The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages

Abstract

Purpose To investigate the sonoactivity of hypericin (HY), together with its sonodynamic effect on THP-1 macrophages and the underlying mechanism. Materials and methods CCK-8 was used to examine cell viability. Confocal laser scanning microscopy was performed to assess the localization of HY in cells, reactive oxygen species (ROS) generation, and opening of the mitochondrial permeability transition pore (mPTP) after different treatments. Apoptosis was analyzed using Hoechst–propidium iodide and transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) collapse was detected via fluorescence microscopy. Lipoprotein oxidation was determined in malondialdehyde (MDA) assays. Western blotting was conducted to determine the translocation of BAX and cytochrome C and the expression of apoptosis-related proteins. Results HY was sublocalized among the nuclei and the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosome in the cytosol of THP-1 macrophages. Under low-intensity ultrasound irradiation, HY significantly decreased cell viability and induced apoptosis. Furthermore, greater ROS generation, higher MDA levels, and greater ΔΨm loss were observed in the sonodynamic therapy (SDT) group. Both ROS generation and MDA levels were significantly reduced by the ROS scavenger N-acetyl cysteine (NAC) and the singlet oxygen scavenger sodium azide. Most of the loss of ΔΨm was inhibited by pretreatment with NAC, sodium azide, and the mPTP inhibitor cyclosporin A (CsA). mPTP opening was induced upon SDT but was reduced by pretreatment with bongkrekic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium, CsA, and NAC. Western blot analyses revealed translocation of BAX and cytochrome C, downregulated expression of Bcl-2, and upregulated expression of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase in the SDT group, which were reversed by NAC. Conclusion HY mediated SDT-induced apoptosis in THP-1 macrophages via ROS generation. Then, the proapoptotic factor BAX translocated from the cytosol to the mitochondria, increasing the ratio of BAX/Bcl-2, and the mPTP opened to release cytochrome C. This study demonstrated the great potential of HY-mediated SDT for treating atherosclerosis.

ROS‐Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A‐Sonodynamic Therapy in THP‐1 Macrophages

“…The rapidly expanding body of literature on SDT has shown that the development of sonosensitizers is one of the most essential factors in this treatment. In recent years, our group has identified sonosensitizers derived from extracts of Chinese herbs, including hypericin, curcumin, and hydroxyl acetylated curcumin [ 2 , 20 , 21 ]. However, further animal experiments and clinical translation of these sonosensitizers are limited by their lipophilic properties and rare application in the clinic.…”

“…At 30 min after HSYA-SDT, the cells were harvested by centrifugation and analyzed as previously described [ 20 ]. The images were observed and obtained with a transmission electron microscope (JEM-1220, Japan).…”

“…An initial clinical application of SDT showed therapeutic benefits in cancer patients [ 19 ]. Moreover, our group demonstrated that SDT had a beneficial effect on atherosclerosis as it could induce apoptosis of macrophages in vitro [ 20 – 22 ] and effectively stabilized the atherosclerotic plaques of the rabbit femoral artery [ 23 ]. Reactive oxygen species (ROS) were found to be the most important factors in the biological effects induced by SDT in these studies.…”

“…The application of sonosensitizers is one of the most critical factors for SDT. In recent years, our group has identified various sonosensitizers derived from the extracts of Chinese herbs for future clinical use [ 20 , 21 , 29 ]. Hydroxysafflor yellow A (HSYA) is a major constituent of the hydrophilic fraction of the safflower plant, a traditional Chinese herbal medicine with a long history of clinical treatment of cardiovascular diseases by intravenous injection [ 30 ].…”

The bright side of sound: perspectives on the biomedical application of sonoluminescence

“…The first evidence about SDT came from a study by Yumita et al in 1989, where various hematoporphyrin derivatives employed in PDT also caused a relevant cell damage after being exposed to US. 61 Since then, it has been shown that SDT can treat solid tumours, 62-65 leukaemia 66 and atherosclerosis 67 and, moreover, remove proliferative scars and kill pathogenic microorganisms. 68,69 Since SDT was developed on the basis of PDT, some suggest that SDT shares a similar therapeutic mechanism where the ultrasonic wave triggers the sonosensitizer inside the target tissue, with production of ROS that kill the target cells.…”