Hypocrellin B in hepatocellular carcinoma cells: Subcellular localization and sonodynamic damage

Abstract

Purpose: To study subcellular localization of hypocrellin B in hepatocellular carcinoma cells, and hypocrellin B-mediated sonodynamic action-induced cell damage.

Materials and methods: After incubation with 2.5 μM of hypocrellin B, human hepatocellular carcinoma HepG2 cells were exposed to ultrasound waves for 8 sec at an intensity of 0.46 W/cm². Clonogenic survival of HepG2 cells was measured using a colony forming assay and light microscope. Ultrastructural morphology was observed using transmission electron microscope (TEM) and mitochondrial membrane potential (MMP) was assessed using confocal laser scanning microcope (CLSM) after rhodamine 123 staining. Additionally, subcellular localization of hypocrellin B in HepG2 cells with organelle probe staining was also observed using CLSM.

Results: The colony forming units of HepG2 cells decreased substantially after sonodynamic treatment. The results of TEM showed microvilli disappearance, apoptotic body formation, swollen mitochondria with loss of cristae and mitochondrial myelin-like features (or membrane whorls). Collapse of MMP was found in the treated cells. Hypocrellin B was distributed in mitochondria and lysosomes as well as in endoplasmic reticulum and Golgi apparatus.

Conclusions: The findings demonstrated that sonodynamic action of hypocrellin B induced mitochondrial damage, survival inhibition, and apoptosis of HepG2 cells. Additionally, other subcellular organelles such as endoplasmic reticulum, Golgi apparatus and lysosomes were also the targets of hypocrellin B-mediated sonodynamic action as well as mitochondria.

Keywords::

• Sonodynamic therapy
• hypocrellin B
• hepatocellular carcinoma cells
• subcellular localization
• clonogenic survival
• mitochondrial damage
• apoptosis

Acknowledgements

This work was supported by the general research fund (GRF) from Hong Kong Research Grants Council (RGC) (476912), Natural Science Foundation of Shannxi Province (2011K12–83), Special Research Project of Shannxi Provincial Department of Education (12JK0753), and Innovation and Technology Fund of Shenzhen (CXZZ20120619150627260). We would also like to express our sincere thanks to Professor Zheng Huang, Professor Faqi Li, Dr Jin Zhu, Mr Eric Pun, Miss Xin Pan, Mr Gabilan Sivapatham, Mr Guoyang Li, Mr Jianyong Wu, Mr Jingchuan Fan and Mr Kejian Wang for their helpful assistance.

Declaration of interest

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