Research Article: Analyzing bioactive effects of the minor hop compound xanthohumol C on human breast cancer cells using quantitative proteomics

Date Published: March 15, 2019

Publisher: Public Library of Science

Author(s): Simon Roehrer, Verena Stork, Christina Ludwig, Mirjana Minceva, Jürgen Behr, Suman S. Thakur.

http://doi.org/10.1371/journal.pone.0213469

Abstract

Minor prenylated hop compounds have been attracting increasing attention due to their promising anticarcinogenic properties. Even after intensive purification from natural raw extracts, allocating certain activities to single compounds or complex interactions of the main compound with remaining impurities in very low concentration is difficult. In this study, dose-dependent antiproliferative and cytotoxic effects of the promising xanthohumol (XN) analogue xanthohumol C (XNC) were evaluated and compared to XN and a XN-enriched hop extract (XF). It was demonstrated that the cell growth inhibition of human breast cancer cell line (MCF-7) significantly increases after being treated with XNC compared to XN and XF. Based on label-free data-dependent acquisition proteomics, physiological influences on the proteome of MCF-7 cells were analyzed. Different modes of action between XNC and XN treated MCF-7 cells could be postulated. XNC causes ER stress and seems to be involved in cell-cell adhesion, whereas XN influences cell cycles and DNA replication as well as type I interferon signaling pathway. The results demonstrate the utility of using quantitative proteomics for bioactivity screenings of minor hop compounds and underscore the importance of isolating highly pure compounds into their distinct forms to analyze their different and possibly synergistic activities and modes of action.

Partial Text

Hop (Humulus lupulus L.) is well known as a medical plant with many bioactive effects. Due to the trend of evaluating natural compounds for its diverse bioactive effects, hop has been attracting increasing interest as a natural resource for promising biologically active phenolic compounds. In this context, natural compounds have shown promise due to their antimicrobial, antiviral and also anticarcinogenic or (chemo)preventive effects [1, 2]. Hop contains a huge variety of prenylated phenolic compounds [3–6]. The most abundant prenylated chalcone in hops is xanthohumol (XN) [7]. Great importance has been placed on this compound due to its multiple biological activities, including anti-inflammatory [8], neuro-protective [9, 10], anti-microbial [11, 12], anticarcinogenic [13–15] and even radio-sensitizing [16] effects. Furthermore, promising results from in vivo and in vitro studies with several cancer cell lines, including MCF-7 breast cancer cells, have shown its antiproliferative activity [13–15, 17]. Xanthohumol shows co-action with several antibiotics [11, 12], but is less active in combination with other hop compounds [12] against gram-positive bacteria. Moreover, XN showed anti-obesity effects by inhibiting the differentiation of preadipocytes and inducing apoptosis in mature adipocytes by using the mouse cell line 3T3-L1 [18, 19]. All these explorations strongly suggest XN and its analogues as potential compounds for the prevention and treatment of many diseases [2, 3, 6, 7, 14, 16, 20–23]. Especially naturally occurring minor chalcones from hops gained interest due to their different investigated properties. Dietz et al. showed an efficacy improvement of bioactive hop compounds linked to the concentration of minor compounds in the natural extract [5]. Interestingly, many of these seem to be even more bioactive in comparison to XN [4, 7, 24, 25]. 8-prenylnaringenin is known as one of the most potent phytoestrogen [6], desmethylxanthohumol shows e.g. anti-oxidant activity [26], isoxanthohumol inhibits angiogenesis [27], and 6-prenylnaringenin has anti-fungal activities against Trichophyton spp. [28]. Another recently upcoming minor chalcone is xanthohumol C (XNC), which has lately received much attention due to its antiproliferative, cytotoxic [15, 17], neuro-protective [9], and anti-oxidative activities [24, 29, 30]. Xanthohumol C, also called dehydrocycloxanthohumol, was first identified by Stevens et al. [31] and its molecular structure differs to xanthohumol due to a ring closure of the prenyl-side chain with the hydroxyl group at position 4’ (see Fig 1A). Miranda et al. [15] already showed a significant antiproliferative and cytotoxic effect of various prenylated flavonoids and assumed an inhibition capacity only of XNC at lower concentrations compared to XN and iso-xanthohumol for the growth of MCF-7 breast cancer cells. In contrast, Popłoński et al. [17] recently showed a lower in vitro antiproliferative activity of XNC compared to XN in prostate, colon, and breast cancer cell lines. In another study, XNC was identified as the most active compound within a group of hop-derived prenylflavonoids for the differentiation of neuronal precursor cells [9]. While XN and other major phenolic hop compounds are already well characterized in literature, pharmacological data concerning minor compounds such as XNC are scarce due to its limited availability via isolation from natural sources and therefore is still in the process of investigation [32]. In addition, in natural products studies it is often difficult to link a certain bioactivity to a single compound due to the presence of many minor impurities and their unknown influence on the analyzed target compound.

In this study, we explored the effect of xanthohumol C (XNC) compared to xanthohumol (XN) and the commercial XN -enriched hop extract Xantho-Flav (XF) on human breast cancer cell line MCF-7 in vitro. All three showed dose-dependent antiproliferative and cytotoxic effects after 2, 4, and 6 days of incubation. Remarkably, XN was less cytotoxic than the XN -enriched extract XF, while the activity of XNC was highest. All three compounds clearly differed in their half maximal inhibitory concentration (IC50 after two days XNC: 4.18 μM, XF: 8.84 μM, XN: 12.25 μM). This promises the bioactive potential of minor prenylated hop chalcones for further applications in cancer research and indicates hop as a versatile natural resource of active minor compounds with potential anticarcinogenic effects. The results demonstrate a higher anticarcinogenic or chemopreventive potential of the studied hop compounds compared to other phenolic compounds such as resveratrol (IC50: 100μM) or curcumin (IC50: 35μM) [55]. The study indicates the need of highly pure minor compounds to clearly associate certain effects with single compounds.

 

Source:

http://doi.org/10.1371/journal.pone.0213469

 

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