Abstract
Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI50; 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.
Highlights
The plant metabolite, plumbagin (5-hydroxy-2 methyl-1,5naphthoquinone), is a naphthoquinone derivative that was originally identified from the roots of plant Plumbago and belongs to one of the largest and diverse groups of plant metabolites [1,2,3]
We first confirmed the functional role of reactive oxygen species (ROS) in MCF-7 cells because this cell line was most sensitive to this agent among various types of human cancer cells tested in our study (Figure S1 &S2)
These patterns were observed in other cancer cells such as HeLa human cervical cancer cells and A549 human lung carcinoma cells (Figure S3). These results indicated that ROS are critical mediators of plumbagin cytotoxicity on human cancers and that defining the molecular insights of ROS generation by plumbagin might provide therapeutic benefits for drug discovery as well as understanding its mode of action
Summary
The plant metabolite, plumbagin (5-hydroxy-2 methyl-1,5naphthoquinone), is a naphthoquinone derivative that was originally identified from the roots of plant Plumbago and belongs to one of the largest and diverse groups of plant metabolites [1,2,3]. Plumbagin has potent anti-proliferative and apoptotic activities in various types of human cancers, but the mechanisms underlying the anticancer activity are only partially understood. This compound dysregulates multiple pathways that play a crucial role in cancer cell proliferation, survival, invasion and metastasis [4,5,6,7,8,9], in which ROS generation is a critical mediator for cell cycle arrest and apoptosis [6,10,11]. The functional role of PI5K as a molecular target of plumbagin is not known
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