Raddeanin A synergistically enhances the anti-tumor effect of MAP30 in multiple ways, more than promoting endosomal escape.

Biomacromolecules such as proteins and nucleic acids are very attractive due to their high efficiency and specificity as cancer therapeutics. In fact, the endocytosed macromolecules are often trapped in the endosomes and cannot exhibit pharmacological effects well. Many strategies have been used to address this bottleneck, and one promising approach is to exploit the endosomal escape-promoting effect of triterpenoid saponins to aid in the release of biomacromolecules. Here, Raddeanin A (RA, an oleanane-type triterpenoid saponin) was proved to significantly promote endosomal escape as it recruited Galectin-9, an endosomal escape event reporter. As expected, RA effectively enhanced the anti-tumor effect of MAP30 (a type I ribosome-inactivating protein derived from Momordica charantia). However, based on the results of fluorescent colocalization, RA did not significantly promote MAP30 release from endosomes, suggesting that RA enhances MAP30 activity not only by promoting endosomal escape. Furthermore, it was found that the inhibitors of micropinocytosis and caveolae could almost completely inhibit the cytotoxicity of MAP30 combined with RA without affecting the cytotoxicity of MAP30 alone, indicating that RA may regulate the endocytic pathway of MAP30. Meanwhile, the effect of RA is related to the intra vesicular pH and cholesterol content on cell membrane, and is also cell-type dependent. Therefore, RA enhanced the anti-tumor effect of MAP30 in multiple ways, not just by promoting endosomal escape. Our findings will help to further decipher the possible mechanisms by which triterpenoid saponins enhance drug activity, and provide a new perspective for improving the activity of endocytosed drugs.

Overexpression of alfalfa Orange gene in tobacco enhances carotenoid accumulation and tolerance to multiple abiotic stresses.

The multifunctional Orange (Or) protein plays crucial roles in carotenoid homeostasis, photosynthesis stabilization, and antioxidant activity in plants under various abiotic stress conditions. The Or gene has been cloned in several crops but not in alfalfa (Medicago sativa L.). Alfalfa is widely cultivated across the world; however, its cultivation is largely limited by various abiotic stresses, including drought. In this study, we isolated the Or gene from alfalfa (MsOr) cv. Xinjiang Daye. The amino acid sequence of the deduced MsOr protein revealed that the protein contained two trans-membrane domains and a DnaJ cysteine-rich zinc finger domain, and showed a high level of similarity with the Or protein of other plants species. The MsOr protein was localized in leaf chloroplasts of tobacco. The expression of MsOr was the highest in mature leaves and was significantly induced by abiotic stresses, especially drought. To perform functional analysis of the MsOr gene, we overexpressed MsOr gene in tobacco (Nicotiana benthamiana). Compared with wild-type (WT) plants, transgenic tobacco lines showed higher carotenoid accumulation and increased tolerance to various abiotic stresses, including drought, heat, salt, and methyl viologen-mediated oxidative stress. Additionally, contents of hydrogen peroxide and malondialdehyde were lower in the transgenic lines than in WT plants, suggesting superior membrane stability and antioxidant capacity of TOR lines under multiple abiotic stresses. These results indicate the MsOr gene as a potential target for the development of alfalfa cultivars with enhanced carotenoid content and tolerance to multiple environmental stresses.