International Journal of
Molecular Sciences Article
Wedelolactone Acts as Proteasome Inhibitor in Breast Cancer Cells Tereza Nehybová 1,2 , Jan Šmarda 1 , Lukáš Daniel 2,3 , Marek Stiborek 4 , Viktor Kanický 4,5 , Ivan Spasojeviˇc 6 , Jan Preisler 4,5 , Jiˇrí Damborský 2,3 and Petr Beneš 1,2, * 1
2
3 4 5 6
*
Laboratory of Cell Differentiation, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;
[email protected] (T.N.);
[email protected] (J.Š.) International Clinical Research Center, Center for Biological and Cellular Engineering, St. Anne’s University Hospital, Pekarska 53, 656 91 Brno, Czech Republic;
[email protected] (L.D.);
[email protected] (J.D.) Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic Department of Chemistry, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;
[email protected] (M.S.);
[email protected] (V.K.);
[email protected] (J.P.) CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, 11030 Belgrade, Serbia;
[email protected] Correspondence:
[email protected]; Tel.: +420-54949-3125; Fax: +420-54949-5533
Academic Editor: Toshio Morikawa Received: 1 February 2017; Accepted: 25 March 2017; Published: 29 March 2017
Abstract: Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells. Keywords: breast cancer; copper; proteasome; reactive oxygen species; wedelolactone
1. Introduction The ubiquitin-proteasome system (UPS) controls a highly complex and tightly regulated process of cellular protein degradation. In contrast to rather non-specific extracellular/membrane protein degradation by lysosomes, the proteasomes destroy proteins labelled with polyubiquitin chains. UPS consists of numerous protein components (E1, E2, E3 enzymes, proteasome, deubiquitinases). The mammalian cytosolic 26S proteasome complex contains the core 20S proteasome capped with one or two 19S regulatory subunit(s). The proteolytic activities are located in β1, β2, and β5 subunits of Int. J. Mol. Sci. 2017, 18, 729; doi:10.3390/ijms18040729
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the core 20S proteasome complex [1–3]. The eukaryotic proteasome possesses at least three distinct protease activities: chymotrypsin-like (cleavage after hydrophobic residues, located in β5 subunit), trypsin-like (cleavage after basic residues, located in β2 subunit), and caspase-like (cleavage after acidic residues, located in β1 subunit) [4]. The chymotrypsin-like activity is usually the strongest one [5]. Deregulation of UPS has severe effect on cellular function and homeostasis. Over 80% of cellular proteins are degraded via UPS including those involved in regulation of cell proliferation, differentiation, immune signaling, and cell response to stress [3,6]. Emerging evidence show that the targeting of UPS degradation pathway might be a viable anticancer strategy. Due to increased rate of genomic mutations, transformed cells accumulate large quantities of misfolded or overexpressed proteins. In response to such accumulation, malignant cells enhance the expression and activity of UPS [5,7]. Preclinical studies have confirmed a higher susceptibility of malignant cells to cytotoxic effects of UPS inhibitors when compared to normal cells [6,8]. Pertinent to this, several proteasome inhibitors have entered clinical trials, and some have already been approved for the treatment of aggressive hematopoietic tumors [9,10]. However, tumor cell resistance and high toxicity remain an issue in solid tumors leading to search for new UPS inhibitors [5]. Wedelolactone, a natural coumestan, is one of the bioactive compounds found in extracts of Eclipta alba and Wedelia calendulacea [11]. Recently, in vitro and in vivo anti-cancer properties of wedelolactone in solid tumors including breast, colon, prostate, hepatocellular, pituitary cancers, and neuroblastoma were described in a number of reports [12–19]. Wedelolactone is clearly a multi-target compound and its anti-cancer properties were primarily attributed to the inhibition of multiple kinases, androgen receptor, 5-lipoxygenase, and the c-Myc protein [13,15,17–21]. However, it was found recently that wedelolactone also inhibits topoisomerase IIα activity and blocks DNA synthesis in the breast cancer cells, and that these effects are promoted by copper ions, at least partially via redox interactions [12,22]. This study shows that wedelolactone acts as inhibitor of 20S/26S proteasome chymotrypsin-like and to lesser extent also trypsin-like and caspase-like activities. Treatment of breast cancer cells with wedelolactone resulted in accumulation of ubiquitinated proteins and proteins representing typical proteasomal targets, such as p21, p27, p53, and Bax. Molecular docking revealed a productive binding of wedelolactone to the active sites of β1, β2, and β5 proteasomal subunits with a stronger preference for β5 subunit. The proteasome inhibition by wedelolactone is not dependent on cellular copper level in breast cancer cells. This study concludes that wedelolactone acts as copper-independent inhibitor of proteasome. 2. Results 2.1. Wedelolactone Inhibits Proteolytic Activities of Proteasome in Breast Cancer Cell Lines MDA-MB-231, MDA-MB-468, and T47D cells were exposed to increasing concentrations of wedelolactone to study its effect on proteasome in breast cancer cells. Chymotrypsin-like, trypsin-like and caspase-like activities of proteasome were evaluated in cell extracts using the activity-specific fluorogenic substrates. Wedelolactone inhibited all three proteolytic activities of proteasome with the highest potency for the chymotrypsin-like activity (IC50 values 27.8 µM for MDA-MB-231, 12.78 µM for MDA-MB-468 and 19.45 µM for T47D) (Figure 1).
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Figure 1. Wedelolactone inhibitschymotrypsin-like, chymotrypsin-like, trypsin-like and caspase-like activities in breast Figure 1. Wedelolactone inhibits trypsin-like and caspase-like activities in breast cancer cells. MDA-MB-231 (A); MDA-MB-468 (B); and T47D (C) cells were treated with various cancer cells. MDA-MB-231 (A); MDA-MB-468 (B); and T47D (C) cells were treated with various concentrations of wedelolactone (w) for 10 h. Proteasome activities were evaluated in cell extracts concentrations of wedelolactone (w)chymotrypsin-like, for 10 h. Proteasome activitiesand were evaluatedactivities in cell extracts Figure 1. Wedelolactone inhibits trypsin-like caspase-like in breastusing using the activity-specific fluorogenic substrates (Suc-LLVY-AMC for testing chymotrypsin-like, cancer cells. MDA-MB-231 (A); MDA-MB-468 (B); and T47D (C) cells were treated with various the activity-specific fluorogenic substrates (Suc-LLVY-AMC for testing chymotrypsin-like, Z-LLE-AMC Z-LLE-AMC for caspase-like, and Boc-LRR-AMC for trypsin-like activities). Treatment with MG132 concentrations of wedelolactone (w) for 10 h. Proteasome activities were evaluated in cell extracts for caspase-like, Boc-LRR-AMC trypsin-like activities). Treatment served as served as a and positive control. The for data represent the mean values from with three MG132 independent usingcontrol. the activity-specific fluorogenic substrates (Suc-LLVY-AMC for testingexperiments. chymotrypsin-like, a positive The data represent the mean values from three independent Error experiments. Error bars indicate the SD. * indicates a significant (p < 0.05) difference between bars Z-LLE-AMC for caspase-like, and Boc-LRR-AMC for trypsin-like activities). Treatment with MG132 indicate the SD. * indicatesand a significant (p
