Induction of Apoptosis by Radix Paeoniae Alba Extract through Cytochrome c Release and the Activations of Caspase-9 and Caspase-3 in HL-60 Cells

Abstract

Radix Paeoniae Alba (RPA) is the root of Paeonia lactiflora Pallas, which belongs to the Ranunculaceae family. RPA has been commonly used in traditional medicine to tonify the blood, to alleviate pain, and to treat inflammation and cancer. A recent study reported that RPA inhibits hepatocellular carcinoma cell growth and induces their apoptosis. 1)Several constituents isolated from RPA have also been found to have immunologically active.2,3) However, details of the mechanism underlying the activity of RPA are not understood.Apoptosis is a highly regulated process that involves the activation of a series of molecular events that lead to cell death, and is characterized by morphologic changes, chromatin condensation, and apoptotic bodies, which are associated with DNA cleavage and ladder formation. 4,5) There are two major apoptotic pathways, namely, intrinsic and extrinsic apoptosis signaling, in cells responsive to apoptotic stimuli. [6][7][8][9] The intrinsic apoptosis pathway involves mitochondrial signaling, and caspase-9 has been suggested to be the predominant initiator caspase in this pathway. 7,9) In contrast, the extrinsic apoptosis pathway is mediated by death receptors, such as, Fas receptors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and the extrinsic pathway is initiated by caspase-8. 6,8) The activated initiator caspases-8 and -9 starts a proteolytic cascade by cleaving downstream effector caspases, such as, caspase-3, -6, and -7.7) Of these, caspase-3 is believed to be the main executioner caspase and its activation has been shown to be essential for both intrinsic and extrinsic apoptotic cell death. 10,11) To study the mechanism underlying the anticancer activities of RPA, we examined the antiproliferative effect of RPA extracts (RPAE) in HL-60, a human promyelocytic leukemia cell line. Our results demonstrated that RPAE induces apoptosis in a dose-dependent manner in HL-60 cells. We further demonstrate that the apoptotic effects induced by RPAE are accompanied by intrinsic pathway features, i.e., the release of cytochrome c into cytosol and the activation of procaspase-9, but not by extrinsic pathway features, and eventual caspase-3 activation. MATERIALS AND METHODS Culture ConditionsHuman leukemia HL-60 cells were purchased from the American Type Culture Collection. Cells were placed in 75 cm 2 tissue culture flasks and grown at 37°C under a humidified, 5% CO 2 atmosphere in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum and 2 mM glutamine, 10000 units/ml of penicillin, 10 mg/ml of streptomycin, and 2.5 mg/ml of amphotericin B.Preparation of Herbal Extract Radix Paeoniae Alba was identified by Min-Kyo Shin, keeper of the herbarium at the Department of Physiology, School of Oriental Medicine, Wonkwang University (Voucher samples were preserved for reference; Ref. No. Omcphy 2001-80). To produce the extract, 200 g of RPA was added to 1800 ml of water, boiled for 2 h, filtered, and concentrated to 200 ml. The sterile extract (41.97 g) was store...

Aloe-emodin, an anthraquinone, in vitro inhibits proliferation and induces apoptosis in human colon carcinoma cells

“…Thus, the inhibition of casein kinase II activity following aloe-emodin treatment may lead to the activation of caspase-3 through the phosphorylation of Bid and release of apoptosis-inducing factor and cytochrome c from mitochondria. A similar pathway was described for Radix Paeoniae Alba extract-induced apoptosis in HL-60 leukemic cells and for quercitin-mediated cell apoptosis in murine melanoma B16-BL6 cells (27,28). Ahmad et al found that the down-regulation of casein kinase II by antisense RNA elicited intracellular hydrogen peroxide production, which was associated with caspase-3 activation (29).…”

Investigation of the Chemical Changes from Crude and Processed Paeoniae Radix Alba‐Atractylodis Macrocephalae Rhizoma Herbal Pair Extracts by Using Q Exactive High‐Performance Benchtop Quadrupole‐Orbitrap LC‐MS/MS

“…Paeoniae Radix Alba nourishes blood and liver, and Atractylodis Macrocephalae Rhizoma helps invigorate spleen and eliminate dampness [8–12]. Thus, the compatibility of these two medicines could help achieve the goal of purging wood from the earth, regulating the functions of liver and spleen, benefiting qi, and nourishing blood [13–15]. Although the compositions of these two medicines have been extensively studied, the appropriate processing method of them, such as frying, which is believed by the practitioners of traditional medicine to have the effects for enhancing the efficacy of the medicine, and their underlying compatibility mechanism are still under investigation.…”

Paeonia lactiflora Pall inhibits bladder cancer growth involving phosphorylation of Chk2 in vitro and in vivo

“…It has been frequently used as an important ingredient in many traditional prescriptions and is commonly used for nourishing blood, alleviating pain, reducing irritability, as well as treating liver disease and cancer. A recent study reported that the extract of RPA inhibits the growth of hepatocellular carcinoma and HL-60 leukemic cells and induces their apoptosis (Lee et al, 2002;Kwon et al, 2006). Several constituents isolated from RPA have also been found to have immunologically activities (Tomoda et al, 1993(Tomoda et al, , 1994.…”