Induction of apoptosis by Hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression

Hibiscus protocatechuic acid (PCA), a phenolic compound isolated from the d ried flower of Hibiscus sabdariffa L. (Malvaceae), demonstrated antioxidant and antitumor promotion effects in our previous study. In the present stud y, Hibiscus PCA was found to inhibit the survival of human promyelocytic le ukemia 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 w ith Hibiscus PCA (2 mM). Flow cytometric analysis of the DNA content of cel ls 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 tre atment caused an increase in the level of hypophosphorylated retinoblastoma (RB; 180% of control at the 6-hr time point) and, on the contrary, a decli ne in hyperphosphorylated RB. A rapid loss of RE was observed when the trea tment period was extended. Further studies showed that Hibiscus PCA applica tion reduced Bcl-2 protein expression to 47%, and increased Bax protein exp ression 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 leuk emia cells, and that RE phosphorylation and Bcl-2 protein may play a crucia l role in the early stage. BIOCHEM PHARMACOL 60;3:307-315, 2000. (C) 2000 E lsevier Science Inc.