18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways

Abstract

Abstract:In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC 50 value of 100 µM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (∆Ψ m ) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL‐60 cancer cell xenograft growth in vivo

“…Human leukemia cell line (HL‐60) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and maintained in suspension culture in RPMI‐1640 (GIBCO/Invitrogen Life Technologies, Carlsbad, California) containing 10% heat‐inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, Utah), 2 mM l ‐glutamine and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) (Sigma‐Aldrich, Chemical Co.) in a 75 cm 2 tissue culture flasks in a humidified atmosphere of 5% CO 2 incubator at 37°C …”

“…Cells were resuspended in Annexin V binding buffer, followed by incubation with Annexin V‐FITC/PI in the dark for 15 minutes as the manufacturer's instructions for labeling of apoptotic cells . At 10 000 cells from each treatment were analyzed using BD FACSCalibur (BD Biosciences) for percentage of cell apoptosis as described previously …”

Combinational treatment of all‐trans retinoic acid (ATRA) and bisdemethoxycurcumin (BDMC)‐induced apoptosis in liver cancer Hep3B cells

“…All membranes were detected by the proper primary antibodies (1:1,000) overnight at 4°C, and reacted with HRP-conjugated secondary antibody (1:5,000), and immunoreactivities were detected by chemiluminescence reagents (Millipore Corp., Billerica, MA, USA) as described previously (Huang et al, 2016;Li et al, 2017;Ni et al., 2012). Cells were collected and lysed in lysis buffer containing 1% protease inhibitor mixtures (Sigma-Aldrich Corp.).…”

“…The extracted proteins were loaded onto an SDS-polyacrylamide gel and then transferred to a PVDF membrane (Merck KGaA, Darmstadt, Germany). All membranes were detected by the proper primary antibodies (1:1,000) overnight at 4°C, and reacted with HRP-conjugated secondary antibody (1:5,000), and immunoreactivities were detected by chemiluminescence reagents (Millipore Corp., Billerica, MA, USA) as described previously (Huang et al, 2016;Li et al, 2017;Ni et al, 2012).…”

The Mechanistic Antitumor Study of Myricanol 5-Fluorobenzyloxy Ether in Human Leukemic Cell Hl-60

“…Further, it significantly upregulated the expression levels of caspase-9, Bax, Fas, FasL and p21, and downregulated the levels of Bcl-2 and survivin in a dose-dependent manner. These changes associated with apoptosis [26,27].…”