Hibiscus protocatechuic acid (PCA), a phenolic compound isolated from the dried flower of Hibiscus sabdariffa L. (Malvaceae), demonstrated antioxidant and antitumor promotion effects in our previous study. In the present study, Hibiscus PCA was found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration- and time-dependent manner. The study revealed that HL-60 cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after a 9-hr treatment with Hibiscus PCA (2 mM). Flow cytometric analysis of the DNA content of cells treated with PCA for 12 hr showed that the cells were distributed mainly in the hypodiploid phase (apoptotic peak, 46.7%), less in the G(1) (34.2%) and S phase (14.0%), and few in the G(2)/M phase (5.1%). Moreover, PCA treatment caused an increase in the level of hypophosphorylated retinoblastoma (RB; 180% of control at the 6-hr time point) and, on the contrary, a decline in hyperphosphorylated RB. A rapid loss of RB was observed when the treatment period was extended. Further studies showed that Hibiscus PCA application reduced Bcl-2 protein expression to 47%, and increased Bax protein expression to 181% after 1.5 hr as compared with time 0. Overexpression of Bcl-2 in HL-60 cells delayed the occurrence of Hibiscus PCA-induced apoptosis. These data suggest that Hibiscus PCA is an apoptosis inducer in human leukemia cells, and that RB phosphorylation and Bcl-2 protein may play a crucial role in the early stage.
