Cloning, expression and functional analysis of MAP30 from Momordica charantia reveals its induction of apoptosis of the BGC-823 cells

Abstract

Momordica Anti-HIV Protein 30 (MAP30) can induce many tumour cells apoptosis, but the mechanism is still unclear. Since the content of MAP30 in Momordica charantia is limited, in this study, the MAP30 gene was cloned and expressed and the induction of the recombinant MAP30 protein on the apoptosis of BGC-823 cells was investigated. Morphological changes were observed by microscopy. The normal, apoptotic and necrotic cells were identified by fluorescence staining. The apoptosis percentage and the mitochondria membrane potential level was displayed by flow cytometry analysis and JC-1 fluorescent probe assay, respectively. The mRNA expression of caspase-9 and Apaf-1 was investigated by reverse transcription polymerase chain reaction (RT-PCR). The content of Cyt C and the activity of Caspase-3 were investigated by ELISA and spectrophotometric method, respectively. In this study, the MAP30 gene was expressed and the recombinant MAP30 protein was purified successfully. The results of morphology, the fluorescence staining and the flow cytometry analysis indicated that MAP30 could induce cells apoptosis. When treated with MAP30, the mitochondria membrane potential level decreased, while the expression of caspase-9 and Apaf-1 mRNA, the Cyt C content, and the caspase-3 activity all increased. All these indicated that the recombinant MAP30 protein could induce the BGC-823 cells apoptosis in vitro via the mitochondrial pathway.

Key words: Momordica charantia, MAP30, BGC-823 cells, apoptosis, mitochondria membrane potential, Cyt C, caspase-9, Apaf-1, caspase-3.