Phytochemistry 140 (2017) 108e117
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Neolignans isolated from Nectandra leucantha induce apoptosis in melanoma cells by disturbance in mitochondrial integrity and redox homeostasis Fernanda S. de Sousa a, Simone S. Grecco b, Natalia Girola c, Ricardo A. Azevedo d, ~o Henrique G. Lago a, b, * Carlos R. Figueiredo c, **, Joa ~o Paulo, Sa ~o Paulo, 09972-270, Brazil Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sa Center of Natural Sciences and Humanities, Federal University of ABC, Santo Andre, 09210-580, Brazil ~o Paulo, Sa ~o Paulo, 04021-001, Brazil Department of Microbiology, Immunology and Parasitology, Federal University of Sa d ~o Paulo, Sa ~o Paulo, 05508-900, Brazil Laboratory of Tumor Immunology, Institute of Biomedical Sciences, University of Sa a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2017 Received in revised form 27 April 2017 Accepted 29 April 2017
Six neolignans including three previously undescribed metabolites: 1-[(7R)-hydroxy-8-propenyl]-3-[30 methoxy-10 -(80 -propenyl)-phenoxy]-4,5-dimethoxybenzene, 4-hydroxy-5-methoxy-3-[30 -methoxy-10 (80 -propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene and 4,5-dimethoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene were isolated from twigs of Nectandra leucantha Nees & Mart (Lauraceae) using bioactivity-guided fractionation. Cytotoxic activity of isolated compounds was evaluated in vitro against cancer cell lines (SK BR-3, HCT, U87-MG, A2058, and B16F10), being dehydrodieugenol B and 4-hydroxy-5-methoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1-(7-oxo-8propenyl)benzene the most active metabolites. These compounds displayed IC50 values of 78.8 ± 2.8 and 82.2 ± 3.5 mM, respectively, against murine melanoma. Different in vitro mechanism of induced cytotoxicity for this cell line is proposed for both compounds. Obtained results indicated a remarkable effect during the induction of morphological, biochemical and enzymatic features of apoptosis, such as disruption of mitochondrial membrane potential (DJm), exposure of phosphatidylserine in the outer cell membrane, and genomic DNA condensation and fragmentation. Dehydrodieugenol B induced caspase-3 and PARP activation and 4-hydroxy-5-methoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1(7-oxo-8-propenyl)benzene downregulated the levels of Bcl-2 protein. These effects were accompanied by increased levels of reactive oxygen species as a consequence of mitochondrial damage, followed by Factin aggregation during the cell death process. Dehydrodieugenol B showed oxidative properties and both compounds, especially 4-hydroxy-5-methoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1-(7-oxo8-propenyl)benzene, displayed potential to alkylate nucleophiles, suggesting an accessory mechanism of tumor-induced cytotoxicity by these metabolites. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Nectandra leucantha Lauraceae Neolignans Cytotoxic activity Melanoma Mechanism of action
1. Introduction Cancer is one of the major causes of morbidity and death worldwide. In 2012, the WHO notiﬁed about 14 million new cases
* Corresponding author. Center of Natural Sciences and Humanities, Federal University of ABC, Santo Andre, 09210-580, Brazil. ** Corresponding author. Department of Microbiology, Immunology and Parasi~o Paulo, Sa ~o Paulo, 04021-001, Brazil. tology, Federal University of Sa E-mail addresses: c.ﬁ[email protected]
(C.R. Figueiredo), [email protected]
edu.br (J.H.G. Lago). http://dx.doi.org/10.1016/j.phytochem.2017.04.024 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
and those numbers are intended to increase to 22 million in the next two decades (Martel et al., 2012). Despite its low incidence rates, advanced melanoma is the major cause of death among skin cancers and the mortality rate has signiﬁcantly increased over the last 30 years (Erdei and Torres, 2010). It is derived from melanocytes, a kind of cell that have enhanced motility and survival properties, presenting low levels of apoptosis in vitro and in vivo, and for this reason, melanoma is resistant to drug-induced apoptosis, and is one of the most aggressive metastatic cancer (Gray-Schopfer et al., 2007; Soengas and Lowe, 2003). Resistance to apoptosis occurs due to a breakdown of cell death control
F.S. de Sousa et al. / Phytochemistry 140 (2017) 108e117
associated to activation of anti-apoptotic factors, inactivation of pro-apoptotic effectors, and reinforcement of survival signals (Soengas and Lowe, 2003). Therefore, the search for new molecules that induce apoptosis on tumor cells, especially against melanoma, is encouraged. Anticancer agents derived from natural sources are known to display substantial structural diversity (Newman and Cragg, 2007). Several plant-based compounds are important to induce intrinsic apoptosis in melanoma cells by oxidative and endoplasmic reticulum stress (Matsuo et al., 2011; Massaoka et al., 2012; Girola et al., 2015; Cordova et al., 2011). Nectandra leucantha Nees & Mart (Lauraceae) is a large tree found in tropical and subtropical areas of America (The Plant List, 2016). In previous works, was reported the antiparasitic activity of neolignans isolated from twigs (Costa-Silva et al., 2015) as well as the cytotoxic potential of crude essential oils from leaves, chemically composed by terpenoids (Grecco et al., 2015). As part of our continuous work, the hexane extract of twigs of N. leucantha displayed cytotoxic activity and was subjected to a bioactivity-guided fractionation procedure. Using this approach were isolated six neolignans (1e6), from which 4e6 are previously undescribed. All isolated compounds were evaluated for cytotoxic potential against a panel of different cancer cell lines. Our ﬁndings suggest, for the ﬁrst time, the cytotoxic proﬁle of neolignans 1 and 5 to melanoma cells with substantial evidence of triggering apoptotic features by impairing cytoskeleton organization, oxidative damages and DNA degradation by alkylating reactions, which overcome the high apoptotic resistant proﬁle of melanoma cells, and elect both compound as promising therapeutic molecules against high resistant apoptotic cancer cells. 2. Results and discussion 2.1. Chemical characterization of compounds 1e6 In the present work, six natural metabolites (1e6, Fig. 1) were obtained from twigs of N. leucantha of which three are previously undescribed neolignans (4e6). Compounds 1 and 2 were identiﬁed as dehydrodieugenol B (1-(8-propenyl)-3-[10 -(80 -propenyl)-30 methoxyphenoxy]-4-hydroxy-5-methoxybenzene) and 1-(8propenyl)-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-4,5dimethoxybenzene, respectively, by comparison of reported NMR and HRESIMS data. Additionally, NMR, HRESIMS, optical rotation, and ECD data of compound 3 were identical of those reported to 1(7R-hydroxy-8-propenyl)-3-[30 -methoxy-10 -(80 -propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (Costa-Silva et al., 2015; Diaz et al., 1980).
Fig. 1. Chemical structures of isolated compounds 1e6.
Compound 4, isolated as brownish oil, showed virtually identical 1H and 13C NMR spectra of those recorded from compound 3 (Costa-Silva et al., 2015), but displayed additional peaks at dH 3.88 and dC 61.0, attributed to a hindered methoxy group at C-4. These information associated to HRESIMS spectrum which showed the [M þ Na]þ peak at m/z 379.1506 and the [M e H2O þ H]þ peak at m/ z 339.1597, indicated the occurrence of a methylated derivative of compound 3. Both compounds 4 and (R)-phenylethanol (Reetz, 2000) displayed negative Cotton effect at 260 nm, suggesting R conﬁguration to C-7. Therefore, the structure of compound 4 was deﬁned as 1-[(7R)-hydroxy-8-propenyl]-3-[30 -methoxy-10 -(80 propenyl)-phenoxy]-4,5-dimethoxybenzene. Compounds 5 and 6 were isolated as brownish oils. Their IR spectra showed the presence of conjugated carbonyl ketone at 1680 cm1, double bonds of aromatic ring at 1610 cm1 and, in the case of compound 5, a hydroxyl broad band at 3353 cm1. 1H NMR spectra of both compounds displayed similarities to those recorded to compounds 3 and 4, except for the absence of the signals attributed to H-7 and the observance of a deshielded peaks assigned to H-8 at dH 7.03/6.97 (dd, J ¼ 17.1 and 10.6 Hz, 1H) as well as those to H-9 at dH 6.36/6.38 (dd, J ¼ 17.1 and 1.8 Hz) and at dH 5.82/5.83 (dd, J ¼ 10.6 and 1.8 Hz). These data suggested the occurrence of related compounds containing an acryloyl group linked to aromatic ring. This proposal could be conﬁrmed by the presence of peaks at dC 188.8 and 189.3 in the 13C NMR spectra of compounds 5 and 6, respectively. Positioning of acryloyl group at C1 was conﬁrmed by analysis of the HMBC spectra (Fig. 2), which showed correlations between the signal at dH 7.36/7.30 (H-2), 7.21/ 7.04 (H-6), 6.38/6.36 (H-9a) and 5.82/5.83 (H-9b) to those at dC 188.8/189.3 (C-7) as well as that at dH 3.38 (H-70 ) and dC 113.1/113.2 (C-20 ), 121.1/120.9 (C-60 ) and dC 116.2/116.0 (C-90 ). HMBC spectrum of compound 6 (Fig. 2) showed also cross-peaks between the signal at dH 3.98, 3.95 and 3.82 (OCH3) and dC 144.2 (C-4), 150.6 (C-5) and 150.4 (C-30 ), conﬁrming the position of methoxy groups at C-4, C-5 and C-30 . As in the NMR spectra of compound 5, were not observed signals attributed to a hindered methoxy group at C-4, the occurrence of a free hydroxyl group at C-4 was proposed. Finally, HRESIMS spectra of compounds 5 and 6 displayed the [M þ H]þ ion peaks at m/z 341.1389 and 355.1543, conﬁrming the molecular formulas C20H20O5 and C21H22O5, respectively. Therefore, the structures of compounds 5 and 6 were deﬁned as 4-hydroxy-5methoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1-(7-oxo-8propenyl)benzene and 4,5-dimethoxy-3-[30 -methoxy-10 -(80 -propenyl)phenoxy]-1-(7-oxo-8-propenyl) benzene, respectively. Compounds 1e3 were previously described in N. leucantha (Costa-Silva et al., 2015) and compound 2 was also found in Ocotea
Fig. 2. Important HMBC correlations in the structures of compounds 4, 5 and 6.
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cymbarum (Diaz et al., 1980), both from Lauraceae. Nectandra genus consist in a source of different lignoids, especially furofuran, benzofuran, tetrahydrofuran, dihydrofuran, and 3,30 -neolignan derivatives (Grecco et al., 2016). However, despite that more than 30 species of Nectandra genus had chemically been studied, neolignans 1e6 were found exclusively in N. leucantha. 2.2. In vitro antitumor potential In vitro antitumor potential of isolated compounds 1e6 was initially evaluated against murine melanoma B16F10 cells at 50 mg/ mL in a 24 h cytotoxic assay, and tumor cells viability was further accessed using the colorimetric MTT assay. Results indicate that compounds 1, 4 and 5 are the most cytotoxic metabolites against B16F10 cells, with similar cytotoxic potential of positive control Taxol at 1 mM (Fig. 3). Further, the cytotoxic proﬁle of these compounds was evaluated against different human tumor and nontumorigenic cell lines and the IC50 values were quantiﬁed (Table 1). Compounds 1 and 5 showed the highest cytotoxic activities against most of the tumor cells compared with compounds 4 and 2 (further used as inactive metabolite control). Compounds 1 and 5 showed strong cytotoxicity against B16F10 cells (IC50 values
Table 1 IC50 (in mM) of neolignans 1e6 isolated from twigs of N. leucantha against murine and human cells (24 h of incubation). Cell line
B16F10 SKBR-3 HCT U87-MG A2058 T75
IC50 (mM) 1
78.8 ± 2.8 62.3 ± 19.6 103.1 ± 13.8 126.9 ± 21.7 123.0 ± 25.5 137.7 ± 10.1
>300 >300 >300 >300 >300 >300
>300 >300 >300 >300 >300 >300
210.9 187.1 >300 >300 189.9 105.1
± 3.1 ± 23.6
± 5.1 ± 8.4
82.2 ± 3.5 110.0 ± 8.8 118.8 ± 3.8 134.7 ± 14.7 94.4 ± 11.2 144.4 ± 17.1
>300 >300 >300 >300 >300 >300
IC50: 50% inhibitory concentration; murine Melanoma (B16F10); human breast cancer (SK BR-3); human colon carcinoma (HCT); human glioblastoma (U 87-MG); human melanoma (A2058) and human ﬁbroblasts (T75).
of 78.8 and 82.2 mM, respectively) and SKBR-3 (IC50 values of 62.3 and 110.0 mM, respectively), and compound 5 was the most active metabolite against the high drug-resistant human melanoma A2058 cells (IC50 value of 94.4 mM). Structurally, the obtained data indicated that the presence of a free hydroxyl group at C-4 in the neolignans 1 and 5 play an important role in antitumor activity since was observed a methoxy group at C-4 in inactive compounds
Fig. 3. (A) Cytotoxicity of compounds 1e6 in murine melanoma B16F10 cells. Compounds were incubated with tumor cells at each tested concentration for 24 h and viability was accessed by MTT method. (B) Tumor cells morphology after incubation with compounds 1 (76.6 and 153.2 mM) and 5 (73.5 and 147.0 mM). Scale bars: 50 mm. All experiments were made in triplicate.
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(2, 4 and 6). However, this must be associated to the presence of allyl (1) or acryloyl (5) groups at C-1, since a reduction in the activity was observed for neolignan 3, which presents hydroxyl group at C-7. Additionally, the lowest cytotoxic activity of compounds 1 and 5 was observed for the non-tumorigenic cell line T-75 (human ﬁbroblast), with IC50 values of 137.7 and 144.4 mM, respectively, suggesting that selectivity and cytotoxicity induced by both compounds regarding cellular and biochemical mechanisms are more effective in tumor cells compared to non-tumorigenic cells. For these reasons, compounds 1 and 5 were chosen for the study of their antitumor cytotoxic activities using the murine melanoma model B16F10 cells. 2.3. Determination of antitumor mechanism of compounds 1 and 5 against B16F10 cells Morphological changes in B16F10 cells at higher and intermediary concentration induced by compounds 1 (at 153.2 and 76.6 mM) and 5 (at 147.0 and 73.5 mM) showed that both compounds could remarkably reduce cell volume (shrinkage of cytoplasm), round-up the cell structure, retract pseudopods, and also induce loss of substrate attachment and shrivel up without cell lysis. These results were observed mainly at the highest tested concentrations (Fig. 3B). Genomic DNA condensation and fragmentation are also important morphological features to be considered for better comprehension of tumor-induced cell death, and they were both accessed by ﬂuorescence microscopy as previously described in experimental part. Compounds 1 and 5 similarly enhanced genomic DNA condensation as indicated by increased Hoechst dye ﬂuorescence intensity at the highest tested concentrations for both compounds, when compared to negative control (Fig. 4). Together, speciﬁc DNA fragmentation was also observed by TUNEL assay, as
indicated by green positive ﬂuorescence in treated B16F10 cells (Fig. 4A), and the surface plot analysis indicated that the fragmentation pattern was higher for compound 5 when compared to compound 1 and the negative control (Fig. 4). The evaluation of phosphatidylserine externalization in the outer plasma membrane of dying cells is an important feature to determine if treated cells are undergoing apoptosis. Single staining with Annexin-V indicates early apoptosis and double staining with Annexin-V and PI indicates late apoptosis, as previously described (Matsuo et al., 2011). The obtained results indicated that compound 1 induced early and late apoptosis in melanoma cells at both tested concentrations (Fig. 5A). In addition, compound 5 induced early and late apoptosis only at 147.0 mM but not at 73.5 mM, suggesting that the IC50 observed for compound 5 (82.2 mM) may be a combination of induced cell death process at the highest tested concentration, together with cytostatic growth inhibition at the intermediary concentration of 73.5 mM (Fig. 5B). To evaluate the cytostatic potential of compound 5 over the growth of melanoma cells, B16F10 cells (1 106) were incubated with compound 5 at 73.5 mM for 24 h and cell cycle was evaluated as previously described (Figueiredo et al., 2015). Compound 5 signiﬁcantly increased the rate of cells in S and G2/M phases, together with a signiﬁcant decrease at the G1 phase (Fig. 5C). The ability of compounds 1 and 5 to disturb the mitochondrial membrane potential was investigated on live B16F10 cells in a time-lapse ﬂuorescence microscope using the TMRE probe as previously described (Figueiredo et al., 2015). Compound 1 rapidly reduced TMRE ﬂuorescence on tumor cells, indicating an early mitochondrial dependent cytotoxicity at the highest tested concentration until 2 h of incubation. Compound 5 also reduced TMRE ﬂuorescence in tumor cells, and a signiﬁcant reduction of TMRE was observed after 4 h of incubation (Fig. 6A). Changes in cytoskeletal organization were observed for
Fig. 4. Evaluation of chromatin condensation and fragmentation proﬁle of treated cells with compounds 1 and 5. B16F10 cells were incubated with both compounds at 153.2 and 147.0 mM, respectively, for 18 h and analyzed through ﬂuorescence microscopy (scale bars. 100 mm).
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Fig. 5. Annexin V and PI labeling. B16F10 cells were incubated with compounds 1 (153.2 and 76.6 mM e panel A) and 5 (147.0 and 73.5 mM e panel B) for 18 h and stained with Annexin V-FITC and PI. Analysis was performed by image cytometry using the Cytell apoptosis program. Compound 2 was used as inactive metabolite control. (C) Cell cycle arrest induced by compound 5 (73.5 mM) in 5 105 of B16F10 cells after 24 h treatment. The values are percentages obtained from three independent experiments. *p < 0.05 compared to negative control.
compounds 1 and 5 treated B16F10 cells. Compound 1 caused an increase of F-actin ﬂuorescence at 76.6 mM, which is related to Factin aggregation in the cytoplasm, together with cell blebbing formation, as indicated by white arrows (Fig. 6B). Quantiﬁcation of ﬂuorescence intensity for each group is shown in the surface plot, and compound 1 reached the highest RFU levels, ranging from 60 to 100. At this concentration, no F-actin aggregate was observed for compound 5 treated cells. Furthermore, at the highest tested concentration, rounded cells with condensed actin, treated with compounds 1 and 5, were also observed (data not shown). Compound 2 was used as non-active metabolite control. The induction of cytosolic reactive oxygen species (ROS) in treated cells with compounds 1 and 5, together with their redox reactive potential was investigated. Using the dihydroethidium (DHE) probe (Sigma Aldrich, USA), was observed that both compounds do not induce early generation of ROS, but signiﬁcantly increased ROS levels on tumor cells after 18 h of incubation at both tested effective concentrations (Fig. 7A). Compounds 1 and 5 were checked for their 4-(4-nitro-benzyl) pyridine (NBP) alkylating activity by the procedure described in experimental section. The NBP reacts with alkylating agents to form conjugate adducts providing a purple color upon basiﬁcation (Balazs et al., 1970). The obtained data indicates a high alkylation
Fig. 6. (A) Disruption of the mitochondrial transmembrane potential (Djm) in B16F10 cells during treatment with compounds 1 (153.2 mM) and 5 (147.0 mM) for 4 h (*p < 0.001 in relation to untreated control). (B) Aggregation of F-actin in B16F10 cells after treatment with compounds 1 (153.2 mM) and 5 (147.0 mM) for 6 h and further stained with phalloidin-FITC. Scale bars: 100 mm. Intensity surface-plots were acquired from the selected ﬁelds using NIS-elements software (Nikon. Tokyo).
rate for compound 5, compared with other compounds and negative control (Fig. 7B). Compound 2 was used as negative functional metabolite control. Melanoma cells were treated with both compounds 1 (153.2 and 306.4 mM) and 5 (147.0 and 294.0 mM) and total protein was then analyzed to look for the expression levels of total and cleaved PARP, caspase-3, Bcl-2 and Bcl-xl. The obtained results indicated that compound 1 increases the cleavage of caspase-3 and PARP after 2 h of incubation at 153.2 mM, and compound 5 reduced the expression levels of Bcl-2 after 6 h of incubation at 147.0 mM (Fig. 8). Evidences indicate that these compounds damage mitochondria in early stages, with further increase in ROS levels of treated cells and activation of caspases and PARP-c, resulting in several apoptotic hallmarks, such as shrinkage of cytoplasm, rounding-up of cell shape, pseudopods retraction, together with plasma membrane bleb formation, genomic DNA condensation and fragmentation, and extracellular exposure of phosphatidylserine without cell lysis (Kroemer et al., 2009; Galluzzi et al., 2012; Bortner and Cidlowski, 2002). In addition, the disruption of mitochondrial membrane potential by both compounds at early stages suggests that apoptosis may occur by the intrinsic apoptotic pathway (Fulda and Debatin, 2006). The release of ROS from mitochondria led to changes to the actin cytoskeleton dynamics, with aggregation of Factin as a hallmark and subsequent cell death (Gourlay and Ayscoug, 2005). This effect was also observed for compounds 1
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Fig. 7. (A) ROS generation in B16F10 cells treated with compounds 1 (153.2 mM) and 5 (147.0 mM) after 1 and 18 h of incubation. (B) Alkylating proﬁle of compounds 1 (306.4 mM) and 5 (294.0 mM) accessed against NBP substrate. Alkylated NBP swift to blue absorbance was recorded at 570 nm.
Fig. 8. Expression levels of total and cleaved caspase-3 and PARP. Bcl-2 and Bcl-xl in melanoma cells treated with compounds 1 (153.2 mM) and 5 (147.0 mM) at different times of incubation.
and 5 inducing cytotoxicity in melanoma cells. When the intrinsic pathway is initiated, mitochondria releases apoptogenic factors such as cytochrome C and endonucleases from the inter membrane space, which once in the cytosol may trigger caspase-3 activation through the formation of the apoptosome complex (Cande et al., 2002; Saelens et al., 2004). Several natural products induce intrinsic apoptosis in tumor cells via oxidative stress and acting as pro-oxidant agents
(Massaoka et al., 2012; Martin-Cordero et al., 2012; Mates and Sanchez-Jimenez, 2000). Oxidative stress can induce severe damage to organelle membranes such as mitochondria and DNA, leading to apoptosis (Trachootham et al., 2009). Based in these aspects, was propose that both compounds 1 and 5 trigger intrinsic apoptosis on melanoma cells. Compound 1 may behave mainly as a pro-oxidizing agent on tumor cells, supported by the enhanced DHE oxidative proﬁle. In addition, both compounds have alkylating properties, especially compound 5, as observed in the NBP assay. Cancer cells are dependent on cellular antioxidant systems and the redox-sensitive pro-survival signal to maintain viability because they are constantly under ROS generation and increased intrinsic oxidative stress. For this reason, they are more vulnerable to further oxidative insults induced by compounds that abrogate the key antioxidant systems in the cells, which selectively kill cancer cells with less toxicity to normal cells (Schumacker, 2006). This may explain why compounds 1 and 5 were less cytotoxic to non-tumorigenic T75 cells. DNA damage induced by chemotherapeutic agents includes the alkylating agents, such as dacarbazine and temozolomide, which show moderate antitumor efﬁcacy in metastatic melanoma in vivo and comprises the ﬁrst class of drugs approved by the Food and Drug Administration (FDA) (Bhatia et al., 2009). DNA degradation proﬁle, NBP reactivity and chemical characteristics (basically the presence of Michael acceptor such as acryloyl group in compound 5 structure as well as free hydroxyl group in aromatic ring) (Ahn and Sok, 1996), suggest that both compounds 1 and 5 could be responsible for alkylating reactive properties. Because of similar reactivity of NBP and guanine to DNA, NBP serves as a DNA model for identiﬁcation of alkylating agents in a
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colorimetric assay (Provencher and Love, 2015). Results obtained with NBP assay suggest that compounds 1 and 5 may promote alkylating reactions in tumor cells, which could damage DNA or other nucleophiles in the cells, such as thiol-groups, proteins, imidazole groups and nitrogenous bases. In addition, alkylating agents may increase intracellular levels of ROS in tumor cells, which also enhance DNA damage and intrinsic apoptosis (Mates and Sanchez-Jimenez, 2000; Viswesh et al., 2010). This mechanism of cytotoxicity is also selective to proliferative tissues such as cancer cells, because alkylation can occur in both cycling and resting cells, but proliferating cells are more sensitive. This higher sensitivity induces arrest in both S and G2/M phases of the cell cycle, as observed in treated melanoma cells exposed to compound 5, where abnormal base pairing and breaks during DNA replication eventually result in cell death by apoptosis (Harrap and Hill, 1969; Warwick, 1963; Puyo et al., 2014; Bignold, 2006). Results collected here indicate for the ﬁrst time the antitumor proﬁle of the compounds 1 and 5 isolated from N. leucantha against different tumor cell lines and suggest the mechanism of action for inducing apoptosis on melanoma cells. Therefore, it was possible to conclude that compounds 1 and 5 are promising scaffold molecules as prototypes for the development of new drugs for cancer therapy, especially for the class of apoptosis-inducing drugs. Furthermore, IC50 values of compounds 1 and 5 against tumorigenic (B16F10) and non-tumorigenic (T75) cells show increased selective cytotoxicity of both compounds to tumor cells. Compound 1 induced caspase dependent intrinsic-apoptosis by activation of caspase-3 and PARP, and ROS mediated imbalance in the redox rates seems to be essential to this process, in addition to a possible enhancement mechanism of alkylating reactions that may contribute to the apoptotic processes. For compound 5, cytotoxicity seems to be primarily dependent on the DNA alkylating process, without activation of caspase pathway. The mechanisms involved in the induction of apoptosis by alkylating agents are largely mediated by the mitochondrial apoptotic pathway (Debatin et al., 2002), and sometimes, it involves both caspase-dependent and caspaseindependent processes (Artus et al., 2010; Cregan et al., 2004; Liang et al., 2004). 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Na ﬁlter, l ¼ 588 nm) and electronic circular dichroism (ECD) analysis was performed using MeOH on a JASCO J-815 spectropolarimeter. UV spectra were recorded on an UV/visible Shimadzu 1650-PC spectrophotometer. IR spectra were obtained on a Perkin-Elmer 1750 spectrophotometer. NMR spectra were recorded at 300 and 75 MHz in a Bruker Ultrashield 300 Avance III spectrometer. CDCl3 (Aldrich) was used as the solvent with TMS as the internal standard. HRESIMS spectra were measured on a Bruker Daltonics MicroTOF QII spectrometer. High performance liquid chromatography (HPLC) analysis was performed in a Dionex Ultimate 3000 chromatograph, using a Luna Phenomenex RP-18 columns (5 mm, 150 4.6 mm to analytical and 250 10 mm to semipreparative) and an UV-diode array detector (DAD). Silica gel (Merck, 230e400 mesh) and Sephadex LH-20 (Sigma-Aldrich) were used for column chromatography (CC). For all extraction and chromatography procedures, analytical grade solvents (Labsynth Ltd) were used. 3.2. Plant material Twigs of Nectandra leucantha Nees & Mart (Lauraceae) were
~o, Sa ~o Paulo, Brazil, in March 2014, and the plant collected in Cubata species was identiﬁed by Euder G. A. Martins. A voucher specimen (EM357) has been deposited in the Herbarium of the Institute of Biosciences, University of S~ ao Paulo, SP, Brazil. 3.3. Extraction and isolation The air-dried twigs of N. leucantha (220 g) were powdered and exhaustively extracted with n-hexane. This material was ﬁltered and concentrated under vacuum to afford 7.9 g of n-hexane extract. Part of this material (7.4 g) was subjected to column chromatography over SiO2 using increasing amounts of EtOAc in n-hexane to afford ten fractions (A to J). After evaluation of cytotoxic activity (B16F10 cells at 50 mg/mL), fractions C (1584 mg), E (1920 mg) and G (2070 mg) displayed activity. Fraction C was composed by pure compound 1 (1584 mg). Part of fraction E (1500 mg) was puriﬁed by Sephadex LH-20 (52 2 cm, 300 mg of material was applied each time), eluted with MeOH to afford compound 2 (1150 mg). Part of the active fraction G (1450 mg) was chromatographed over SiO2 eluted with increasing amounts of EtOAc in n-hexane to give eight fractions (G1 e G8). Bioactive fraction G4 was composed by pure compound 4 (865 mg). As the cytotoxicity was also detected on the group G3 (385 mg), part of this material (350 mg) was subjected to CC over SiO2 using increasing amounts of EtOAc in n-hexane to afford ﬁve fractions (G3/1 - G3/4). Part of the active fraction G3/2 (268 mg) was puriﬁed over Sephadex LH-20 (MeOH, 51 2 cm) to afford seven fractions (G3/2-I e G3/2-VII). Bioactive fraction G3/2IV afforded compound 3 (149 mg) while part of the active fraction G3/2-V (53 mg) was subjected to the column over SiO2 (n-hexane:EtOAc 7:3, 1:1 and 3:7 as eluent) to afford eight fractions (G3/ 2eV1 to G3/2-V8). Part of the active fraction G3/2-V3 (18 mg) was puriﬁed by semi-prep RP-HPLC (MeOH:H2O 7:3, ﬂow rates 2.0 mL/ min, UV 230 nm) to afford compounds 5 (1.0 mg) and 6 (1.2 mg). 3.3.1. 1-[(7R)-hydroxy-8-propenyl]-3-[30 -methoxy-10 -(80 propenyl)-phenoxy]4,5-dimethoxybenzene (4) Brownish oil. [a]25 D þ 1.6 (c 0.15, MeOH); UV (MeOH) lmax (log ε) 222 (3.7), 278 (2.9) nm; IR (ﬁlm) nmax 3300, 2850, 1640, 1508, 1463, 1365, 996, 918, 745, 540 cm1; 1H NMR (CDCl3, 300 MHz) d 6.82 (1H, d, J ¼ 8.1 Hz, H-50 ), 6.80 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.70 (1H, dd, J ¼ 8.1 and 1.8 Hz, H-60 ), 6.69 (1H, d, J ¼ 1.8 Hz, H-2), 6.43 (1H, d, J ¼ 1.8 Hz, H-6), 5.96 (2H, m, H-8 and H-8'), 5.27 (1H, dt, J ¼ 17.1 and 1.4 Hz, H-9a), 5.15 (1H, dt, J ¼ 10.8 e 1.4, H-9b), 5.09 (2H, m, H-90 ), 5.03 (1H, d, J ¼ 6.0 Hz, H-7), 3.88 (3H, s, 4-OCH3), 3.87 (3H, s, 30 OCH3), 3.82 (3H, s, 5-OCH3), 3.36 (2H, d, J ¼ 6.6 Hz, H-70 ). 13C NMR (CDCl3, 75 MHz) d 153.8 (C, C-30 ), 150.8 (C, C-5), 150.6 (C, C-40 ), 143.8 (C, C-3), 139.9 (CH, C-8), 139.1 (C, C-4), 138.1 (CH, C-80 ), 137.4 (C, C10 ), 136.3 (C, C-1), 120.8 (CH, C-60 ), 119.6 (CH, C-50 ), 115.9 (CH2, C-90 ), 115.2 (CH2, C-9), 113.1 (CH, C-20 ), 109.2 (CH, C-6), 104.8 (CH, C-2), 75.1 (CH, C-7), 61.0 (CH3, 4-OCH3), 56.1 (CH3, 5-OCH3), 56.0 (CH3, 30 OCH3), 40.0 (CH2, C-70 ); HRESIMS m/z 379.1506 [M þ Na]þ (calculated for C21H24O5Na, 379.1521) and 339.1597 [M e H2O þ H]þ (calculated for C21H23O4, 339.1596). 3.3.2. 4-Hydroxy-5-methoxy-3-[30 -methoxy-10 -(80 -propenyl) phenoxy]-1-(7-oxo-8-propenyl)benzene (5) Brownish oil. UV (MeOH) lmax (log ε) 205 (3.7), 284 (2.8), 321 (3.0) nm; IR (ﬁlm) nmax 2840, 1682, 1613, 1517, 1450, 1414, 1150, 968, 751 cm1; 1H NMR (CDCl3, 300 MHz) d 7.36 (1H, d, J ¼ 1.8 Hz, H-2), 7.21 (1H, d, J ¼ 1.8 Hz, H-6), 7.03 (1H, dd J ¼ 17.1 and 10.6, H-8), 6.95 (1H, d, J ¼ 8.1 Hz, H-50 ), 6.82 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.76 (1H, dd, J ¼ 8.1 and 1.8 Hz, H-60 ), 6.38 (1H, dd, J ¼ 17.1 and 1.8 Hz, H-9a), 6.00 (1H, m, H-80 ), 5.82 (1H, dd, J ¼ 10.6 e 1.8, H-9b), 5.10 (2H, m, H-90 ), 3.98 (3H, s, 5-OCH3), 3.86 (3H, s, 30 -OCH3), 3.38 (2H, d, J ¼ 6.6 Hz, H70 ). 13C NMR (CDCl3, 75 MHz) d 188.8 (C, C-7), 150.5 (C, C-30 ), 148.1
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(C, C-5), 144.5 (C, C-3), 143.4 (C, C-40 ), 141.9 (C, C-4), 137.3 (CH, C-80 ), 131.8 (CH, C-8), 129.5 (CH2, C-9), 128.7 (C, C-1), 121.1 (CH, C-60 ), 120.0 (CH, C-50 ), 116.2 (CH2, C-90 ), 113.2 (CH, C-6), 113.1 (CH, C-20 ), 107.1 (CH, C-2), 56.5 (CH3, 5-OCH3), 56.0 (CH3, 30 -OCH3), 40.0 (CH2, C-70 ); HRESIMS m/z 341.1384 [M þ H]þ (calculated for C20H21O5, 341.1389). 0
3.3.3. 4,5-Dimethoxy-3-[3 -methoxy-1 -(8 -propenyl)phenoxy]-1(7-oxo-8-propenyl)benzene (6) Brownish oil. UV (MeOH) lmax (log ε) 214 (3.7), 284 (3.2); IR (ﬁlm) nmax 3353, 2843, 1681, 1612, 1508, 1452, 1416, 1153, 970, 750 cm1; 1H NMR (CDCl3, 300 MHz) d 7.30 (1H, d, J ¼ 1.8 Hz, H-2), 7.04 (1H, d, J ¼ 1.8 Hz, H-6), 6.97 (1H, dd J ¼ 17.1 and 10.6, H-8), 6.83 (1H, d, J ¼ 8.1 Hz, H-50 ), 6.82 (1H, d, J ¼ 1.8 Hz, H-20 ), 6.72 (1H, dd, J ¼ 8.1 and 1.8 Hz, H-60 ), 6.36 (1H, dd, J ¼ 17.1 and 1.8 Hz, H-9a), 6.02 (1H, m, H-80 ), 5.83 (1H, dd, J ¼ 10.6 e 1.8, H-9b), 5.11 (2H, m, H-90 ), 3.98 (3H, s, 4-OCH3), 3.95 (3H, s, 5-OCH3), 3.82 (3H, s, 30 -OCH3), 3.38 (2H, d, J ¼ 6.6 Hz, H-70 ). 13C NMR (CDCl3, 75 MHz) d 189.3 (C, C-7), 153.7 (C, C-1), 150.6 (C, C-5), 150.4 (C, C-30 ), 144.2 (C, C-4), 143.4 (C, C-3), 137.2 (CH, C-80 ), 132.2 (C, C-40 ), 131.9 (CH, C-8), 129.9 (CH2, C9), 120.9 (CH, C-60 ), 119.7 (CH, C-50 ), 116.0 (CH2, C-90 ), 113.2 (CH, C20 ), 112.5 (CH, C-6), 107.1 (CH, C-2), 61.1 (CH3, 4-OCH3), 56.3 (CH3, 5OCH3), 55.9 (CH3, 30 -OCH3), 40.0 (CH2, C-70 ); HRESIMS m/z 355.1543 [M þ H]þ (calculated for C21H23O5, 355.1545). 3.4. Cell lines and culture conditions The murine melanoma cell line B16F10 was established at the ~o Paulo Experimental Oncology Unit, Federal University of Sa (UNIFESP), and deposited in the Banco de C elulas do Rio de Janeiro (BCRJ), reg. 0342. The following human cancer cell lines (melanoma A2058, breast cancer SKBR-3, colorectal cancer HCT) and nontumorigenic cell human ﬁbroblast T75 were provided by the Ludwig Institute for Cancer Research. Prof. O. Keith Okamoto, from ~o Paulo, provided the human glioblastoma U87-MG University of Sa cell line. Cells were cultivated as previously described (Figueiredo et al., 2014). 3.5. Cell viability assay Crude extracts, fractions and metabolites from twigs of N. leucantha were incubated with 1 104 tumor and nontumorigenic cells for 24 h at different concentrations ranging from 0 to 100 mg/mL, where the IC50 was determined in mM to each pure compound based on their respective molecular weight. Cell viability was quantiﬁed using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich) and was shown as percent values in comparison with untreated cells as previously described (Santana et al., 2012). Cisplatin was used for comparison of positive toxicity. All experiments were performed in triplicates. 3.6. DNA condensation and fragmentation assay TUNEL assay was performed to access the induced DNA fragmentation in melanoma cells. B16F10 cells (1 104 cells/well) were cultured on 96-well plates and incubated with 25 and 50 mg/mL of compounds 1 (76.6 and 153.2 mM) and 5 (73.5 and 147.0 mM) at 37 C for 24 h. Cells were then processed as described elsewhere (Massaoka et al., 2012). In addition, cells were also stained with 10 mg/mL for 15 min at 37 C to access DNA condensation events. Green-FITC represents DNA fragmentation process and blueHoechst33342 represents single nucleus events in each ﬁeld. After the reaction staining process, images of ﬂuorescent tumor cells were acquired and quantiﬁed using Cytell Image System cytometer
(GE Healthcare, Little Chalfont, UK) at 200 magniﬁcation. Quantiﬁcation of ﬂuorescence intensity was performed by the surface plot algorithm in ImageJ software. 3.7. Mitochondrial transmembrane potential (Djm) B16F10 cells (1 104) were incubated with 5 nM of tetramethylrhodamine ethyl ester (TMRE, Molecular Probes, OR, USA), a ﬂuorescent probe for intact mitochondrial transmembrane potential, for 30 min at 37 C with 5% CO2 in a four chamber dishes. Further, cells were incubated with 50 mg/mL of compounds 1 (153.2 mM), 2 (146.9 mM) and 5 (147.0 mM) for 4 h and ﬂuorescence kinetics were measured in a Time-lapse ﬂuorescence microscope (Biostation, Nikon) and quantiﬁed using the NIS-Elements imaging software (Nikon, Tokyo). 3.8. Annexin V assay B16F10 cells (1 106) were cultured in 6-well plates and further incubated with 1 mL of compounds 1 (153.2 mM), 2 (146.9 mM) and 5 (147.0 mM) or complete medium (negative control) for 2 h at 37 C. Phosphatidylserine detection was performed using the Annexin VFITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO) as previously described (Obata et al., 1990). Cells were then detached and seeded in a 96-black Costar plate with transparent ﬂat-bottom and apoptotic cells were analyzed in the Apoptosis algorithm of Cytell Image System following the manufacturer's instructions (GE Healthcare, Little Chalfont, UK). 3.9. Cell protein extraction and western blotting B16F10 cells (6 105) were treated with 50 mg/mL of compound 1 (153.2 mM during 1 and 2 h) and 100 mg/mL of compound 5 (294.0 mM during 1, 2, 4 and 6 h). No treated cells were assayed (control). Total protein lysates were acquired in different incubation periods and analyzed by western blot as described elsewhere (Massaoka et al., 2012). The following antibodies were used: antiPARP, total and cleaved caspase-3, Bcl-2 and Bcl-xl, all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-a-tubulin (Sigma-Aldrich, St. Louis, MO, USA) was used as protein loading control. Secondary antibodies conjugated with IgG horseradish peroxidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Readings were made in Uvitec Alliance 2.7 (UK, Cambridge). 3.10. Cell cycle assay B16F10 cells (5 105) were seeded in 6 well plates and incubated with 25 mg/mL of compound 5 (73.5 mM) and negative control for 24 h. After the incubation period, cells were trypsinized, suspended in complete media and centrifuged at 170 g for 5 min. Pellets were homogenized in cold EtOH 70% through ﬁxation in ice for 15 min. After ﬁxation, cells were centrifuged at 380 g for 5 min, suspended in PI solution (PI 50 mg/mL, RNase A 0.1 mg/mL and Triton X-100 0.05%) and incubated for 40 min at 37 C. PBS was added to each tube and cells were pelleted and suspended in PBS. Cell cycle was quantiﬁed in the Facs CANTO II (BD) and analyzed in FlowJo software V10.07 (Ashland, Oregon, USA). 3.11. Alkylation assay All compounds were checked for their 4-(4-nitro-benzyl) pyridine (NBP) alkylating activity (Balazs et al., 1970). Compounds 1 and 5 (1 mL) at 0.7 mg/mL (2.1 and 2.0 mM, respectively) was incubated with NBP solution (71 mg/mL in acetone) and buffer solution (potassium hydrogen phthalate 10 mg/mL in H2O) in a glass ﬂask. The
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ﬂask was kept at 85 C in a water bath for 30 min. Then, the solution was cooled immediately for 2 min at 4 C and KOH solution (56 mg/ mL in EtOH 95%) was added. The solution was diluted to 5.0 mL with EtOH 95% (140 mg/mL, the ﬁnal concentration of compounds 1 and 5) and the absorbance was immediately recorded in a spectrophotometer at 570 nm (Spectramax M2e, Molecular Devices). 3.12. Redox assay B16F10 cells (1 104) were previously incubated with dihydroethidium probe (DHE) for 20 min at 37 C in a 96 wells plate, and then, cells were washed and incubated with 50 mg/mL of compounds 1 (153.2 mM) and 5 (147.0 mM). Fluorescent oxidized DHE proﬁle was quantiﬁed after 1 and 18 h of incubation at 485 nm and 538 nm (excitation/emission) in a plate spectrometer (Spectramax M2e, Molecular Devices). 3.13. Statistical analysis All experiments were performed in triplicates. Values are expressed as means ± standard deviations (S.D.). For statistical analysis, Student's t-test was used for all experiments using the GraphPad Prism 4.0 software (La Jolla, CA), and p < 0.05 was adopted for signiﬁcant differences. Acknowledgments ~o de Amparo a The present work was supported by Fundaça ~o Paulo - FAPESP (project number 2015/ Pesquisa do Estado de Sa 11936-2). The authors are grateful for the CNPq Scientiﬁc Research Award given to J.H.G.L. and to a FAPESP scholarship given to F.S.S. (project number 2015/04143-6). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.04.024. References Ahn, B.Z., Sok, D.E., 1996. Michael acceptors as a tool for anticancer drug design. Curr. Pharm. Des. 2, 247e262. Artus, C., Boujrad, H., Bouharrour, A., Brunelle, M.N., Hoos, S., Yuste, V.J., Lenormand, P., Rousselle, J.C., Namane, A., England, P., Lorenzo, H.K., Susin, S.A., 2010. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 29, 1585e1599. Balazs, M.K., Anderson, C.A., Iwamoto, R.H., Lim, P., 1970. Synthesis of 4-[p-[(2chloroethyl)-(2-hydroxyethyl)amino]phenyl]butyric acid and its behavior in the 4-(4-nitrobenzyl)pyridine assay procedure. J. Pharm. Sci. 59, 563e565. Bhatia, S., Tykodi, S.S., Thompson, J.A., 2009. Treatment of metastatic melanoma: an overview. Oncol. Willist. Park) 23, 488e496. Bignold, L.P., 2006. Alkylating agents and DNA polymerases. Anticancer Res. 26, 1327e1336. Bortner, C.D., Cidlowski, J.A., 2002. Apoptotic volume decrease and the incredible shrinking cell. Cell Death Differ. 9, 1307e1310. Cande, C., Cecconi, F., Dessen, P., Kroemer, G., 2002. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J. Cell Sci. 115, 4727e4734. Cordova, C.A., Locatelli, C., Assuncao, L.S., Mattei, B., Mascarello, A., Winter, E., Nunes, R.J., Yunes, R.A., Creczynski-Pasa, T.B., 2011. Octyl and dodecyl gallates induce oxidative stress and apoptosis in a melanoma cell line. Toxicol. Vitro. 25, 2025e2034. Costa-Silva, T.A., Grecco, S.S., Sousa, F.S., Lago, J.H., Martins, E.G., Terrazas, C.A., Varikuti, S., Owens, K.L., Beverley, S.M., Satoskar, A.R., Tempone, A.G., 2015. Immunomodulatory and antileishmanial activity of phenylpropanoid dimers isolated from Nectandra leucantha. J. Nat. Prod. 78, 653e657. Cregan, S.P., Dawson, V.L., Slack, R.S., 2004. Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene 23, 2785e2796. Debatin, K.M., Poncet, D., Kroemer, G., 2002. Chemotherapy: targeting the mitochondrial cell death pathway. Oncogene 21, 8786e8803. Diaz, A.M.P., Gottlieb, H.E., Gottlieb, O.R., 1980. Dehydrodieugenols from Ocotea cymbarum. Phytochemistry 19, 681e682.
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