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.

Keywords:

• apoptosis
• hypericin
• sonodynamic therapy
• mitochondria–caspase pathway
• atherosclerosis

Acknowledgments

This study was supported by the National Natural Science Foundation of China (81000688, 81271734), the Heilongjiang Provincial Youth Science Foundation (QC2011C002), the Foundation of Science and Technology Innovation Talent of Harbin Science and Technology Bureau (2011RFQXS072), the Wu Lian de Youth Science Foundation of Harbin Medical University (WLD-QN1104), the Postdoctoral Science-Research Developmental Foundation of Heilongjiang Province (LBHQ12049), the China Postdoctoral Science Foundation funded project (20090460911), the Ministry of Education, and the Research Foundation of the Health Department of Heilongjiang Province (2009-217).

Disclosure

There are no conflicts of interest among the authors of this work.