Hematoporphyrin Monomethyl Ether Enhances the Killing of Ultrasound on Osteosarcoma Cells Involving Intracellular Reactive Oxygen Species and Calcium Ion Elevation

Objective: The present study aims to investigate the possible mechanisms of hematoporphyrin monomethyl ether (HMME) enhancing the cytotoxicity of ultrasound in osteosarcoma cells. Methods: Osteosarcoma cell line UMR-106 was treated by HMME and ultrasound radiation, with the HMME concentration kept at 20 μg/mL and ultrasound radiation for 10 seconds at the intensity of 0.5 W/cm2. Cell proliferation was investigated at 12, 24, 36, and 48 hours using MTT assay after ultrasound and HMME treatment. Ultrastructural morphology was observed using transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) was measured using a flow cytometry with DCFH-DA staining and intracellular free calcium ion (Ca2+) with Fluo-3-AM staining. Results: The UMR-106 cells proliferated rapidly in the sham radiation and HMME treatment alone group, but ultrasound-treated cells and HMME-ultrasound-treated cells proliferated slowly. There was a significant difference between HMME-ultrasound treatment and the controls, including ultrasound radiation, HMME treatment alone, and sham radiation (P < .05). TEM showed endoplasmic reticulum and mitochondrial swelling in the ultrasound-treated cells, and more cells presented apoptosis and necrosis after treatment with ultrasound and HMME together. Intracellular ROS and Ca2+ in the cells increased more significantly after both ultrasound and HMME treatment than after ultrasound treatment alone. Conclusions: HMME could effectively enhance the inhibition effect of ultrasound on osteosarcoma cells. Intracellular ROS and Ca2+ in the UMR-106 cells increased more significantly after the treatment of HMME and ultrasound together, indicating that the enhancement of HMME on ultrasound cytotoxicity to osteosarcoma cells possibly involves both intracellular ROS and Ca2+ elevation.