Apoptosis-inducing and antiangiogenic activity of partially purified protein from the pericarp of Zanthoxylum rhetsa in vitro and in vivo

Abstract

Background: Biological activities of Zanthoxylum rhetsa have been well studied, and its various parts have been reported to possess anticancer activities. The reports on anticancer activities of proteins from the fruits of Z. rhetsa are limited. Objectives: The study assessed the proapoptotic and antiangiogenic activity of partially purified proteins from the pericarp of Z. rhetsa. Methods: MCF-7, MDA MB 231, HeLa, and HCT 116 cells treated with partially purified protein fractions of Z. rhetsa pericarp were assessed for cytotoxicity by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-diphenyl tetrazolium bromide (MTT) assay. Fraction ZR3 showed high cytotoxicity against the MCF-7 cells; hence, it was chosen for further assays. The proapoptotic activity was scrutinized by Giemsa staining, acridine orange–ethidium bromide staining, and DNA fragmentation assay. The study was supported by wound healing assay and colony formation assay. Cell cycle analysis was performed by fluorescence-activated cell sorter. Ehrlich ascitic carcinoma-bearing Swiss albino mice were used as in vivo model. Angiogenesis was studied by peritoneal angiogenesis in mice and chorioallantoic membrane assay in fertilized eggs. Results: MTT assay showed the inhibition of MCF-7 cells (IC50 = 21.5 μg/mL) by ZR3 fraction. Reduction in proliferation and failure to produce large cell colonies were observed. Proapoptotic activity was evident from the DNA fragmentation and staining methods. ZR3 blocked the cells in the G2/M phase of the cell cycle. In vivo studies suggested the antiproliferative and proapoptotic activities of ZR3. ZR3 exhibited antiangiogenic properties in vivo. Conclusion: This study confirmed the role of Z. rhetsa partially purified proteins as a potential proapoptotic and antiangiogenic agent against the MCF-7 cell line.