma cells

Abstract

To date, there is limited data on the biological effects of low-intensity pulsed ultrasound (LIPUS) on primary malignant bone tumors. The purpose of the present study was to investigate the antitumor effects of LIPUS on osteosarcoma cells. The effects of LIPUS on cell viability, induction of apoptosis, mitochondrial membrane potential and intracellular signaling molecules in the LM8 osteosarcoma cell line were investigated. LIPUS inhibited cell viability (P=0.0022) and mitochondrial membrane potential (P=0.0019) in LM8 cells. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed significantly higher numbers of apoptotic (P<0.0001) and necrotic cells (P=0.0091) compared with cells without treatment. LIPUS treatment significantly increased phosphorylated Akt (P<0.0001) and IκBα (P=0.0001) levels, and reduced phosphorylated mitogen-activated protein kinase 7 (P<0.0001) and phosphorylated checkpoint kinase 1 (P=0.0008) levels. These results suggest that LIPUS is a non-invasive adjuvant therapy that is able to inhibit cellular proliferation in osteosarcoma cells.

Keywords: LM8; apoptosis; low-intensity pulsed ultrasound; osteosarcoma; sonication.

Figure 1.

Viability of LM8 cells treated with LIPUS. Treatment with LIPUS for 18 or 24 h significantly inhibited the growth of LM8 cells (18 h, P=0.0133; 24 h, P=0.0022), compared with no treatment. No significant differences in cell growth between cells treated with LIPUS for 1 or 12 h and untreated cells (1 h, P=0.3762; 12 h, P=0.1858). LIPUS, low-intensity pulsed ultrasound.

Figure 2.

Viability of MC3T3-E1 cells treated with LIPUS. Treatment with LIPUS for 1 or 12 h significantly inhibited the growth of MC3T3-E1 cells (1 h, P=0.0048 and 12 h, P<0.0001), compared with no treatment. No significant difference in cell growth between cells treated with LIPUS for 18 or 24 h compared with untreated cells (18 h, P=0.6574; 24 h, P=0.3606). LIPUS, low-intensity pulsed ultrasound.

Figure 3.

Effects on mitochondrial membrane potential of 48 h LIPUS treatment. LM8 cells subjected to LIPUS treatment for 48 h had a significantly lower mitochondrial membrane potential compared with untreated cells (P=0.0019). No significant differences between MC3T3-E1 cells with and without LIPUS treatment (P=0.2437). LIPUS, low-intensity pulsed ultrasound.

Figure 4.

Representative image of flow cytometry detection of apoptosis and necrosis in LM8 cells treated with low-intensity pulsed ultrasound for 48 h. PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 5.

Quantification of flow cytometry detection of apoptosis and necrosis in LM8 cells treated with LIPUS for 48 h. Flow cytometry analysis indicated that LM8 cells treated with LIPUS had significantly higher numbers of apoptotic and necrotic cells compared with untreated cells (apoptotic cells, P<0.0001; necrotic cells, P=0.0091). LIPUS, low-intensity pulsed ultrasound; PI, propidium iodide.

Figure 6.

Representative image of terminal deoxynucleotidyl transferase dUTP nick end labeling staining of LM8 cells, with or without low-intensity pulsed ultrasound treatment.

Figure 7.

Rate of TUNEL-positive cells. TUNEL staining revealed that LM8 cells treated with LIPUS had significantly higher numbers of apoptotic cells compared with untreated cells (P<0.0001). TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; LIPUS, low-intensity pulsed ultrasound.

Figure 8.

Representative image of a stress and apoptosis signaling protein array following the treatment of LM8 cells with or without low-intensity pulsed ultrasound for 48 h. X, blank spot; p-, phosphorylated; ERK, mitogen-activated protein kinase; MAPK, mitogen-activated protein kinase; SAPK/JNK, mitogen-activated protein kinase 8; IκBα, NFκB inhibitor α; eIF2α, eukaryotic translation initiation factor 2α; TAK1, mitogen-activated protein kinase 7; Chk1, checkpoint kinase 1; Bad, Bcl-2-associated agonist of cell death.

Figure 9.

Effects on comprehensive intracellular signaling following LIPUS treatment for 24 h. LM8 cells were treated with LIPUS for 24 h, resulting in a significant increase in p-ERK1/2, p-Akt, and IκBα levels (p-ERK1/2, P<0.0001; p-Akt, P<0.0001; IκBα, P<0.0001) and a decrease in p-TAK1 and p-Chk1 levels (p-TAK1, P<0.0001; p-Chk1, P=0.0006). There was no effect on p-Bad levels (P=0.6926). LIPUS, low-intensity pulsed ultrasound; ERK1/2, mitogen-activated protein kinase 3/1; IκBα, NFκB inhibitor α; TAK1, mitogen-activated protein kinase 7; Chk1, checkpoint kinase 1; Bad, Bcl-2-associated agonist of cell death.

Figure 10.

Effects on intracellular signaling following LIPUS treatment for 48 h. LM8 cells were treated with LIPUS for 48 h, resulting in a significant increase in p-Akt and IκBα levels (p-Akt, P<0.0001; IκBα, P=0.0001) and a decrease in p-TAK1 and p-Chk1 levels (p-TAK1, P<0.0001; p-Chk1, P=0.0008). There were no effects on p-ERK1/2 or p-Bad levels (p-ERK1/2, P=0.2437; Bad, P=0.9837). LIPUS, low-intensity pulsed ultrasound; IκBα, NFκB inhibitor α; TAK1, mitogen-activated protein kinase 7; Chk1, checkpoint kinase 1; ERK, mitogen-activated protein kinase; Bad, Bcl-2-associated agonist of cell death.