Accumulation of camptothecin and 10-hydroxycamptothecin and the transcriptional expression of camptothecin biosynthetic genes in Camptotheca acuminata cambial meristematic and dedifferentiated cells

摘要

Camptotheca acuminata Decne (Nyssaceae) is a major natural source of the anticancer drug camptothecin (CPT) and its derivatives. The leaves and stems are two of the main regions that accumulate CPT in C. acuminata. In this study, we successfully isolated cambial meristematic cells (CMCs) and dedifferentiated cells (DDCs) from C. acuminata stems and leaves, identified their characteristic features using micrograph analysis, and assessed their hypersensitivity to γ-irradiation and zeocin. The growth and kinetics curves of CMCs and DDCs were also studied. Furthermore, the accumulation of CPT and 10-hydroxycamptothecin (HCPT)—a more potent and less toxic CPT derivative—was assessed by liquid chromatography, and the transcriptional levels of nine genes encoding key enzymes involved in CPT and HCPT biosynthesis was assessed using quantitative real-time polymerase chain reaction (PCR) in CMCs and DDCs. The results showed that CMCs induced were particularly hypersensitive to γ-irradiation and zeocin and presented with abundant and small vacuoles, whereas DDCs were not. The above morphological and physiological characteristics of CMCs were consistent with previous reports. B₅ medium was determined as the best growth medium for CMCs and DDCs. The accumulation of CPT and HCPT was higher in CMCs than DDCs, and the expression of IPI, G10H, ASA1, TSB, TDC1, TDC2, and STR was significantly upregulated in CMCs compared with DDCs; HMGR2 and HMGR3 were downregulated in CMCs. We speculate that the high accumulation of CPT and HCPT in CMCs was due to the upregulation of these seven genes.