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The effect of casticin was investigated by focusing on cell viability, apoptosis induction and cell cycle arrest in HL-60 cells. Casticin induced a dose- and time-dependent decrease in cell viability associated with apoptosis induction and G2/M cell cycle arrest. The addition of SB203580, an inhibitor for p38 mitogen-activated protein kinase (MAPK), but not SP600125 [c-Jun NH2-terminal protein kinase (JNK) inhibitor] and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor], abrogated casticin-induced cell cycle arrest and apoptosis associated with the activation of caspases including caspase-8, -9 and -3. Endogenous p38 MAPK activation was observed in untreated cells based on the detection of the expression levels of phospho-p38 MAPK, whereas casticin did not affect the degree of p38 MAPK activation. Interestingly, the addition of SB203580 suppressed casticin-induced phosphorylation of histone H3, a downstream molecule of the p38 MAPK signaling pathway and known to be involved in chromosome condensation during mitosis. More importantly, casticin induced upregulation of intracellular ATP levels. Although casticin induced intracellular reactive oxygen species, antioxidants failed to block casticin-mediated cytotoxicity, indicating that casticin-induced cytotoxicity appears to be independent of reactive oxygen species generation. Based on the fact that SB203580 has been reported to compete with ATP for binding to the active form of p38 MAPK, and consequently blocks the p38 MAPK activity in activating downstream molecules, these results suggest that casticin induces cytotoxicity associated with apoptosis and cell cycle arrest in HL-60 cells through the p38 MAPK pathway, in which intracellular ATP levels and phosphorylation of histone H3 play critical roles.

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We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D₃ (VD₃)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

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