Elsevier

Toxicology in Vitro

Volume 23, Issue 2, March 2009, Pages 259-265
Toxicology in Vitro

Naringenin-induced apoptosis is attenuated by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human leukemia U937 cells

https://doi.org/10.1016/j.tiv.2008.12.005Get rights and content

Abstract

Naringenin, a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth and there is increasing interest in its therapeutic applications. In this study, the effect of ectopic Bcl-2 expression on naringenin-induced apoptosis was investigated. We found that Bcl-2 overexpression markedly protected human leukemia U937 cells from time- and dose-dependent induction of apoptosis by naringenin, as did caspase-3 and caspase-9 inhibitors. Additionally, Bcl-2 overexpression attenuated naringenin-induced Bax translocation and cytosolic release of cytochrome c. Our results also indicated that co-administration of HA14-1 and naringenin increased apoptosis in Bcl-2 overexpressing U937 cells by restoring mitochondrial dysfunction and activation of caspase-9 and caspase-3, as well as by cleavage of poly (ADP-ribose) polymerase. Taken together, these observations indicate that Bcl-2 confers apoptosis resistance to naringenin by inhibiting a mitochondrial amplification step in U937 cells.

Introduction

Flavonoids are a diverse group of plant natural products synthesized from phenylpropanoid and acetate-derived precursors, which play important roles in growth and development, and in defense against microorganisms and pests (Dixon and Steele, 1999). Naringenin is a flavonoid that is considered to have a bioactive effect on human health (Szejtli and Szente, 2005, Chen et al., 2007). Previous several studies have shown that naringenin inhibit CYP3A4 activity, and exhibit aorta dilatory, antioxidant, anitproliferative effects (Knowles et al., 2000, Galati et al., 2001). Stimulation with naringenin reduces HCV secretion in infected cells by 80%. Moreover, that naringenin is effective at concentrations that are an order of magnitude below the toxic threshold in primary human hepatocytes and in mice (Nahmias et al., 2008). Result of in vitro anticarcinogenesis assay indicated that naringenin, but not naringin, inhibited aflatoxin B1-induced carcinogenesis (Guengerich and Kim, 1990) and that naringenin caused cytotoxicity and apoptosis via a transient induction of caspase-3/CPP32 activity, in the human promyeloleukemia cell line HL-60 (Chen et al., 2003). Although numerous studies on the antioxidant and anticancer effects of naringenin have been reported, the cellular and molecular mechanisms underlying naringenin-induced apoptosis are not clear in human leukemia U937 cells.

New experimental evidence suggests that flavopiridol causes early mitochondrial damage in chronic lymphocytic leukemia cells was not blocked by caspase inhibitors, but was reduced by Bcl-2 overexpression (Hussain et al., 2008). Mitochondria-mediated apoptosis is regulated by the Bcl-2 family of proteins, which can promote (Bax and Bid) or inhibit (Bcl-2 and Bcl-xL) apoptosis (Dlugosz et al., 2006, Jeong and Seol, 2008). Our previous reports indicate that overexpression of Bcl-2 apparently inhibits induction of apoptosis in response to a variety of chemical agents such as esculetin through inactivation of caspases and poly(ADP-ribose)polymerase (PARP) degradation in human leukemia U937 cells (Park et al., 2008). Although the effects of Bcl-2 on the release of proapoptotic factors have been described in detail, the role of Bcl-2 in naringenin-mediated apoptosis is not clear.

In this study, we investigated naringenin-induced apoptosis in human leukemic U937 cells. We further investigated whether ectopic expression of Bcl-2 or caspases inactivation could attenuate naringenin-induced loss of MMP and apoptosis.

Section snippets

Reagents and antibodies

Propidium iodide (PI), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindile (DAPI) were obtained from Sigma chemical Co. (St. Louis, MO). Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN). An enhanced chemiluminescence (ECL) kit was purchased from GE Healthcare (Arlington Heights, IL). The caspase-3 inhibitor, z-DEVD-fmk, the caspase-9 inhibitor, z-LEHD and the specific mitochondrial dye,

Effect of naringenin and naringin on the cell growth inhibition and induction of apoptosis

To examine the effects of naringenin and its glycoside, naringin, on cell growth, we conducted a dose escalation experiment (Fig. 1A). The results of this experiment indicated that U937 cells were refractory to naringin-induced cytotoxic effects at doses as high as 500 μM, whereas treatment with 50–500 μM naringenin resulted in a 5%–80% inhibition of cell growth. Next, the growth inhibitory effects of naringenin were evaluated to determine if they were correlated with the induction of apoptosis.

Discussion

Naringenin (not to be confused with naringin) is a flavonoid that is considered to have a bioactive effect on human health as antioxidant, free radical scavenger, anti-inflammatory, carbohydrate metabolism promoter, and immune system modulater. This substance has also been shown to reduce oxidative damage to DNA in vitro. Scientists exposed cells to 80 micromoles of naringenin per liter, for 24 h, and found that the amount of hydroxyl damage to the DNA was reduced by 24% in that very short

Acknowledgments

Cheng-Yun Jin is the recipient of postdoctoral fellowship from the Ministry of Education, Science and Technology through the Brain Korea 21 Project. This study was supported by the Technology Development Program for Agriculture and Forestry (105113-03-2-SB010) and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-521-E00015), Republic of Korea.

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