Functional Analysis of Mature Activin A Produced by Enterokinase in Plant Cells

Molecular farming for producing biopharmaceuticals in plants is considered an excellent method to replace some of the production methods currently used, and a significant number of recombinant proteins have already shown the potential to facilitate this. In particular, production of activin A, which has a variety of important biological functions in humans, is limited. The purpose of this study was to develop a safe, stable, and efficient plant-based in vitro production system for activin A, assess its biological activity in cancer cells, and demonstrate its potential for use in cancer research. We evaluated the expression and production of activin A in plant cells through a mass culture and secretion system. The formation of mature activin A homodimers, produced by enterokinase, was also assessed. Southern blot and inverse PCR were performed to investigate the gene insertion sites in the plants, and the stability of activin A was evaluated over six months under various pH conditions. The activity of plant-derived activin A was analyzed in HEK293T, Huh7, MCF7, and MDA-MB-231 cancer cell lines using luciferase reporter, migration, phosphorylation, and gelatin zymography assays. We developed cell line #71, which showed the highest levels of mature activin A expression (8.44 μg/g calli fresh weight) and had multicopy gene insertions. Pro-activin A was converted to mature activin A using enterokinase. We demonstrated that the optimal stability of plant-derived activin A was maintained for six months at pH 7 below 4 °C. Plant-derived activin A significantly enhanced activin A signaling activity in HEK293T, Huh7, and MCF7 cancer cells. Additionally, we confirmed that plant-derived activin A inhibited the growth of Huh7 cancer cells by activating the Smad pathway without affecting the MAPK pathway. Contrastingly, in MDA-MB-231 breast cancer cells, plant-derived activin A promoted cell migration. Our results confirm that plant-derived activin A, produced using a mass production system, exhibits full biological activity and affects cancer cell behavior in a manner similar to activin A derived from traditional mammalian systems. Furthermore, this study highlights the importance of considering cellular context when determining the functional outcomes of activin A treatment.