Anticancer effects of alloxanthoxyletin and fatty acids esters – In vitro study on cancer HTB-140 and A549 cells

Biomedicine & Pharmacotherapy 110 (2019) 618–630

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Anticancer effects of alloxanthoxyletin and fatty acids esters – In vitro study on cancer HTB-140 and A549 cells

T

⁎

Michał Jóźwiaka,b,c, Marta Strugab,c, , Piotr Roszkowskid, Agnieszka Filipeke, Grażyna Nowickaa,c, Wioletta Olejarza,c a

Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02-097, Warsaw, Poland Chair and Department of Biochemistry, First Faculty of Medicine, Medical University of Warsaw, 1 Banacha Street, 02-097, Warsaw, Poland c Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 1 Banacha Street, 02-097, Warsaw, Poland d Faculty of Chemistry, University of Warsaw, 1 Pasteura Street, 02-093, Warsaw, Poland e Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02-097, Warsaw, Poland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Alloxanthoxyletin derivatives Fatty acids Cytotoxicity Apoptosis Interleukin-6

Alloxanthoxyletin, a natural occurring pyranocoumarin isolated from a number of plant sources, such as family of Rutaceae, and its synthetic derivatives show cytotoxic and antitumor activities. In the present study new eleven esters of alloxanthoxyletin and fatty acids were synthesized and evaluated for their anticancer toxicity. The structures of the compounds were confirmed by Proton Nuclear Magnetic Resonance (1H NMR), Carbon-13 Nuclear Magnetic Resonance (13C NMR) and High Resolution Mass Spectrometry (HRMS) analyses. For all compounds 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxic effect on human melanoma cells (HTB-140), human epithelial lung carcinoma cells (A549) and human keratinocyte line (HaCaT). For the most active compounds (8–11) lactate dehydrogenase (LDH) assay to assess the level of cell damage as well as migration inhibition assay were performed. To explain the basic mechanism of cell death induction, the effect of derivatives 8–11 on early and late apoptosis in Annexin V-FITC/7-AAD flow cytometry analysis was investigated. The results indicate that human melanoma cells (HTB-140) and human epithelial lung carcinoma cells (A549) were more sensitive to new alloxanthoxyletin derivatives exposure compared to human keratinocytes (HaCaT). Both, the cytotoxicity and the migration tests showed a concentration-dependent inhibition of cell growth, although with a different degree of efficacy. Tested compounds induced apoptosis in cancer cells, however, derivatives 8, 9, 10 and 11 were found to be much more potent inducers of early apoptosis in HTB-140 cells than in A549 and HaCaT cells. To establish the potent mechanism of action of alloxanthoxyletin derivatives 8, 9, 10 and 11 on HaCaT, A549 and HTB-140 cells, the level of IL-6 was measured. Our results indicate, that tested compounds significantly decrease the release of IL-6 for all cancer cell lines.

1. Introduction Coumarins are natural products widely distributed in plants, mainly belonging to the Rutaceae and Apiaceae families, as well as, in many species of bacteria and fungi [1]. The plant extracts containing coumarin-related heterocycles, which were used as herbal remedies in early days, have been recognized as interesting and valuable natural products exhibiting a broad spectrum of biological activities [2–4] including properties such as antitumor [5,6], anti-inflammatory [7,8], antimicrobial [9,10]. As they are bioactive compounds of nature origin

there has also been a growing interest in their synthesis due to their useful and diverse pharmaceutical and biological properties [11,12]. Numerous efforts, including the separation and purification of naturally occurring coumarins from a variety of plants as well as artificial synthesis of novel coumarin derivatives, have been focusing on the evaluation of these compounds as potential drugs [13,14]. Pyranocoumarins are complex coumarin derivatives containing a pyran ring in their structure forming a linear arrangement (xanthyletin structure) or angular (seselin and alloxanthoxyletin structures) [15]. Some naturally occurring pyranocoumarins such as seselins and

⁎ Corresponding author at: Chair and Department of Biochemistry, First Faculty of Medicine, Medical University of Warsaw, 1 Banacha Street, 02-097, Warsaw, Poland. E-mail address: [email protected] (M. Struga).

https://doi.org/10.1016/j.biopha.2018.12.005 Received 4 September 2018; Received in revised form 28 November 2018; Accepted 2 December 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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triethylamine and appropriate acids were supplied from Sigma Aldrich. All chemicals were of analytical grade and were used without any further purification. The Nuclear Magnetic Resonance (NMR) spectra were recorded on a Varian VNMRS 300 Oxford NMR spectrometer, operating at 300 MHz for 1H NMR and at 75.4 MHz for 13C NMR. Chemical shifts (δ) were expressed in parts per million (ppm) relative to TMS. Mass spectral ESI measurements were carried out on Waters ZQ Micro-mass instruments with quadruple mass analyzer. The spectra were performed in the negative ion mode at a declustering potential of 40–60 V. The sample was previously separated on a Ultra-Performance Liquid Chromatography (UPLC) column (C18) using UPLC ACQUITYTM system by Waters connected with DPA detector. Thin Layer Chromatography (TLC) analyses were performed on silica gel plates (Merck Kiesegel GF254) and visualized using UV light or iodine vapour. Column chromatography was carried out at atmospheric pressure using Silica Gel 60 (230–400 mesh, Merck) and using dichloromethane/methanol (0–2%) mixture as eluent.

xanthyletins display cytotoxic activity [16–18] or have been reported to act as DNA-damaging agents [19]. Seselin and alloxanthoxyletin derivatives display a wide range of cytotoxic properties, for example Oaminoalkyl derivatives exhibit toxic activity against human melanoma cell line (HTB-140) [20]. Alloxanthoxyletin and avicennin were found to inhibit isoenzymes of carbonic anhydrase – one of the targets for cancer treatment [21]. Moreover, 5-methoxyseselin derivatives act as potent anti-HIV agents [22] and 2,2-dimethylpyranocoumarins show antibacterial activity [23]. Coumarin is considered unsuitable for therapeutic use due to its low solubility and high toxicity [24]. To increase the uptake of compounds through natural membrane barriers in cells and hence increasing their activity, numerous modifications of chemical structure were performed [25]. Introduction of hydrophobic fragment into the active molecule’s scaffold causes increase of non-polar solvent solubility. Fatty acids are natural products, which display antibacterial, antifungal and antitumor activity, especially unsaturated ones [26–30]. Their chain length and the level of unsaturation affects permeability of the lipid membranes [31]. They can also act as penetration enhancers as they impact the lipid model membrane by integrating within the phospholipid bilayer [32]. Considering the properties of fatty acids, their hydrophobic character, biocompatibility and antiproliferative properties, it appears prudent to use these compounds to make conjugates with pyranocoumarins to increase their lipophilicity and cytotoxicity against tumor cell lines. Studies show, that khellactone (seselin-type derivative) with attached hydrophobic hydrocarbon chain in the pyran ring structure possess anticancer activity expressing at high concentration caspase-dependent apoptosis in breast and cervical cell lines [33]. Accumulating evidence suggests, that cancer cells overexpress Interleukin 6 (IL-6) in the tumor microenvironment [34]. It was shown, that patients suffering from various cancers had increased levels of IL6 in serum samples [35]. IL-6 play an important role in cell migration, invasion, growth of malignancies [36], apoptosis progression [37], angiogenesis [38] and differentiation of tumor cells [39]. IL-6 aids tumor growth by inhibiting cancer cell apoptosis and inducting tumor angiogenesis [40] and contributes to the proliferation cancer cells especially at the advanced stage of development [41]. IL-6 has also been shown to enhance endothelial cell migration [42] a key step in angiogenesis, and dissemination of solid tumors. IL-6 has a broad spectrum of biological activity relating to regulation of inflammation, cell proliferation, immunomodulation, haematopoiesis and tumourigenesis. IL-6 transcription and downstream signalling have significant roles in melanoma progression, especially in the context of angiogenesis [43]. IL-6 is also involved in the proliferation and differentiation of lung cancer [44,45]. IL-6 has been correlated with cancer drug resistance where modulating the IL-6 pathway directly affects the cellular resistance to drug treatments. Exogenous IL-6 treatment rendered tumor cells resistant to apoptosis induced by a number of cytotoxic agents including doxorubicin and cisplatin [46]. IL-6 is also promoting tumor cell to escape cell death induced by chemotherapy drugs and increases the expression of antiapoptotic proteins. These data strongly support that IL-6 is important regulator of anti-apoptotic gene expression and drug resistance [47]. The present study focuses on the synthesis of novel conjugations of alloxantof the length and unsaturation level of hydrocarbon chain, as well as on evaluation of cytotoxicity, migration and apoptosis induction of newly obtained compounds.

2.1.1. Procedure of synthesis of esters of 5-Hydroxy-2,2,10-trimethyl-2Hpyrano[2,3-f]chromen-8-one (1–11) To a magnetically stirred at 22–23 °C solution of A (0.15 g; 0.58 mmol) and appropriate acid (0.58 mmol) in dry 1,4dioxane:CH2Cl2 mixture (2:1, 12 mL) a BOP (0.26 g; 0.58 mmol) and triethylamine (0.12 mL; 0.87 mmol) were added. The resulting solution was stirred at 22–23 °C for 1 h and concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (25 mL) and washed with 1% HClaq solution (4 x 12 mL) and distilled water (2 x 15 mL). The organic layer was dried over MgSO4 and after evaporation of the solvent under reduced pressure the product was isolated using column chromatography on silica gel and CH2Cl2:MeOH mixture (0–1% MeOH) as an eluent (1-11) (Table 1). 2.1.1.1. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl Table 1 Cytotoxic activity (IC50, μM) of tested compounds 1–11 determined by the MTT assaya,b. Compound

Cancer cells

Normal cells

HTB-140c

1 2 3 4 5 6 7 8 9 10 11 cisplatind doxorubicind

A549

HaCaT

IC50

SI

IC50

SI

IC50

41.2 ± 3.9 46.2 ± 9.5 37.3 ± 8.1 47.7 ± 6.2 50.5 ± 4.2 41.9 ± 7.9 43.4 ± 7.9 35.4 ± 5.3 32.3 ± 4.4 23.2 ± 5.1 14.4 ± 8.5 1.1 ± 0.2 0.5 ± 0.2

1.5 1.4 1.8 1.5 1.3 1.5 1.4 1.7 1.8 1.7 1.9 2.5 2.3

52.3 ± 6.4 63.0 ± 7.7 64.7 ± 8.7 66.8 ± 1.6 61.2 ± 3.4 46.8 ± 3.7 50.3 ± 5.7 39.4 ± 7.6 38.2 ± 1.2 24.9 ± 3.5 17.0 ± 3.2 1.9 ± 0.8 0.6 ± 0.2

1.2 1.1 1.1 1.1 1.1 1.3 1.3 1.5 1.6 1.6 1.6 1.5 1.7

60.6 ± 7.9 65.9 ± 8.4 67.9 ± 4.9 71.1 ± 3.6 64.3 ± 5.4 60.8 ± 3.5 62.7 ± 3.6 61.1 ± 1.4 59.9 ± 7.3 39.4 ± 2.7 26.9 ± 9.6 2.8 ± 1.1 1.1 ± 0.2

a Data are represented as IC50 (μM), i.e. the concentration of the compound that corresponds to a 50% growth inhibition of a given cell line (as compared to the control) after having cultured the cells for 72 h with the compound of interest. b Three independent experiments were performed, each in triplicate. Data are expressed as mean ± SD. The SI (Selectivity Index) was calculated for each of compound using a formula: SI = IC50 for normal cell line / IC50 cancer cell line. Compounds with SI greater or equal 1.5 for both tumor cell lines have been bolded. c Human melanoma cell line (HTB-140), Human epithelial lung carcinoma cell line (A549) and Human immortal keratinocyte cell line from adult human skin (HaCaT). d The selected reference compounds commonly used in cancer treatment.

2. Material and methods 2.1. Chemistry Dichloromethane, 1,4-dioxane, methanol, BOP ((Benzotriazol-1yloxy)tris(dimethylamino) phosphonium hexafluorophosphate), 619

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propionate (1). White solid, 155 mg (85%). Mp. 111 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 1.29 (t, J = 7.5 Hz, 3″-3 H), 1.51 (s, 2′6 H), 2.58 (d, J = 1.2 Hz, 10′-3 H), 2.65 (q, J = 7.5 Hz, 2″-2 H), 5.63 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1H,), 6.31 (d, J = 10.2 Hz, 41H,), 6.65 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 9.0 (C-3″), 24.2 (C-10′), 27.5 (C2″), 27.8 (C-2′), 78.0 (C-2), 103.4 (C-4a), 108.5 (C-10a), 110.8 (C-6), 113.9 (C-9), 115.8 (C-4), 129.2 (C-3), 148.4 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.4 (C-10), 160.3 (C-8), 171.8 (C-1″). HRMS (ESI) m/z 336.9618 (calcd for C18H18O5Na [M + Na]+, 336.9630). 2.1.1.2. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl hexanoate (2). White solid, 150 mg (72%). Mp. 63 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.94 (t, J = 6.9 Hz, 6″-3 H), 1.38–1.43 (m, 4″,5″4 H), 1.51 (s, 6H, 2′-H), 1.78 (quint, J = 7.2 Hz, 3″-2 H), 2.58–2.63 (m, 10′,2″-5 H), 5.62 (d, J = 10.2 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.31 (d, J = 9.9 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 13.9 (C-6″), 22.3 (C-5″), 24.3 (C-10′), 24.5 (C-3″), 27.9 (C-2′), 31.2 (C-4″), 34.1 (C-2″), 78.0 (C-2), 103.5 (C-4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C-9), 115.9 (C-4), 129.2 (C-3), 148.5 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.4 (C-10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 379.0415 (calcd for C21H24O5Na [M + Na]+, 379.0427).

Scheme 1. Synthesis of starting compounds.

HRMS (ESI) m/z 491.2542 (calcd for C29H40O5Na [M + Na]+, 491.2554). 2.1.1.6. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl palmitate (6). White solid, 160 mg (55%). Mp. 57 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 16″-3 H), 1.26–1.41 (m, 4″-15″24 H), 1.51 (s, 2′-6 H), 1.77 (quint, J = 7.2 Hz, 3″-2 H), 2.57–2.62 (m, 10′,2″-5 H), 5.62 (d, J = 10.2 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.31 (d, J = 9.9 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-16″), 22.7 (C-15″), 24.3 (C-10′), 24.8 (C-3″), 27.8 (C-2′), 29.1 (C-13″), 29.2 (C-12″), 29.3 (C11″), 29.4 (C-10″), 29.6 (C-9″), 29.6 (C-8″), 29.6 (C-7″), 29.7 (C-6″), 29.7 (C-4″,5″), 31.9 (C-14″), 34.2 (C-2″), 78.0 (C-2), 103.4 (C-4a), 108.5 (C-10a), 110.8 (C-6), 113.9 (C-9), 115.9 (C-4), 129.2 (C-3), 148.5 (C-5), 152.3 (C-6a), 153.4 (C-11a), 154.4 (C-10), 160.2 (C-8), 171.2 (C1″). HRMS (ESI) m/z 519.3074 (calcd for C31H44O5Na [M+Na]+, 519.3086).

2.1.1.3. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl octanoate (3). White solid, 150 mg (67%). Mp. 62 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.90 (t, J = 6.9 Hz, 8″-3 H), 1.31–1.44 (m, 4″,5″,6″,7″-8 H), 1.51 (s, 2′-6 H), 1.77 (quint, J = 7.2 Hz, 3″-2 H), 2.58–2.63 (m, 10′,2″-5 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.31 (d, J = 9.9 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.0 (C-8″), 22.6 (C-7″), 24.3 (C-10′), 24.8 (C-3″), 27.8 (C-2′), 28.8 (C-5″), 29.0 (C-4″), 31.6 (C-6″), 34.2 (C-2″), 78.0 (C-2), 103.4 (C-4a), 108.5 (C-10a), 110.8 (C-6), 113.9 (C-9), 115.9 (C-4), 129.2 (C-3), 148.5 (C-5), 152.3 (C-6a), 153.4 (C11a), 154.4 (C-10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 407.0947 (calcd for C23H28O5Na [M+Na]+, 407.0959).

2.1.1.7. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl stearate (7). White solid, 160 mg (52%). Mp. 45 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (bt, J = 6.9 Hz, 18″-3 H), 1.26–1.43 (m, 4″17″-28 H), 1.50 (s, 2′-6 H), 1.77 (quint, J = 7.2 Hz, 3″-2 H), 2.57–2.62 (m, 10′,2″-5 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.31 (d, J = 9.9 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-18″), 22.7 (C-17″), 24.2 (C-10′), 24.8 (C-3″), 27.8 (C-2′), 29.1 (C-15″), 29.2 (C-14″), 29.3 (C13″), 29.4 (C-12″), 29.6 (C-11″), 29.6 (C-10″), 29.6 (C-9″), 29.7 (C8″,7″), 29.7 (C-6″,5″,4″), 31.9 (C-16″), 34.2 (C-2″), 78.0 (C-2), 103.4 (C-4a), 108.5 (C-10a), 110.8 (C-6), 113.9 (C-9), 115.9 (C-4), 129.2 (C3), 148.5 (C-5), 152.3 (C-6a), 153.4 (C-11a), 154.4 (C-10), 160.2 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 547.3609 (calcd for C33H48O5Na [M+Na]+, 547.3621).

2.1.1.4. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl dodecanoate (4). White solid, 145 mg (57%). Mp. 46 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 12″-3 H), 1.27–1.43 (m, 4″-11″-16 H), 1.50 (s, 2′-6 H), 1.76 (quint, J = 7.5 Hz, 3″-2 H), 2.57–2.62 (m, 10′,2″-5 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.31 (d, J = 10.2 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-12″), 22.7 (C-11″), 24.3 (C-10′), 24.9 (C-3″), 27.9 (C-2′), 29.1 (C-9″), 29.2 (C-8″), 29.3 (C-7″), 29.4 (C-6″), 29.6 (C-5″,4″), 31.9 (C-10″), 34.2 (C-2″), 78.0 (C-2), 103.5 (C-4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C-9), 115.9 (C-4), 129.2 (C3), 148.5 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.5 (C-10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 463.2010 (calcd for C27H36O5Na [M+Na]+, 463.2022).

2.1.1.8. (Z)-2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5yl octadec-9-enoate (8). Colorless oil, 160 mg (53%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.90 (bt, J = 6.9 Hz, 18″-3 H), 1.29–1.43 (m, 4″7″,12″-17″-20 H), 1.53 (s, 2′-6 H), 1.77 (quint, J = 7.5 Hz, 3″-2 H), 2.00–2.06 (m, 8″,11″-4 H), 2.59–2.64 (m, 10′,2″-5 H), 5.33–5.43 (m, 9″,10″-2 H), 5.64 (d, J = 9.9 Hz, 3-1 H), 6.07 (q, J = 1.2 Hz, 9-1 H), 6.32 (d, J = 9.9 Hz, 4-1 H), 6.66 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-18″), 22.7 (C-17″), 24.3 (C-10′), 24.8 (C-3″), 27.1 (C-8″), 27.2 (C-11″), 27.9 (C-2′), 29.1 (C-15″), 29.1 (C-14″), 29.1 (C-13″), 29.3 (C-12″,7″), 29.5 (C-6″), 29.7 (C-5″), 29.8 (C-4″), 31.9 (C-16″), 34.2 (C-2″), 78.0 (C-2), 103.5 (C-4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C-9), 115.9 (C-4), 129.2 (C-3), 129.7 (C9″), 130.1 (C-10″), 148.5 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.4 (C-

2.1.1.5. 2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl tetradecanoate (5). White solid, 140 mg (51%). Mp. 52 °C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 14″-3 H), 1.26–1.43 (m, 4″-13″-20 H), 1.50 (s, 2′-6 H), 1.76 (quint, J = 7.5 Hz, 3″-2 H), 2.57–2.62 (m, 10′,2″-5 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.30 (d, J = 10.2 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-14″), 22.7 (C-13″), 24.3 (C-10′), 24.9 (C-3″), 27.9 (C-2′), 29.1 (C-10″), 29.2 (C-9″), 29.4 (C-8″), 29.5 (C-7″), 29.6 (C-6″), 29.6 (C-5″,4″), 29.7 (C-11″), 31.9 (C-12″), 34.2 (C-2″), 78.0 (C-2), 103.5 (C-4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C9), 115.9 (C-4), 129.2 (C-3), 148.5 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.5 (C-10), 160.3 (C-8), 171.2 (C-1″). 620

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Scheme 2. Synthesis of esters of alloxanthoxyletin (1–11). Fig. 1. Determination of cell death using LDH assaya. a LDH release as a marker of cell death in the HTB-140, A549 and HaCaT cells, treated for 72 h with different concentrations of compounds (10, 20, 40 and 60 μM for derivatives 8, 9, 10 and 2.5, 5, 10 and 20 μM for derivative 11). Data are expressed as the mean ± SD from three independent experiments performed in triplicate. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control.

29.1 (C-7″,6″), 29.1 (C5″), 29.6 (C4″), 34.2 (C2″), 78.0 (C-2), 103.5 (C4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C-9), 115.9 (C-4), 127.1 (C-15″), 127.8 (C-10″), 128.2 (C-12″), 128.3 (C-13″), 129.2 (C-3), 130.2 (C-9″), 132.0 (C-16″), 148.5 (C-5), 152.3 (C-6a), 153.5 (C-11a), 154.5 (C-10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 541.3129 (calcd for C33H42O5Na [M + Na]+, 541.3141).

10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 545.3447 (calcd for C33H46O5Na [M + Na]+, 545.3459).

2.1.1.9. (9Z,12Z,15Z)-2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f] chromen-5-yl octadeca-9,12,15-trienoate (9). Colorless oil, 160 mg (53%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.97 (t, J = 7.5 Hz, 18″3 H), 1.35–1.42 (m, 4″-7″-8 H), 1.50 (s, 2′-6 H), 1.76 (quint, J = 7.5 Hz, 3″-2 H), 1.98–2.12 (m, 8″,17″-4 H), 2.57–2.62 (m, 10′,2″-5 H), 2.72–2.83 (m, 11′,14″-4 H), 5.27–5.46 (m, 9″,10″,12″,13″,15″16″6 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.30 (d, J = 10.2 Hz, 4-1 H), 6.64 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.3 (C-18″), 20.6 (C-17″), 24.3 (C-10′), 24.8 (C-3″), 25.5 (C14″), 25.6 (C11″), 27.2 (C-8″), 27.9 (C-2′),

2.1.1.10. Conjugated linoleic acids (CLA) – mixture of isomers: (9Z,11E)2,2,10-trimethyl-8-oxo-2,8-dihydropyrano[2,3-f]chromen-5-yl octadeca9,11-dienoate and (10E,12Z)-2,2,10-trimethyl-8-oxo-2,8-dihydropyrano [2,3-f]chromen-5-yl octadeca-10,12-dienoate (10). Colorless oil, 170 mg (56%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.86-0.92 (m, 18″-3 H), 1.27–1.40 (m, 4″-7″,14″-17″-16 H), 1.51 (s, 2′-6 H), 1.77 (quint, J = 7.5 Hz, 3″-2 H), 2.06–2.17 (m, 8″,13″-4 H), 2.57–2.62 (m, 621

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Fig. 2. The effect of compounds 8, 9, 10 and 11 on HaCaT cell migration.

J = 9.9 Hz, 3-1 H), 6.05 (q, J = 1.2 Hz, 9-1 H), 6.30 (d, J = 10.2 Hz, 4-1 H), 6.63 (s, 6-1 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.2 (C-22″), 20.5 (C-21″), 22.6 (C-20″), 24.2 (C-10′), 25.5 (C-9″), 25.6 (C-12″), 25.6 (C-15″), 25.6 (C6″,18″), 27.8 (C-2′), 34.0 (C-2″), 78.0 (C-2), 103.4 (C-4a), 108.5 (C10a), 110.7 (C-6), 114.0 (C-9), 115.9 (C-4), 126.9 (C-19″), 127.1 (C-5″), 127.8 (C-7″), 127.8 (C-8″), 128.0 (C-10″,11″), 128.2 (C-13″,14″), 128.4 (C-16″), 128.5 (C-17″), 129.2 (C-3), 130.1 (C-4″), 132.0 (C-20″), 148.4 (C-5), 152.3 (C-6a), 153.4 (C-11a), 154.4 (C-10), 160.2 (C-8), 171.5 (C1″). HRMS (ESI) m/z 591.3716 (calcd for C37H44O5Na [M+Na]+, 591.3728).

10′,2″-5 H), 5.25–5.35 (m, 12″-1 H), 5.62 (d, J = 9.9 Hz, 3-1 H), 5.63–5.71 (m, 9″-1 H), 5.91–5.99 (m, 11″-1 H), 6.05 (q, J = 1.2 Hz, 91 H), 6.25–6.35 (m, 10″-1 H), 6.30 (d, J = 9.9 Hz, 4-1 H), 6.64 (s, 61 H). 13 C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-18″), 14.1 (C-18″), 22.5 (C-17″), 22.6 (C-17″), 24.3 (C-10′), 24.8 (C-3″), 27.6 (C-8″), 27.7 (C13″), 27.9 (C-2′), 28.9 (C-4″), 29.0 (C-14″), 29.1 (C-14″), 29.2 (C-7″), 29.3 (C-7″), 29.4 (C-6″), 29.4 (C-6″), 29.6 (C-5″), 29.7 (C-5″), 31.5 (C15″), 31.7 (C-15″), 32.8 (C-16″), 32.9 (C-16″), 34.2 (C-2″), 78.0 (C-2), 103.5 (C-4a), 108.5 (C-10a), 110.8 (C-6), 114.0 (C-9), 115.9 (C-4), 125.5 (C-10″), 125.7 (C-10″), 128.5 (C-11″), 128.7 (C-11″), 129.2 (C-3), 129.8 (C-9″), 130.2 (C-9″), 134.5 (C-12″), 134.8 (C-12″), 148.5 (C-5), 152.3 (C-6a), 153.4 (C-11a), 154.4 (C-10), 160.3 (C-8), 171.2 (C-1″). HRMS (ESI) m/z 543.3288 (calcd for C33H44O5Na [M+Na]+, 543.3300).

2.2. Cytotoxicity assessment 2.2.1. Cell cultures Human immortal keratinocyte cell line from adult human skin (HaCaT), Human melanoma cell line (HTB-140) and Human epithelial lung carcinoma cell line (A549) were purchased from American Type Culture Collection (Rockville, USA), and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% antibiotics (penicillin and streptomycin) and 10% heat-inactivated FBS-fetal bovine serum (Gibco Life Technologies, USA) at 37 °C and 5% CO2

2.1.1.11. (4Z,7Z,10Z,13Z,16Z,19Z)-2,2,10-trimethyl-8-oxo-2,8dihydropyrano[2,3-f]chromen-5-yl docosa-4,7,10,13,16,19-hexaenoate (11). Colorless oil, 190 mg (58%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.96 (t, J = 7.5 Hz, 22″-3 H), 1.50 (s, 2′-6 H), 2.06 (quint, J = 7.5 Hz, 3″-2 H), 2.49–2.58 (m, 10′,2″-5 H), 2.65–2.70 (m, 21″2 H), 2.78–2.89 (m, 6″,9″,12″,15″,18″-10 H), 5.25–5.53 (m, 4″,5″,7″,8″,10″,11″,13″, 14″,16″,17″,19″20″-12 H), 5.61 (d, 622

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Fig. 3. The effect of compounds 8, 9, 10 and 11 on HTB-140 cell migration.

2.2.3. LDH assay – cell damage assessment As a marker of cell death the release of lactate dehydrogenase (LDH) from the cytosol to culture medium (cellular membrane integrity assessment) was used. The assay was performed after 72 h incubation of cells in 96-well plates with investigated compounds as described before [20,25]. The activity of lactate dehydrogenase (LDH) released from cytosol of damaged cells to the supernatant was measured according to the protocol of the cytotoxicity detection kit LDH test described by the manufacturer (Roche Diagnostics, Germany). An absorbance was measured at 490 nm using a microplate reader (using Epoch microplate reader, BioTek Inc., USA) equipped with Gen5 software (BioTech Instruments, Inc., Biokom). Compounds mediated cytotoxicity expressed as the LDH release (%) was determined by the following equation: [(A test sample − A low control)/(A high control − A low control)] × 100% (A-absorbance); where “low control” were cells in DMEM or FGM with 2% FBS without tested compounds and “high control” were cells incubated in DMEM or FGM with 2% FBS with 1% Triton X-100 (100% LDH release).

atmosphere. Cells were passaged using trypsin-EDTA (Gibco Life Technologies, USA) and cultured in 96-well plates (1 × 104 cells per well). Experiments were conducted in DMEM with 2% FBS.

2.2.2. MTT assay – cell viability assessment The cell viability was assessed by determination of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) conversion by mitochondrial dehydrogenase [20], [25]. The cells were incubated for 72 h in 96-well plates with different concentrations of tested compounds and subsequently for another 4 h with 0.5 mg/mL of MTT solution, which was converted in living cells by mitochondrial dehydrogenase into insoluble formazan. The dye was then solubilized in 0.04 M HCl in absolute isopropanol. Absorbance of solubilized formazan was measured spectrophotometrically at 570 nm (using Epoch microplate reader, BioTek Inc., USA) equipped with Gen5 software (BioTech Instruments, Inc., Biokom). Cell viability was presented as a percent of reduced MTT in treated cells versus control cells (incubated in serum-free DMEM without tested compounds). The relative MTT level (%) was calculated as [A]/[B] × 100, where [A] is the absorbance of the test sample and [B] is the absorbance of control sample containing the untreated cells. Decreased relative MTT level indicates decreased cell viability.

2.2.4. Cell migration assay Cells were seeded in 96-well plates, next after reaching a 70% confluence a cutting line in the middle of the plate was made and the tested compounds were added at two (non-toxic to cells) concentrations 623

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Fig. 4. The effect of compounds 8, 9, 10 and 11 on A549 cell migration.

– 20 μM and 40 μM. Cells were incubated for 72 h and then a microscope Nikon Eclipse TS 100 pictures were taken. The total number of cells and the number of cells that crossed the cutting line were counted and the percent of inhibition of cell migration as compared to control (without the tested compounds) was calculated.

AAD negative were identified as early apoptotic. Cells which were Annexin V:FITC positive and 7-AAD positive were identified as late apoptotic or necrotic. 2.2.7. Interleukin-6 analysis Interleukin IL-6 ELISA kit was purchased from Diaclon SAS (Besancon Cedex, France). HaCaT, A549 and HTB-140 cells were treated with IC50 concentration of alloxanthoxyletin derivatives 8, 9, 10, 11 and with IC50 cytostatic drugs for 72 h. IL-6 level in cell culture supernatant was measured using enzyme-linked immunosorbent assay in accordance with the manufacturer's protocol.

2.2.5. In vitro drug sensitivity of cells assessment Cells were preincubated with tested compounds for 24 and 48 h and then incubated with a chemotherapeutic agent for a total time of treatment 72 h. The cell viability was determined by MTT assay for the IC50 concentrations of the compounds as compared to 72 h exposition to cisplatin or doxorubicin.

2.2.8. Statistical analysis The results are expressed as the mean ± SD from the indicated number of experiments. Comparisons were made using Student’s t-test. Differences between experimental groups were considered to be statistically significant at p ≤ 005.

2.2.6. Annexin V binding assay – apoptosis assessment To assess apoptosis a commercially available kit (FITC:Annexin V Apoptosis Detection Kit I; BD Biosciences Pharmingen) was used. Cells were preincubated for 72 h with IC50 concentrations of tested compounds. The effect of the exposure of HaCaT, HTB-140 and A549 cells to tested compounds was determined by dual staining with Annexin V:FITC and 7-AAD. Annexin V:FITC and 7-AAD were added to the cellular suspension as described in the manufacturer’s instructions and a fluorescence sample of 10,000 cells was analyzed by flow cytometry (Becton Dickinson). Cells which were Annexin V:FITC positive and 7-

3. Results and discussion 3.1. Chemistry The starting compound (A) was obtained in the reaction of 5,7624

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Fig. 5. Effect of compounds 8, 9, 10 and 11 on cisplatin and doxorubicin activities on HTB-140, A549 and HaCaT cells – sensitivity testsa. a Cell viability was assessed by MTT mitochondrial conversion in cells treated with IC50 concentration of acids: A8, A9, A10, A11 and alloxanthoxyletin derivatives: 8, 9, 10, 11 for 24 h (24) and 48 h (48) (upper row) followed by an exposition of cells for 48 h and 24 h to cisplatin (C) (Fig. 5.a.) and doxorubicin (D) (Fig. 5.b.), respectively (lower row). The relative MTT level (%) was calculated as [A]/[B]×100, where [A] is the absorbance of the test sample and [B] is the absorbance of control sample containing the untreated cells. Decreased relative MTT level indicates decreased cell viability. Data are expressed as means ± SD. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to cisplatin or doxorubicin (72 h treatment).

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Fig. 6. The effect of compounds 8, 9, 10 and 11 on early and late apoptosis or necrosis in HaCaT cells as detected by flow cytometrya. a Cells were incubated for 72 h with compounds 8, 9, 10 and 11 used in their IC50 concentrations, then cells were harvested, stained with Annexin V-FITC and 7-AAD, and analyzed using flow cytometry. a) Data are expressed as % of cells at early stage of apoptosis and as % of cells at late stage apoptosis or necrotic cells. Data are expressed as means ± SD. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control. b) Diagrams of FITC-Annexin V/7-AAD flow cytometry. The lower right quadrant represent early apoptotic cells (Annexin V-FITC positive and 7-AAD negative staining). The upper right and upper left quadrants contain late stage apoptotic cells or necrotic cells (Annexin VFITC positive and 7-AAD positive and Annexin V-FITC negative and 7-AAD positive staining, respectively).

normal cells: human immortal keratinocyte cell line from adult human skin (HaCaT) and two tumor cell lines: human melanoma cell line (HTB-140) and human epithelial lung carcinoma cell line (A549). The cells viability was determined by MTT assay. As reference compounds, two commonly used antitumor drugs cisplatin and doxorubicin were used. The cytotoxic activity was established by determination of the inhibitory concentration IC50 for all derivatives, which is defined as the concentration of a compound that corresponds to a 50% cells’ viability and growth inhibition compared to the control. In addition, a cytotoxic capacity against tumor cells for each tested compound was determined and expressed as a selectivity factor (Selectivity Index, SI) [48]. SI value for compounds having higher toxicity against the tumor cells than against normal cells exceeds 1.0. Tested alloxanthoxyletin derivatives (1–11) were less toxic to all three evaluated cell lines (HTB-140, A549 and HaCaT) than selected positive reference compounds (cisplatin and doxorubicin) and displayed higher cytotoxic activity against tumor cells than against normal cells (in all cases SI > 1.0). Saturated fatty acid derivatives (1–7) were less active against studied cell lines than unsaturated (8–11) with IC50 within the range of 37.3–71.1 μM and 14.1–61.1 μM, respectively. HTB140 cells were more sensitive to saturated hydrocarbon chain compounds (37.3–50.5 μM) than A549 cells (46.8–66.8 μM) as well as to unsaturated: 14.4–35.4 μM vs 17.0–39.4 μM, respectively. No correlation between the length of saturated chain and cytotoxic activity was

dihydroxy-4-methylcoumarin with 4,4-dimethoxy-2-methylbutan-2-ol (Scheme 1). As a result of this reaction, three products were obtained. Application of the two-step crystallization allowed two products: 5Hydroxy-2,2,10-trimethyl-2H-pyrano[2,3-f]chromen-8-one (A) from benzene and 5-Hydroxy-4,8,8-trimethyl-8H-pyrano[2,3-f]chromen-2one from methanol (B). The excess of 4,4-dimethoxy-2-methylbutan-2ol used in the reaction yields dipetalolactone as an unwanted [20]. In the next step the corresponding ester derivatives 1–11 were obtained by the reaction of the hydroxyl group of alloxanthoxyletin with appropriate fatty acid (Scheme 2). The synthesis was carried out under room temperature in a solvents mixture of 1,4-dioxane/CH2Cl2 and BOP ((Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate) as a coupling reagent. Structures of compounds were determined using different spectroscopic methods (1H NMR, 13C NMR and HRMS). Spectral data (NMR, HRMS) of all compounds were in full agreement with the proposed structures. 3.2. Biological screening 3.2.1. Cytotoxic activity The aim of this study was to assess cytotoxic activities of the obtained alloxanthoxyletin derivatives. Relevant tests were conducted on 626

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Fig. 7. The effect of compounds 8, 9, 10 and 11 on early and late apoptosis or necrosis in HTB140 cells as detected by flow cytometrya. a Cells were incubated for 72 h with compounds 8, 9, 10 and 11 used in their IC50 concentrations, then cells were harvested, stained with Annexin V-FITC and 7-AAD, and analyzed using flow cytometry. a) Data are expressed as % of cells at early stage of apoptosis and as % of cells at late stage apoptosis or necrotic cells. Data are expressed as means ± SD. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control. b) Diagrams of FITC-Annexin V/7-AAD flow cytometry. The lower right quadrant represent early apoptotic cells (Annexin V-FITC positive and 7-AAD negative staining). The upper right and upper left quadrants contain late stage apoptotic cells or necrotic cells (Annexin VFITC positive and 7-AAD positive and Annexin V-FITC negative and 7-AAD positive staining, respectively).

approach because these compounds delay tissue invasion and the formation of metastases by cancer cells emerging from primary tumor sites [49–51]. The most active derivatives (8–11) were used in two concentrations (20 μM and 40 μM) to examine the inhibition of cell migration. As shown in Figs. 2–4, compounds 8, 9, 10 and 11 significantly (p < 0.001) inhibited cell migration. Compounds 8, 9, 10 and 11 at a concentration of 20 μM after 72 h inhibited the HaCaT cell migration on average by 44.2%. Tumor cell migration was inhibited on average by 9.8% more than normal keratinocyte cell line in HTB-140 cells, and by 20.5% more in A549 cells. When compounds were used at 40 μM concentration the HaCaT cells migration was inhibited on average by 58.0%. HTB-140 cell migration was inhibited by 10.2% and 15.9% more than normal cells in HTB-140 and A549 cells, respectively (Figs. 2–4).

established. The increasing level of unsaturation of hydrocarbon chain for compounds 8–11 was associated with enhanced compound’s activity. Derivatives 8, 9, 10 and 11 showed the highest cytotoxic potential against HTB-140 cells with IC50 of 14,4–35,4 μM, with compound 11 being the most effective. LDH assay was performed for unsaturated fatty acid derivatives (8–11), as they expressed the highest cytotoxic potential against tumor cells. All four tested compounds were used in four concentrations: 10, 20, 40 and 60 μM, and three independent experiments were performed for each cell line in triplicate. DHA derivative (11) was additionally tested in 5 and 2.5 μM concentration because of its high activity. Alloxanthoxyletin derivatives expressed grater cytotoxic effect against tumor cell lines than against normal keratinocyte cells (HaCaT). However, the effect was stronger in case of melanoma cell line (HTB140) compared to lung carcinoma cell line (A549). Compound 8 showed statistically significant effect against both tumor cell lines when used in the concentration of 40 μM, compounds 9 and 10 were cytotoxic in concentration of 20 μM and 11 when 10 μM concentration was used (Fig. 1).

3.2.3. In vitro drug sensitivity Interaction of biologically active agents describes the effect that results from the presence of several agents at the same time. The purpose of using the combination of cytotoxic drugs in the treatment of cancer or infections is to lower doses of each drug to obtain better therapeutic effects with less side-effects including toxicity. In this study

3.2.2. Cell migration inhibition The design of anti-migratory compounds is a particularly promising 627

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Fig. 8. The effect of compounds 8, 9, 10 and 11 on early and late apoptosis or necrosis in A549 cells as detected by flow cytometrya. a Cells were incubated for 72 h with compounds 8, 9, 10 and 11 used in their IC50 concentrations, then cells were harvested, stained with Annexin V-FITC and 7-AAD, and analyzed using flow cytometry. a) Data are expressed as % of cells at early stage of apoptosis and as % of cells at late stage apoptosis or necrotic cells. Data are expressed as means ± SD. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control. b) Diagrams of FITC-Annexin V/7-AAD flow cytometry. The lower right quadrant represent early apoptotic cells (Annexin V-FITC positive and 7-AAD negative staining). The upper right and upper left quadrants contain late stage apoptotic cells or necrotic cells (Annexin VFITC positive and 7-AAD positive and Annexin V-FITC negative and 7-AAD positive staining, respectively).

Fig. 9. Effects of alloxanthoxyletin derivatives (8, 9, 10, 11), cisplatin (C) and doxorubicin (D) on IL-6 levelsa. a IL-6 levels in culture supernatant were measured by ELISA. Data are expressed as the mean ± SD from three independent experiments performed in triplicate. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control.

a chemotherapeutic drug. In addition, free fatty acids (A8, A9, A10, A11) used to obtain compounds 8-11 were also tested to confirm the effect of alloxanthoxyletin derivatives using the same methodology. The cell viability was determined by MTT assay; the tested compounds were used in IC50 concentrations.

in vitro drug sensitivity testing on HTB-140, A549 and HaCaT cells was performed. To examine the effect of derivatives 8, 9, 10 and 11 on altering cells’ sensitivity to cisplatin and doxorubicin the cells were preincubated with these derivatives in two variants: for 24 h and 48 h; then incubated with

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effect of reference compounds used – cisplatin or doxorubicin.

Preincubation of cells with compounds 8, 9, 10 and 11 increased a sensitivity of tumor cells to a tested cytostatic drug through induction of cell death after 24 h derivative- and 48 h drug- treatment. Under such conditions, normal keratinocyte cell line (HaCaT) expressed increased viability during cisplatin and doxorubicin treatment for all tested derivatives. There was no clear correlation between the tested alloxanthoxyletin derivatives and cell drug sensitivity change during 24 h compound exposition followed by 48 h drug treatment in case of all three cell lines. HaCaT cells showed higher viability during this treatment than HaCaT cells exposed to cisplatin or doxorubicin for 72 h. The strongest effects on cisplatin and doxorubicin cytotoxic activities were found for compound 8 (oleic acid derivative) for 48 h derivative- and 24 h drug-treatment. The free fatty acid preincubation (A8–A11) caused increased viability of all tested cells (HaCaT, HTB-140 and A549) as compared to corresponding derivatives (8–11). Using oleic acid (A8) for 24 h followed by 48 h cell drug exposition did not affect the cisplatin cytotoxicity against tumor cells significantly (HTB-140 from 49.1% to 45.7%; A549 from 50.8% to 62.9%), as well as doxorubicin cytotoxicity (HTB-140 from 50.0% to 44.9%; A549 from 54.9% to 54.9%), but significantly decreased the effect of these drugs on normal cells (viability increased from 51.1% to 84.7% for cisplatin and from 51.8% to 81.5% for doxorubicin). Acids A9, A10 and A11 treatment resulted in higher drug cytotoxicity for all tested cell lines (Fig. 5). The 48 h free fatty acid pretreatment (A8–A11) followed by 24 h drug treatment (cisplatin and doxorubicin) resulted in higher viability of normal keratinocyte cell line than tumor cell lines. Pretreatment cells with acids A8 and A11 for 48 h before 24 h drug treatment did not cause significant change of HaCaT cells viability as compared to drug exposure itself, but had a stronger cytotoxic effect in tumor cells. Acids A9 and A10, in general, did not increase the cytotoxicity to tumor cells but decreased in normal cells for cisplatin and doxorubicin. The DHA fatty acid (A11) 48 h pretreatment and 24 h doxorubicin treatment resulted in lower tumor cell viability as referred to drug-only treatment in HTB-140 cells (viability decreased from 50.0% to 25.7%) and A549 cells (decrease from 54.9% to 32.2%), and higher normal keratinocyte cell line viability (increase from 51.8% to 68.9%).

4. Conclusion In conclusion, the results of this study clearly indicate that derivatives 8, 9, 10 and 11 showed the highest cytotoxic potential against HTB-140 cells with IC50 of 14,4–35,4 μM, with compound 11 being the most effective (MTT assay). The increasing level of unsaturation of hydrocarbon chain for compounds 8–11 resulted in increased compound’s activity. Alloxanthoxyletin derivatives expressed grater cytotoxic effect (LDH assay) against tumor cell lines than on normal cells (HaCaT). However, the effect was stronger in case of melanoma cell line (HTB-140) compared to lung carcinoma cell line (A549). Compound 8 showed statistically significant effect against both tumor cell lines when used in the concentration of 40 μM, compounds 9 and 10 in concentration of 20 μM and 11 when 10 μM concentration was used. Compounds 8, 9, 10 and 11 significantly inhibited cell migration of all cell lines. However, stronger inhibitory effects were observed in cancer cells (HTB-140 and A549) than in normal cells (HaCaT). Preincubation of cells with compounds 8, 9, 10 and 11 increased a sensitivity of tumor cells to a tested cytostatic drug through indication of cell death and decreased sensitivity of normal cells (HaCaT). Derivatives 8, 9, 10 and 11 appeared to be more potent inducers of early apoptosis in tumor cells (HTB-140 and A549 cell lines) than in HaCaT cells. The present study demonstrated that alloxanthoxyletin derivatives 8, 9, 10 and 11 treatment reduced secretion of IL-6 by HaCaT, A549 and HTB-140 cells. Our results indicate possible suppressive effect alloxanthoxyletin derivatives on cancer cells. Declaration of interest The authors declare no competing financial interest. Acknowledgments This work was supported by the Polish National Science Centre under the grant PRELUDIUM number 2017/25/N/NZ7/01583 and the statutory founds of Department of Biochemistry and Pharmacogenomics and Chair and Department of Biochemistry, Medical University of Warsaw. This work was supported by the Medical University of Warsaw and carried out with the use of CePT infrastructure financed by the European Union – the European Regional Development Fund within the Operational Programme Innovative Economy for 2007–2013.

3.2.4. Apoptosis induction assessment To explain the basic mechanism of cell death induction, the effect of derivatives 8, 9, 10 and 11 on early and late apoptosis using Annexin VFITC/7-AAD flow cytometry analysis was investigated. Derivatives 8, 9, 10 and 11 appeared to be more potent inducers of early apoptosis in tumor cells (HTB-140 and A549 cell lines) than in HaCaT cells (Figs. 6–8). The % of total cell population in early apoptosis is within the range of 21.3–27.1% for normal cells in comparison to 42.3–52.8% for HTB-140 and 39.3–48.9% for A549 cells. Despite the high percentage of cells being in the stage of early apoptosis, tested compounds do not affect the level of late apoptosis or necrosis in HTB140 cells significantly (14.7–18.3%) as compared to normal keratinocytes (16.7–18.4%). A549 cells apart from the high index of early apoptosis, showed a high index of cells being at late apoptotic or necrotic state (36.0–54.2%), which was 2x higher from HTB-140 or HaCaT cells (Figs. 6–8). Compound 11 in case of A549 cell line caused over 10% higher late apoptosis or necrosis compared to early apoptosis induction than compounds 8, 9 and 10 (Fig. 8).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.12.005. References [1] T.M. Pereira, D.P. Franco, F. Vitorio, A.E. Kummerle, Coumarin compounds in medicinal chemistry: some important examples from the last years, Curr. Top. Med. Chem. 18 (2) (2018) 124–148. [2] A.M. Katsori, H.-L. Dimitra, Coumarin derivatives: an updated patent review (2012 – 2014), Expert Opin. Ther. Pat. 24 (12) (2014) 1323–1347. [3] M.J. Matos, L. Santana, E. Uriarte, O.A. Abreu, E. Molina, E.G. Yordi, R. Venketeshwer (Ed.), “Coumarins — An Important Class of Phytochemicals,” in Phytochemicals - Isolation, Characterisation and Role in Human Health, InTech, 2015. [4] F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity, Curr. Med. Chem. 12 (8) (2005) 887–916. [5] S.A. Morsy, A.A. Farahat, M.N.A. Nasr, A.S. Tantawy, Synthesis, molecular modeling and anticancer activity of new coumarin containing compounds, Saudi Pharm. J. 25 (6) (2017) 873–883. [6] S. Emami, S. Dadashpour, Current developments of coumarin-based anti-cancer

3.2.5. Interleukin-6 analysis Treatment with alloxanthoxyletin derivatives 8, 9, 10, 11 reduced the Interleukin-6 level in culture supernatant by A549 and HTB-140 cells (Fig. 9). All tested compounds (8–11) significantly inhibited the release of IL-6 as compared to the control. The strongest effect was observed for derivative 11, which inhibited IL-6 release by 91.8% for HTB-140 cell line and by 91.4% for A549 cell line. This effect was greater than the 629

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