The role of Beclin 1 in SDT-induced apoptosis and autophagy in human leukemia cells

Abstract

Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis.

Materials and methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4ʹ-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3- phosphatidylethanolamine conjugate/a cytosolic form of LC3 (LC3 II/I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy.

Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with Beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC) staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage.

Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.

Keywords::

• Sonodynamic therapy
• Beclin 1 shRNA
• autophagy
• apoptosis
• human leukemia K562 cells

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by National Natural Science Foundation of China (No. 81472846), Natural Science Foundation of Shaanxi Province, China (No. 2014JQ3092), the Fundamental Research Funds for the Central Universities (No. GK201404004 and No. GK201302022), the Hong Kong Scholars Program (No. XJ2013034), and the general research fund grant from Hong Kong research grant committee (RGC) (476912).