Induction of apoptotic cell death in human Jurkat T cells by a chlorophyll derivative (Cp-D) isolated from Actinidia arguta Planchon

The chloroform and methanol (2:1, v/v) extract from an edible plant, Actinidia arguta Planchon, appeared to possess antitumor activity against human leukemias Jurkat T and U937 cells through inducing apoptosis. The substance in the solvent extract was purified by silica gel column chromatography, preparative TLC, and Sephadex LH-20 column chromatography. Characteristics of the substance analyzed by UV scanning analysis, and ¹H and ¹³C NMR spectra suggested that the substance belongs to the chlorophyll derivatives-like group. The IC₅₀ value of the chlorophyll derivative (Cp-D) determined by MTT assay was 15 μg/ml for Jurkat, 10 μg/ml for U937, and 11.4 μg/ml for HL-60, and was more toxic to these leukemias than to solid tumors or normal fibroblast. In order to elucidate cellular mechanisms underlying the cytotoxicity, the effect of the Cp-D on Jurkat T cells was investigated. When cells were treated with the Cp-D at a concentration of 15 μg/ml, [³H]thymidine incorporation declined rapidly and was undetectable in 1 h. However, no significant changes were made in the cell cycle distribution of the cells by 24 h. The sub-G1 peak representing apoptotic cells began to be detectable in 36 h, at which time apoptotic DNA fragmentation was also detected on agarose gel electrophoresis, demonstrating that the cytotoxic effect of the Cp-D is attributable to the induced apoptosis. Under the same conditions, although the protein level of cyclin-dependent kinases such as cdc4, cdk6, cdk2, and cdc2 was not significantly changed until 24 h, the kinase activity of all cdks rapidly declined and reached a minimum level within 1 -6 h and then recovered to the initial level by 12 h and sustained until 24 h. These results suggest that inactivation of cdks at an inappropriate time during the cell cycle progression in Jurkat T cells following a treatment with the Cp-D leads to induction of apoptotic cell death.