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Involvement of Caspase 8 in Apoptosis Induced by Ultrasound-Activated Hematoporphyrin in Sarcoma 180 Cells In Vitro

College of Life Sciences, Shaanxi Normal University, Xi’an, China.

Address correspondence to Quanhong Liu, PhD, College of Life Sciences, Shaanxi Normal University, 199 Chang-An S Rd, Xi’an, 710062 Shaanxi, China. E-mail: lshaof @snnu.edu.cn

Objective. Sonodynamic therapy (SDT), a novel and promising cancer therapy that uses a combination of ultrasound and hematoporphyrin, can induce apoptosis in some cancer cells. However, the mechanism(s) of SDT-induced cell apoptosis is not well understood. This study investigated SDT-induced apoptosis in sarcoma 180 cells. Methods. Cell suspension were treated by 1.75-MHz continuous focused ultrasound in the presence of hematoporphyrin for 3 minutes, and apoptosis was assessed by flow cytometry, scanning electron microscopy, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end labeling, confocal microscopy, and apoptosis-related protein analysis. Results. DNA breaks, apoptotic bodies, and cleaved poly (adenosine triphosphate-ribose) polymerase were observed 1 hour after SDT. By using laser-scanning confocal microscopy, we found that the Fas-associated death domain and caspase 8 translocated from the cytoplasm to the plasma membrane. Activities of caspase 8 and caspase 3 were detected by an immunohistochemical assay. The results suggested that SDT led to activation of caspase 8, which in turn activated downstream caspase 3. In addition, Z-Ile-Glu-Thr-Asp-fluoromethylketone, a specific inhibitor for caspase 8, was used to confirm the effect of caspase 8 in apoptosis. Conclusions. Our data primarily show that SDT can induce apoptosis in sarcoma 180 cells in vitro, and caspase 8 may play an important role in SDT-induced apoptosis.

Key Words: apoptosis • caspase 8 • hematoporphyrin • sonodynamic therapy • ultrasound

Abbreviations: BSA, bovine serum albumin • DISC, death-inducing signaling complex • dUTP, deoxyuridine triphosphate • FADD, Fas-associated death domain • FITC, fluorescein isothiocynate • HPD, hematoporphyrin • OD, optical density • PARP, poly (adenosine diphosphate–ribose) polymerase • PBS, phosphate-buffered saline • PDT, photodynamic therapy • PMT, photomultiplier • ROS, reactive oxygen species • SDT, sonodynamic therapy • S180, sarcoma 180 • SP, streptavidin/peroxidase • TdT, terminal deoxynucleotidyl transferase • TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end labeling • Z-IETD-FMK, Z-Ile-Glu-Thr-Asp-fluoromethylketone