Moon Ki-Beom, Park Ji-Sun, Kim Han-Gyeul, Jeon Jae-Heung, Kwon Tae-Ho, Chung Kyung-Sook, Lee Hyo-Jun, Kim Hyun-Soon
Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, South Korea.
Center for Gene & Cell Therapy, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, South Korea.
Rice (N Y). 2025 Mar 14;18(1):16. doi: 10.1186/s12284-025-00775-7.
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.
利用植物进行分子农业生产生物制药被认为是取代当前一些生产方法的极佳方式，并且大量重组蛋白已显示出推动这一进程的潜力。特别是，在人类中具有多种重要生物学功能的激活素A的生产受到限制。本研究的目的是开发一种安全、稳定且高效的基于植物的激活素A体外生产系统，评估其在癌细胞中的生物学活性，并证明其在癌症研究中的应用潜力。我们通过大规模培养和分泌系统评估了激活素A在植物细胞中的表达和生产。还评估了由肠激酶产生的成熟激活素A同二聚体的形成。进行了Southern印迹和反向PCR以研究植物中的基因插入位点，并在不同pH条件下评估了激活素A六个月的稳定性。使用荧光素酶报告基因、迁移、磷酸化和明胶酶谱分析在HEK293T、Huh7、MCF7和MDA-MB-231癌细胞系中分析了植物源激活素A的活性。我们开发了细胞系#71，其显示出最高水平的成熟激活素A表达（8.44μg/g愈伤组织鲜重）并且具有多拷贝基因插入。使用肠激酶将前体激活素A转化为成熟激活素A。我们证明植物源激活素A在pH 7且低于4°C的条件下可保持六个月的最佳稳定性。植物源激活素A显著增强了HEK293T、Huh7和MCF7癌细胞中的激活素A信号传导活性。此外，我们证实植物源激活素A通过激活Smad途径抑制Huh7癌细胞的生长，而不影响MAPK途径。相反，在MDA-MB-231乳腺癌细胞中，植物源激活素A促进细胞迁移。我们的结果证实，使用大规模生产系统生产的植物源激活素A具有完整的生物学活性，并以类似于传统哺乳动物系统来源的激活素A的方式影响癌细胞行为。此外，本研究强调了在确定激活素A治疗的功能结果时考虑细胞背景的重要性。
