Inhibition of the proteasome activity, a novel mechanism associated with the tumor cell apoptosis-inducing ability of genistein
Introduction
Genistein is one of the simplest biosynthetic isoflavonoid compounds of the Leguminosae. It is synthesized in plants from the flavanone naringenin by a novel ring migration reaction catalyzed by the cytochrome p450 enzyme isoflavone synthase (IFS) [1]. It is the predominant isoflavone phytoestrogen found in soy. Tofu, soy flour, soy milk, miso, soy sauce, tempeh, etc. are the major dietary sources of isoflavonoids for humans. It has been shown that 1 g of powdered soybean chips contains nearly 800 μg of daidzein and over 500 μg of genistein (primarily as glycosides), whereas 1 g of soy protein has approximately 150 μg of daidzein and 250 μg of genistein [1]. Bioavailability of dietary flavonoid components depends on relative uptake rates of conjugated and free forms, hydrolysis of glycosides by gut bacteria or gut wall enzymes, further metabolism, for example to glucuronides within the liver, and excretion rate. A recent animal study has shown that the principle metabolite of genistein that accumulates in the prostate is 2-(4-hydroxyphenyl)-propionic acid (2-HPPA) [2].
A growing body of epidemiological studies have shown that increased soy consumption is associated with a reduced risk of breast, colon, and prostate cancer in Asian compared with populations in the United States and Western countries [3], [4]. Animal studies also found that both a soy diet and dietary genistein itself may confer a protective effect on mammary, colon, and skin tumors [5]. Different possible mechanisms have been suggested to be responsible for genistein-mediated antitumor activities. These include inhibition of tumor cell proliferation [6], angiogenesis [7], tumor cell invasion [8], and tumor metastasis [9], as well as its antioxidant [10] and apoptosis-inducing activities [11]. Furthermore, several potential molecular targets of genistein have been suggested in the literatures, including tyrosine kinase [12], tumor necrosis factor α [13], topoisomerase II [14], the enzymes involved in steroid metabolism (such as aromatase) [15], 17β-hydroxysteroid dehydrogenase and 5α reductase [16], c-fos[17], and microsomal lipid peroxidase [18]. Like other phytoestrogens, genistein also binds to the classical ER [19]. It has been shown that genistein has higher affinity to ER beta (ERβ) than ER alpha (ERα), which suggests that genistein-mediated ERβ signaling pathways may play an important role for its biological action [19]. However, the detailed molecular mechanisms and targets of genistein remain unclear.
Effects of genistein have been extensively studied in human prostate cancer cells. It has been shown that genistein is involved in cell cycle arrest caused by down-regulation of cyclin B1 and cdc25C, up-regulation of p21Waf1[20], apoptosis induction by activation of caspase-3 [21], NF-κβ inactivation [13], and DNA strand breakage [22]. In a recent study using human prostate cancer cell lines, it has been shown that genistein up-regulates the expression of the antioxidant enzyme glutathione peroxidase gene [23]. Although it appears that genistein elicits pleiotropic molecular effects on tumor cells including prostate cancer cells, a conclusive mechanistic pathway of genistein-mediated cancer prevention has not yet been identified.
The structural and metabolic integrity of cells is maintained by proteolysis. From yeast to man, proteasome is present in all the cells, where it plays the central role for degradation of the vast majority of intracellular proteins [24]. Typically, a protein must be targeted before it is degraded by the proteasome and this targeting process is called ubiquitination. The multi-catalytic 26S proteasome is the ubiquitous proteinase in eukaryotes responsible for the degradation of intracellular proteins [24]. The 20S proteasome, a multicatalytic complex (700 kDa), constitutes the catalytic component of the ubiquitous proteolytic machinery of the 26S proteasome [25], [26], [27], [28]. It has been shown that the ubiquitin/proteasome-dependent protein degradation pathway plays an essential role to promote tumor cell proliferation and to protect tumor cells against apoptosis [25], [26], [27], [28]. It has also been shown that the chymotrypsin-like, but not trypsin-like, activity of the proteasome is associated with tumor cell survival [29], [30]. Cell proliferation and cell death regulators have been identified as targets of the ubiquitin/proteasome-mediated degradation pathway, including p53 [31], pRb [32], p21 [33], p27Kip1[34], IκB-α [35], and Bax [36]. Recently, it has been reported from our laboratory that ester bond-containing tea polyphenols, such as (−)-EGCG, potently and specifically inhibited the chymotrypsin-like activity of the proteasome in vitro (ic50 86–194 nM) and in vivo (1–10 μM) at the concentrations found in the serum of green tea drinkers [37].
Genistein consists of three rings, similar to those of A, C, and B in green tea polyphenol (−)-EGCG (Fig. 1A). Therefore, we hypothesize that genistein might be a proteasome inhibitor. Here we report that indeed genistein inhibits the chymotrypsin-like activity in purified 20S proteasome and 26S proteasome of LNCaP and MCF-7 cell extracts. Furthermore, inhibition of the proteasome by genistein in intact LNCaP and MCF-7 cells is associated with accumulation of ubiquitinated proteins and the known proteasome target proteins, including p27Kip1, IκB-α, and Bax. Following genistein-mediated proteasome inhibition, apoptosis occurred. In addition, genistein also induced p53 protein accumulation, associated with increased levels of p53 downstream target proteins such as p21Waf1. Finally, the proteasome-inhibitory and apoptosis-inducing effects of genistein were observed only in SV40-transformed human fibroblasts (VA-13), but not in their parental normal lung fibroblast counterpart (WI-38).
Section snippets
Materials
Highly purified Gen, Gen-G, Gly, Gly-G, Daid, Daid-G, and ADG were purchased from LC Laboratories and used directly without additional purification. Fetal calf serum, propidium iodide, RNase A, and DMSO were purchased from Sigma–Aldrich. RPMI 1640 medium, Dulbecco’s modified Eagle’s medium, penicillin, and streptomycin were purchased from Invitrogen. Purified 20S proteasome (rabbit) was purchased from Boston Biochem. Fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal
Computational modeling of genistein binding to the chymotrypsin site of 20S proteasome
The proteasomal β5 subunit is responsible for its chymotrypsin-like activity (cleavage after hydrophobic residues), which depends on the presence of the N-terminal Thr (Thr 1) residue [25]. In addition, an S1 pocket of the β5 subunit defined by the hydrophobic residues plays an important role in the substrate specificity and proteasome inhibitor binding [45], [46].
Most recently, we have established, for the first time, a computational molecular model that shows how an ester bond-containing
Discussion
To date, the molecular mechanism for the genistein’s cancer-preventive effects is poorly understood. In this study, we report that genistein was able to inhibit the proteasomal activity both in vitro and in vivo (Fig. 2, Fig. 3, Fig. 4). Among different soy compounds genistein was the most potent inhibitor of the proteasomal chymotrypsin-like activity (Fig. 3B). Inhibition of proteasome activity by genistein in LNCaP and MCF-7 cells was associated with increased levels of p27, IκB-α, Bax, and
Acknowledgements
This work is supported in part by research grants from the National Cancer Institute-National Institutes of Health, the United States Army Medical Research and Material Commend, and H. Lee Moffitt Cancer Center & Research Institute Drug Discovery Program, Cancer Control Division and Moffitt Foundation (to Q.P.D.).
References (61)
- et al.
Genistein
Phytochemistry
(2002) - et al.
Soy isoflavonoids and cancer—metabolism at the target site
Mutat. Res.
(2001) Effect of genistein on in vitro and in vivo models of cancer
J. Nutr.
(1995)- et al.
Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis
J. Nutr.
(1995) - et al.
Soybean isoflavones reduce experimental metastasis in mice
J. Nutr.
(1999) - et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J. Biol. Chem.
(1987) - et al.
Effect of genistein on topoisomerase activity and on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells
Biochem. Biophys. Res. Commun.
(1988) - et al.
Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens
J. Steroid Biochem. Mol. Biol.
(1993) - et al.
Inhibition of in vitro microsomal lipid peroxidation by isoflavonoids
Biochem. Pharmacol.
(1985) - et al.
The mediating role of caspase-3 protease in the intracellular mechanism of genistein-induced apoptosis in human prostatic carcinoma cell lines, DU145 and LNCaP
Biol. Cell
(2000)
Ubiquitin, proteasomes, and the regulation of intracellular protein degradation
Curr. Opin. Cell Biol.
Proteasome inhibitors as potential novel anticancer agents
Drug Resist. Updat.
p53-dependent induction of apoptosis by proteasome inhibitors
J. Biol. Chem.
Proteasome-dependent regulation of p21WAF1/CIP1 expression
Biochem. Biophys. Res. Commun.
Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo
J. Biol. Chem.
Modeling of the binding mode of a non-covalent inhibitor of the 20S proteasome. Application to structure-based analogue design
Bioorg. Med. Chem. Lett.
Proteasome inhibitors: from research tools to drug candidates
Chem. Biol.
Effects of genistein on cell proliferation and cell cycle arrest in nonneoplastic human mammary epithelial cells: involvement of Cdc2, p21(waf/cip1) p27(kip1), and Cdc25C expression
Biochem. Pharmacol.
WAF1, a potential mediator of p53 tumor suppression
Cell
Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (kinako)
J. Nutr.
Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone
J. Biol. Chem.
Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice
J. Nutr.
The role of soy products in reducing risk of cancer
J. Natl. Cancer Inst.
Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States)
Cancer Causes Control
Genistein, a dietary-derived inhibitor of in vitro angiogenesis
Proc. Natl. Acad. Sci. U.S.A.
Genistein and curcumin block TGF-beta 1-induced u-PA expression and migratory and invasive phenotype in mouse epidermal keratinocytes
Nutr. Cancer
Inhibition of tumor promoter-induced hydrogen peroxide formation in vitro and in vivo by genistein
Nutr. Cancer
Genistein elicits pleiotropic molecular effects on head and neck cancer cells
Clin. Cancer Res.
Genistein inhibits NF-kappa B activation in prostate cancer cells
Nutr. Cancer
Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids
J. Endocrinol.
Cited by (144)
-
Potency of copper(II) complexes towards drug-sensitive and -resistant Plasmodium falciparum: Structure-activity relationship, ROS-generation and proteasome inhibition
2022, Journal of Molecular StructureCitation Excerpt :As such, screening of metal complexes for differential inhibition of the proteolytic sites of a selected model proteasome (mammalian and/or Pf) can lead to potential new antimalarial compounds. Such data on selective inhibition of the various active sites of different types of proteasome will lead to greater understanding of the potency of these metal complexes towards normal human cells, cancer cells and the parasitic protozoa [72,73]. In this study, mouse 20S proteasome was used for proteasome inhibition assay.
-
Natural compounds in the regulation of proteostatic pathways: An invincible artillery against stress, ageing, and diseases
2021, Acta Pharmaceutica Sinica B -
Genistein and daidzein
2021, Nutraceuticals and Health Care -
Flavonoids as Inducers of Apoptosis and Autophagy in Breast Cancer
2021, Discovery and Development of Anti-Breast Cancer Agents from Natural Products -
Another look at phenolic compounds in cancer therapy the effect of polyphenols on ubiquitin-proteasome system
2019, European Journal of Medicinal Chemistry