Enhancement of antitumor activity by using 5-ALA–mediated sonodynamic therapy to induce apoptosis in malignant gliomas: significance of high-intensity focused ultrasound on 5-ALA-SDT in a mouse glioma model

Abstract

ABBREVIATIONS DCFH-DA = dichlorodihydrofluorescein diacetate; DPBS = Dulbecco phosphate-buffered saline; FBS = fetal bovine serum; FUS = focused ultrasound; GSL = glioma stemlike; HIFU = high-intensity focused ultrasound; IP = intraperitoneal; NSC = neural stem cell; PDT = photodynamic therapy; PFA = paraformaldehyde; PpIX = protoporphyrin IX; ROS = reactive oxygen species; SDT = sonodynamic therapy; TBS = Tris-buffered saline; US = ultrasound; 5-ALA = 5-aminolevulinic acid; 5-ALA-SDT = 5-ALA-mediated SDT. OBJECTIVE High invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT). METHODS In vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251 SUBMITTEDOct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated. RESULTS The 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact. CONCLUSIONS The 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-...

Participation of MicroRNAs in the Treatment of Cancer with Phytochemicals

“…5-Aminolevulinic acid (5-ALA) can be utilized as a sensitizer for photodynamic and sonodynamic therapies. The generation of ROS contributes to the anti-cancer effects of these 5-ALA-based therapies [ 214 , 215 ]. Moreover, the expression of miRNAs can be changed by 5-ALA-based therapies.…”

Applications of Focused Ultrasound in Cerebrovascular Diseases and Brain Tumors

“…FUS is able to deeply and specifically penetrate brain tissue and to excite sonosensitizer only in the target area. Combining the specificity of 5-ALA accumulation in some types of tumors to the specificity of FUS insonation is possible to obtain necrosis and apoptosis only in the target volume [98]. Numerous studies demonstrated the efficacy in various cancers also comprising malignant glioma [98][99][100].…”

“…Combining the specificity of 5-ALA accumulation in some types of tumors to the specificity of FUS insonation is possible to obtain necrosis and apoptosis only in the target volume [98]. Numerous studies demonstrated the efficacy in various cancers also comprising malignant glioma [98][99][100]. Satoshi Suehiro et al [98] studied the combined application of 5-ALA and FUS demonstrating effective cytotoxicity towards glioma cells thus resulting in prolonged survival of tumor-bearing mice.…”

“…Numerous studies demonstrated the efficacy in various cancers also comprising malignant glioma [98][99][100]. Satoshi Suehiro et al [98] studied the combined application of 5-ALA and FUS demonstrating effective cytotoxicity towards glioma cells thus resulting in prolonged survival of tumor-bearing mice. Growing body of evidences support the superiority of sonodynamic therapy over photodynamic therapy.…”

“…Growing body of evidences support the superiority of sonodynamic therapy over photodynamic therapy. Indeed, this technique is equally effective in generating reactive oxygen species, does not require craniotomy or surgical intervention, is able to activate chemical agents deeply located in the brain, and permits conformal dosage of energy [98,101,102].…”