Evidence-based Complementary and Alternative Medicine : eCAM. Dec/31/2014; 2015

Published online Jul/11/2015

PMID: 26246833

PMC: 4515262

doi: 10.1155/2015/298463

Cytotoxic Activity and Chemical Composition of the Root Extract from the Mexican Species Linum scabrellum: Mechanism of Action of the Active Compound 6-Methoxypodophyllotoxin

Ivonne Alejandre-García

Laura Álvarez

Alexandre Cardoso-Taketa

Leticia González-Maya

Mayra Antúnez

Enrique Salas-Vidal

J. Fernando Díaz

Silvia Marquina-Bahena

María Luisa Villarreal

Abstract

The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC₅₀ = 0.57 μg/mL), MCF7 breast (IC₅₀ = 0.56 μg/mL), PC3 prostate (IC₅₀ = 1.60 μg/mL), and SiHa cervical (IC₅₀ = 1.54 μg/mL), as well as toward the normal fibroblasts line HFS-30 IC₅₀ = 1.02 μg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC₅₀ value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant K_b = 7.6 × 10⁶ M⁻¹). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.

1. Introduction

The genus Linum of the Linaceae family includes more than 200 globally distributed species. The phytochemical profiles that have been reported for the species of Linum suggest the presence of some lignan-type secondary metabolites [1]. These kinds of compounds show several important pharmacological activities such as transcriptase reverse inhibition and HIV activity, effects on cardiovascular system, antileishmaniasis properties, hypoglycemic activities, 5-lipoxygenase inhibition, and antifungal, antirheumatic, antipsoriasis, antimalarial, and antiasthmatic properties [2–14]. However, cytotoxic and antitumoral activities are a major interest on these types of lignans [15]. Podophyllotoxin (PTOX) is the most important aryltetralin-lignan for human health in relation to anticancer and antiviral activities. It is used for the treatment of genital warts (condyloma acuminata) caused by the human papilloma virus [15] and for the semisynthesis of the two important antineoplastic agents etoposide and teniposide, which are actually used in the treatment of several cancers, among others: acute myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, lung cancer (both small cell and non-small cell), gastric cancer, breast cancer, and ovarian cancer [16].

Some species of Linum as L. persicum and L. nodiflorum accumulate PTOX [17], while others as L. flavum, L. mucronatum ssp. armenum, L. capitatum, L. arboretum, L. campanulatum, L. elegans, L. pamphylicum, L. tauricum, L. thracicum, L. sibiricum, and L. nodiflorum accumulate 6-methoxypodophyllotoxin (6MPTOX) [18]. The mechanism of action of PTOX is based on the inhibition of tubulin polymerization through interaction with the binding site of colchicine [19] as well as on the arrest of the cell cycle in metaphase [20–22]. Although the compound 6MPTOX has been reported with cytotoxic activity against murine Ehrlich ascites and HeLa cervix uteri neoplastic cell lines, its mechanism of action is unknown. Others derivatives as desmethoxy-yatein and desoxypodophyllotoxin are responsible for cytotoxic and antitumoral activities [23, 24]. The analogue 4′-demethyl-epipodophyllotoxin inhibits DNA topoisomerase II [16, 24], and several series of nonlactonic podophyllic aldehyde analogues produce apoptosis induction of neoplastic cells without a previous tubulin inhibition [25].

In Mexico 28 species of Linum have been identified, of which 12 are endemic. The species of Linum are used in Mexican Traditional Medicine to treat respiratory infections, snake bites, malaria, leishmaniasis, gastrointestinal affections, inflammation, and cystitis as well as other conditions [26–29].

Linum scabrellum is a perennial herb endemic to Mexico that grows up to 40 or 50 cm, pubescent in its green parts with leaves alternated and flowering from June to December [28]. It is distributed in 7 states of the country: Querétaro, Guanajuato, Hidalgo, Oaxaca, Puebla, Nuevo León, and Tamaulipas. In a previous study, we reported the cytotoxic activity of three extracts (chloroform, butanol, and aqueous) prepared from aerial parts and roots of L. scabrellum against three human cancer cell lines. The obtained results showed that the most toxic activity was observed from the root's chloroformic extract against KB (epidermoid), HF6 (colon), and MCF7 (breast) carcinomas, exhibiting IC₅₀ values that ranged from 0.2 to 4.8 μg/mL [30].

The aim of the present investigation was to study the chemical composition of Linum scabrellum and to identify its cytotoxic constituents. This is the first phytochemical study that reports the complete metabolic content of this species that allowed the identification of 6MPTOX as the active metabolite against human cancer cell lines. In order to explore the mechanism of action of this compound, several studies were conducted to determine its effects on the cell cycle progression of PC3 prostate cancer cells, mitotic arrest, microtubule polymerization, binding affinity to tubulin, and cell death.

2. Materials and Methods

2.1. Plant Material

The wild plant Linum scabrellum was collected by M.S. Ivonne Alejandre García on November 2012 at 5 km to the south of Vizarrón, Cadereyta Queretaro, Mexico. A sample was authentified by BS Alejandro Flores and deposited at the HUMO Herbarium of the Universidad Autónoma del Estado de Morelos (UAEM), with the voucher number 27194.

2.2. Preparation of Extracts

The collected plant material was air dried and grounded using an electric mill (749 g), and four extracts of L. scabrellum were obtained with solvents of different polarity (hexane, ethyl acetate, dichloromethane, and methanol). The extracts of dichloromethane and methanol exhibited the major content of secondary metabolites; nonpolar metabolites were present in the dichloromethane extract while polar metabolites were in the methanol extract. The grounded plant was extracted with CH₂Cl₂:MeOH (50/50 v/v), in order to obtain the majority of the compounds, and sonicated during 30 min (×3). The extract was concentrated in a Buchi rotary evaporator at 40°C and stored at room temperature for further chemical fractionation. The extract yield was 19.4 g.

2.3. Isolation of Compounds

Compounds were isolated by means of open column chromatography (CC). The isolation procedures and purity of compounds were checked by thin layer chromatography (TLC), visualized by means of UV light, and sprayed with Ce(SO₄)₂·2(NH₄)₂SO₄·2H₂O. The UV spectra were recorded on a Hewlett Packard 8452A spectrophotometer. All ¹H, ¹³C, and 2D NMR experiments were recorded in CDCl₃, acetone-d₆, and CD₃OD on a Varian Unity 400 spectrometer at 400 MHz for ¹H NMR, and at 100 MHz for ¹³C NMR. FABMS spectra were recorded on a JEOL JMX-AX 505 HA mass spectrometer. The IR spectrum was recorded on a Perkin Elmer FTIR spectrophotometer (Perkin Elmer, USA) using KBr, measured in cm⁻¹. Melting points were determined on a Fisher-Johns Melting Point apparatus. Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter at 25°C. All the reagents and solvents used were of analytical grade. The CH₂Cl₂:MeOH extract (19.4 g) was fractionated in an open chromatographic column (60 cm × 9 cm) previously packed with silica gel (582 g, 70–230 mesh; Merck) and eluted with a dichloromethane/methanol gradient system, starting with 100% of the less polar solvent and subsequently increasing the methanol. Seventy-eight fractions of 100 mL each were collected and concentrated by vacuum distillation and grouped according to their chemical content as monitored by TLC. Eight groups of fractions were obtained: F-1 (1.3 g, 100 : 0), F-2 (2.5 g, 98 : 2), F-3 (1.0 g, 96 : 4), F-4 (1.82 g, 94 : 6), F-5 (3.71 g, 92 : 8), F-6 (2.88 g, 90 : 10), F-7 (2.75 g, 85 : 15), and F-8 (0.90 g, 0 : 100). In the less polar fraction (F-1) oleic acid (1, 180 mg) and octadecenoic acid (2, 320 mg) were identified. After a successive chromatographic process using CH₂Cl₂:MeOH gradient system, F-2 (100 : 00→97 : 03) yielded stigmasterol (3, 25 mg) and α-amyrin (4, 14 mg), F-5 (100 : 00→85 : 15) afforded pinoresinol (5, 4.9 mg), 6-methoxypodophyllotoxin (6, 40 mg), and coniferin (7, 6.5 mg) and F-6 (100 : 00→80 : 20) yielded 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8, 503.1 mg).

2.4. Cytotoxic Assay

The CH₂Cl₂:MeOH of aerial and root extracts as well as compounds 6, 7, and 8 were subjected to a cytotoxic evaluation using breast (MCF7), colon (HF6), prostate (PC3), and cervical (SiHa) human cancer cell lines from ATCC (American Type Culture Collection, USA), along with normal human fibroblast cells (HSF-30) at passage number 33 (In Vitro Co.). Cell cultures were grown in RPMI-1640 medium (Sigma Aldrich) supplemented with fetal bovine serum 10% (SFB, Invitrogen), NaHCO₃ 7.5%, and cultivated in 96-well plates (10⁴ cells/mL) at 37°C with CO₂ 5% (humidity 100%). Normal human fibroblasts were grown in DMEM medium (Invitrogen) supplemented with fetal bovine serum 10%. The cells in a log phase of their growth cycle were treated in triplicate with various concentrations of the test samples (0.16–20 μg/mL) dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich) and incubated for 72 h in the conditions described above. In order to guarantee that the cells were in exponential growth, the criteria of confluence between 60–70% were adopted. The cell concentration was determined by the NCI sulforhodamine method [31]. The results were expressed as the dose that inhibits 50% control growth after the incubation period (IC₅₀). The values were estimated from a log⁡10 plot of the drug concentration against the percentage of viable cells. The standards included as controls were PTOX and Taxol (Sigma Aldrich). Extracts with IC₅₀ ≤ 20 μg/mL and compounds with IC₅₀ ≤ 4 μg/mL were considered active according to the National Cancer Institute (NCI) guidelines [32]. The data analysis was processed using the program ORIGIN 8.0, IC₅₀ values were obtained by a regression curve with coefficient factors R² between 0.80 and 0.99.

2.5. Cell Cycle Analysis

Prostate cancer cells PC3 (1 × 10⁵) were plated in 6-well plates and allowed to attach overnight at 37°C in 5% CO₂. Exponential growing cells were exposed at four different concentrations of 6MPTOX in accordance with IC₅₀ values (0.011 μM, 0.005 μM, 0.002 μM, and 0.001 μM) for 72 h. Cells from each treatment were trypsinized and collected into single cell suspensions, centrifuged, and fixed in cold ethanol (70%) overnight at −20°C. The cells were then treated with RNase (0.01 M, Sigma Aldrich) and stained with propidium iodide (PI) (7.5 μg/mL, Invitrogen) for 30 min in the dark, PI has the ability to bind to DNA molecules, and then RNase was added in order to allow PI to bind directly to DNA. The percentage of cells in G1, S, and G2 phases was analyzed with a flow cytometer (Becton, Dickinson, FACS Calibur, San Jose, CA); the number of cells analyzed for each sample was 10,000. Data obtained from the flow cytometer were analyzed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA) to generate DNA content frequency histograms, and to quantify the number of cells in the individual cell cycle phases.

2.6. Immunofluorescence of Histone H3 Phosphorylated

PC3 cells were grown in RPMI medium, and 7.5 × 10⁴ cells were added in 24-well culture plates containing slides and allowed to attach overnight at 37°C in 5% CO₂. Then, they were treated with 6MPTOX (0.005 μM), PTOX (0.002 μM), and DMSO (2.25 μM) at 37°C for 72 h. The cells were fixed with PFA (p-formaldehyde) 4% in PEM buffer PIPES (0.1 M), EGTA (2 mM), and MgSO₄ (1 mM), pH 6.95. After 15 min PFA/NaHCO₃ was added and incubated for 45 min at room temperature. The slides were rinsed with PBS, treated with 0.1% Triton X-100 (Sigma Aldrich), and then incubated with the primary antibody antiphospho-histone H3 (1 : 500, Santa Cruz Biotechnology) overnight at 4°C. A secondary antibody anti-rabbit Alexa 488 (1 : 1000, Molecular Probes) was added and incubated for 1 h at 37°C. The cells were stained with 0.4 μg/mL 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR) in PBS for 10 min, then mounted, and imaged by fluorescence microscopy.

2.7. Immunofluorescence of α-Tubulin

PC3 cells were grown in RPMI medium, and 7.5 × 10⁴ cells were added in 24-well culture plates containing slides and allowed to attach overnight at 37°C in 5% CO₂. Then they were treated with 6MPTOX (0.005 μM), PTOX (0.002 μM), and DMSO (2.25 μM-0.02%) at 37°C for 72 h. The cells were fixed with PFA (paraformaldehyde) 4% in PEM (buffer). After 15 min PFA/NaHCO₃ was added and incubated for 45 min at room temperature. The slides were rinsed with PBS and treated with 0.1% Triton X-100 (Sigma Aldrich) and then incubated with the first antibody anti-α-tubulin (1 : 300, Sigma Aldrich) overnight at 4°C. A second antibody anti-mouse Alexa 647 (1 : 1000, Molecular Probes) was added and incubated for two hours at 37°C. The cells were stained with Sytox Green (1 : 5000) for one hour, mounted, and imaged by confocal microscopy.

2.8. Determination of the Binding Site and Affinity Constant K_b

The binding affinity constant (K_b) of 6MPTOX was determined following the method described previously [33]. Displacement of the colchicine analogue MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one], a commercial reversible tubulin ligand in the colchicine site that binds to the heterodimer α/β tubulin, was evaluated. Initially, mixtures of tubulin and MTC (5 μM) were incubated in 10 mM of buffer GAB (NaPi with 0.1 mM GTP and pH 7). Subsequently, increasing concentrations of the above tested compound and controls were added and incubated for 10 min between each concentration at 25°C in the fluorometer Horiba Fluoromax-2, with λ exc = 350 nm and λ em = 423 nm. Binding constants were calculated by the decrease of the fluorescence of MTC due to competition for the same site. Data processing was analyzed using the Equigra v5 software [34].

2.9. Cell Death

PC3 cells (7.5 × 10⁴ cells/mL) were seeded in 6-well plates and incubated for 18 h. Exponentially growing cells were treated for 72 h with RPMI medium added with 0.02% of DMSO (control) and with four different concentrations of 6MPTOX (0.011 μM, 0.005 μM, 0.002 μM, and 0.001 μM) in accordance with the IC₅₀ value of the compound. Cells treated with H₂O₂ were used as apoptotic death control [35] and for the necrotic control PC3 cells were treated with boiling water. Then, each cell culture was washed with 100 mL of PBS and stained with 100 μL AO/EB solution (100 μg/mL AO, 100 μg/mL EB), according to reported procedures [36]. The cells were observed using a fluorescence microscope (Olympus Co., Tokyo, Japan, with emission at 521 nm). AO/EB are intercalating nucleic acid-specific fluorochromes, and when bounded to DNA they emit green and orange fluorescence, respectively. It is well known that AO can pass through cell membranes, but EB cannot. Under the fluorescence microscope, living cells appear green. Necrotic cells stain red but have a nuclear morphology resembling that of viable cells. Apoptotic cells appear green, and morphological changes such as formation of apoptotic bodies are observed. The criteria for identification are as follows: (i) viable cells appear to have green nucleus with intact structure; (ii) early apoptosis cells exhibit a bright-green nucleus showing condensation of chromatin; (iii) late apoptosis appears as dense orange areas of chromatin condensation; and (iv) orange intact nucleus depicts secondary necrosis [37].

3. Results and Discussion

3.1. Cytotoxic Activity and Chemical Composition of Linum scabrellum

The CH₂Cl₂:MeOH extract from aerial parts was noncytotoxic to the tested cell lines (IC₅₀ ≤ 20 μg/mL). The CH₂Cl₂:MeOH roots extract of L. scabrellum presented cytotoxic activity against the four carcinoma cell lines exhibiting IC₅₀ values from 0.56 to 1.54 μg/mL as well as toward the normal cell line with an IC₅₀ value of 1.02 μg/mL (Table 1). The first complete phytochemical profile of L. scabrellum was established in order to identify the cytotoxic metabolites. In the CH₂Cl₂:MeOH extract, eight compounds were identified and characterized (Figure 1), three of them (5, 6, and 8) belong to the lignan group. Four compounds, oleic acid (1), octadecenoic acid (2) stigmasterol (3), and α-amyrin (4), were identified by their spectroscopic data ¹H and ¹³C and cochromatography with authentic samples available on our laboratory. The other four compounds were identified by their spectroscopic data ¹H, ¹³C NMR, spectra mass, mp, UV, IR, and 2D experiments (COSY, HSQC) as follows: F-5 was subjected to column chromatography packed with silica gel (115 g) and eluted with a gradient system of CH₂Cl₂:MeOH (100 : 00→90 : 10) obtaining 46 fractions of 25 mL each and were grouped in six groups according to their similarity in thin layer chromatography (F-5A→ F-5F).

Fraction F-5B eluted with 98:02 CH₂Cl₂:MeOH afforded 4.9 mg of pinoresinol (5), isolated as amorphous powder, mp 121–22°C (lit: 122°C) an optically active powder [α]²⁴_D −82.3° (c 0.012, Me₂CO), and had the formula C₂₀H₂₂O₆ derived from its positive FABMS [M+H]⁺ ion at m/z 359. IR (KBr, υ_max in cm⁻¹): 3210, 2870, 1600, 1465 1050. UV λ_max (nm): 232, and 280. ¹H NMR (400 MHz, CDCl₃) δ_H: 6.89 (d, J = 1.2 Hz; H-2, H-2′), 6.88 (d, J = 8.4 Hz; H-5, H-5′), 6.81 (dd, J = 2.1 and 8.4 Hz; H-6, H-6′), 4.73 (d, J = 4.2 Hz; H-7, H-7′), 3.09 (c, H-8, H-8′), 4.24 (dd, J = 7 and 9.1 Hz; 9-Hβ, 9′-Hβ), 3.87 (dd, J = 3.5 and 9.1 Hz; 9-Hα, 9′-Hα), 3.90 (s, C3′-OMe, C3-OMe). ¹³C NMR (100 MHz, CDCl₃) δ_C: 132.90 (C-1, C-1′), 108.57 (C-2, C-2′), 146.68 (C-3, C-3′), 145.22 (C-4, C-4′), 114.24 (C-5, C-5′), 118.96 (C-6, C-6′), 85.86 (C-7, C-7′), 54.16 (C-8, C-8′), 71.66 (C-9, C-9′). On the basis of the spectral data, compound 5 was identified as (-)-pinoresinol [38].

One of the fractions F-5C eluted with 96:04 CH₂Cl₂:MeOH yielded 40 mg of compound 6, obtained as a white amorphous powder, mp 202-203°C (lit: 203°C). The positive FABMS spectrum showed the [M + H]⁺ ion peak at m/z 445 with molecular formula C₂₃H₂₄O_9. IR (KBr, υ_max in cm⁻¹): 3220, 1768, 1600. UV λ_max (nm): 272, and 282. ¹H NMR (400 MHz, acetone-d₆) δ_H: 6.30 (s, H-3), 5.03 (d, J = 9.8 Hz, H-7), 2.88 (m, H-8), 4.06 (dd, J = 8.4 and 10 Hz; H-9β), 4.64 (t, J = 7.7 and 8.4 Hz; H-9α), 6.44 (s, H-2′, H-6′), 4.53 (d, J = 4.9 Hz, H-7′), 2.75 (dd, J = 4.2 and 14.7 Hz; H-8′), 3.78 (s, C3′-OMe, C5′-OMe), 3.81 (s, C4′-OMe), 3.78 (s, C6-OMe), 5.95 (s, –O-CH₂-O-). ¹³C NMR (100 MHz, acetone-d₆) δ_C: 124.93 (C-1), 137.04 (C-2), 104.36 (C-3), 149.45 (C-4), 132.84 (C-5), 141.57 (C-6), 70.52 (C-7), 39.01 (C-8), 71.92 (C-9), 134.71 (C-1′), 108.09 (C-2′), 152.61 (C-3′), 134.93 (C-4′), 152.61 C-5′), 108.09 (C-6′), 45.11 (C-7′), 44.53 (C-8′), 174.44 (C-9′), 56.18 (C3′-OMe), 60.77 (C4′-OMe), 56.18 (C5′-OMe), 59.93 (C6-OMe), 101.37 (-O-CH2-O-). On the basis of the spectral data, compound 6 was identified as 6-methoxypodophyllotoxin [39].

Fraction F-5C eluted with 90 : 10 CH₂Cl₂:MeOH yielded 6.5 mg of coniferin (7), obtained as an amorphous solid, mp 180–82°C [lit: 182°C]. Positive FABMS m/z 342 [M + H]⁺, with molecular formula C₁₆H₂₂O₈. IR (KBr, υ_max in cm⁻¹): 3020, 1060, 940. UV λ_max (nm): 256 and 292. [α]²⁴_D −66.3 (c 0.010, Me₂CO). ¹H NMR (400 MHz, CDCl₃) δ_H: 7.07 (d, J=2.4 Hz; H-3), 6.89 (dd, J = 1.6 and 8 Hz; H-5), 7.09 (d, J = 8 Hz; H-6), 6.52 (d, J = 15.2 Hz; H-1′), 6.29 (td, J = 5.6 and 16 Hz; H-2′), 4.19 (t, J = 5.6 Hz; H-3′), 4.87 (d, J = 7.7 Hz; H-1′′), 4.23 (t, J = 7.8 Hz, H-2′′), 4.22 (t, J = 7.6 Hz, H-3′′), 4.21 (dd, J = 7.6 and 7.8 Hz, H-4′′), 4.05 (m, H-5′′), 3.54 (t, J = 9.6 Hz, H-6′′a), 3.70 (dd, J = 4.5 and 9.8 Hz, H-6′′b), 3.79 (s, C2-OMe). ¹³C NMR (100 MHz, acetone-d₆) δ_C: 150.01 (C-1), 146.54 (C-2), 110.24 (C-3), 132.36 (C-4), 117.11 (C-5), 119.23 (C-6), 128.88 (C-1′), 128.87 (C-2′), 62.36 (C-3′), 55.53 (C2-OMe), 101.69 (C-1′′), 73.92 (C-2′′), 77.07 (C-3′′), 70.69 (C-4′′), 76.93 (C-5′′), 61.82 (C-6′′). On the basis of spectral data, compound 7 was identified as phenylpropane glycoside coniferin (7) [40].

From the fraction F-6, compound 8 was isolated as an amorphous yellow solid, mp 287°C [lit: 287°C]. The positive FABMS spectrum showed the [M + H]⁺ ion peak at m/z 607, with molecular formula C₂₉H₃₄O_14. IR (KBr, υ_max in cm⁻¹): 3020, 1746, 1620, 1050, 920. UV λ_max (nm): 276 and 284. ¹H NMR (400 MHz, CD₃OD) δ_H: 6.28 (s, H-1), 5.48 (d, 7.2 Hz; H-7), 2.90 (m, H-8), 4.23 (t, J = 8.8 and 9.6 Hz; H-9β), 4.71 (t, J = 8.4 and 8.6 Hz; H-9α), 6.31 (s, H-2′, H-6′), 4.51 (d, J = 4.8 Hz; H-7′), 3.25 (dd, J = 4.8 and 14 Hz; H-8′), 3.72 (s, C3′-OMe, C5′-OMe), 3.99 (s, C4′-OMe), 3.68 (s, C6-OMe), 6.02 (s,-O-CH2-O-), 4.54 (d, J = 7.6 Hz; H-1′′), 3.28 (t, J = 9.2 Hz; H-2′′), 3.35 (t, J= 7.2 Hz; H-3′′), 3.41 (dd, J = 7.2 y 6.8 Hz; H-4′′), 3.43 (m, H-5′′), 3.76 (dd, J = 2.2 and 10 Hz, H-6′′a), 3.84 (dd, J = 5.4 and 10.2 Hz, H-6′′b). ¹³C NMR (100 MHz, CD₃OD) δ_C: 124.26 (C-1), 136.28 (C-2), 105.74 (C-3), 149.68 (C-4), 135.89 (C-5), 140.26 (C-6), 72.78 (C-7), 39.87 (C-8), 72.39 (C-9), 136.73 (C-1′), 108.58 (C-2′), 152.89 (C-3′), 136.54 (C-4′), 152.89 (C-5′), 108.58 (C-6′), 45.86 (C-7′), 46.23 (C-8′), 176.67 (C-9′), 56.24 (C3′-OMe, C5′-OMe), 60.76 (C4′-OMe), 60.76 (C6-OMe), 102.34 (-O-CH2-O-), 100.46 (C-1′′), 74.87 (C-2′′), 77.85 (C-3′′), 71.67 (C-4′′), 78.13 (C-5′′), 63.08 (C-6′′). On the basis of spectral data, compound 8 was identified as 6-methoxypodophyllotoxin 7-O-β-D-glucopyranoside [40].

The obtained data were compared with the literature [41–43] (see Supplementary Material Figures 1–6 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/298463).

Some studies reported that compounds 1, 2, and 4 possess anti-inflammatory activity [42] while compound 5 has chemopreventive properties. [38]. Even though all of the 8 compounds had been previously reported in members of the genus Linum [44–47], this is the first time that they were identified in Linum scabrellum.

3.2. Cytotoxic Activity of Purified Compounds

The isolated compounds 6, 7, and 8 were evaluated for cytotoxic effect against the above mentioned cell lines. Compounds 7 and 8 were nonactive (IC₅₀ ≥ 20 μg/mL), while compound 6 (6MPTOX) showed an important cytotoxic activity with IC₅₀ values ranging from 0.0632 to 2.7433 μg/mL against the cancer tested cell lines (Table 1). It is important to point out that 6MPTOX was also toxic against the normal fibroblasts. These data showed that the inhibitory effect of 6MPTOX as well as that of the CH₂Cl₂:MeOH roots extract of Linum scabrellum is not specific against certain carcinomas but rather exerts a general cell toxic action. Because the important cytotoxic activity demonstrated by 6MPTOX, we decided to investigate the mechanism of action of this compound.

3.3. Effect of 6MPTOX on PC3 Cell Cycle Arrest

The effect of 6MPTOX in the cell cycle progression of PC3 cells was determined at four concentrations: 0.011 μM, 0.005 μM, 0.002 μM, and 0.001 μM. DMSO (2.55 μM-0.02%) and PTOX (0.0024 μM) were used as negative and positive controls, respectively. Figure 2 displays DNA histograms of PC3 cell cycle. The 6MPTOX induced in PC3 a G2/M phase cell arrest. The effect was dose-dependent since cell arrest in G2/M was declining, while the G1 phase cell population was increasing when the 6MPTOX concentration decreased. 6MPTOX and PTOX at 0.0002 μM induced a similar effect on G2/M cell arrest with 39.8% and 41.1% of G2/M cell population, respectively. These results suggest that 6MPTOX efficiently arrest cells at G2/M phase.

3.4. Effect of 6MOTX on Mitotic Arrest

Histone H3 phosphorylated at serine 10 has long been used to identify mitotic cells nuclei in culture cell lines [48]. The proportion of cells exhibiting mitosis in the PC3 cultures was determined using an antibody antiphospho-histone H3, after treatment with 6MPTOX. The mitotic index was calculated by image analysis. Taxol and PTOX were used as positive controls, while DMSO was used as the negative control. 6MPTOX showed a mitotic index of 0.1, Taxol of 0.25, and PTOX of 0.2, while the negative control displayed a mitotic index of 0.035 (Figure 3). These results suggest that 6MPTOX induces mitotic arrest as did PTOX and Taxol, in contrast to the negative control. Inhibition of tubulin has been implicated in G2/M phase of the cell cycle arrest in various cancer cell lines [49]. It is well known that Taxol stabilizes microtubules protecting them from depolymerization, which blocks cells in mitosis and induces apoptosis [50], in contrast with PTOX, which promotes depolymerization by destabilizing microtubules and arrests the cell cycle in mitosis [51].

Our results showed that 6MPTOX induced a mitotic cell arrest by the observed high mitotic index and the G2/M cell arrest, suggesting a similar effect as that displayed by PTOX or Taxol. In order to determine the mechanism of action of 6MPTOX, we analyzed the effect of microtubule polymerization by 6MPTOX through disruption of α/β tubulin binding.

3.5. Effect of 6MPTOX on the α-Tubulin Polymerization

Microtubules are in dynamic equilibrium with tubulin dimers as tubulin is polymerized into microtubules and depolymerized as free tubulin. This dynamic equilibrium is targeted by microtubule disrupting agents [52], which inhibit the organization of the mitotic spindle and arrest chromosomes in metaphase of mitosis [53]. The effect of 6MPTOX on the microtubule polymerization by immunofluorescence of α-tubulin was observed in PC3 cells by confocal microscopy. Figure 4(b) shows that treatment with 6MPTOX inhibits the polymerization of microtubules possibly through binding to tubulin, of which effect was also observed by treatment with PTOX (Figure 4(c)). In the negative control (DMSO 0.02%, Figure 4(a)) no effect was appreciated.

These results suggest that 6MPTOX as well as PTOX act as microtubule-destabilizing agents, while cells treated with DMSO (0.02%) demonstrated a normal and intact tubulin organization (Figure 4(a)).

3.6. Determination of the Binding Site and Binding Constant (K_b) of 6MPTOX

To corroborate whether 6MPTOX binds to α/β-tubulin as does PTOX, 6MPTOX was assayed for its ability to displace MTC from its binding at the colchicine site of α/β-tubulin [54, 55]. Figure 5 shows fluorescence changes produced by the displacement of MTC binding to tubulin by both 6MPTOX and PTOX. The fluorescence spectra of MTC decreased by 43% with 6MPTOX indicating that 6MPTOX binds to the colchicine binding site, while PTOX decreased 65%. The binding constant for 6MPTOX was determined as K_b = 7.26 × 10⁶ M⁻¹ by displacement of MTC in the colchicine binding site. MTC has K_b = 4.7 × 10⁵ M⁻¹ [33], which indicated that 6MPTOX is a stronger inhibitor when compared to MTC.

3.7. Effect of 6MPTOX on Cellular Death of PC3 Cells

Cell death occurs through at least three morphologically distinct subroutines that have been named apoptosis, autophagy cell death (ACD), and necrosis [56]. Apoptosis is morphologically defined by nuclear shrinkage and fragmentation [57], whereas necrosis is defined by early permeabilization of the plasma membrane [58]. The effect of 6MPTOX on cellular death of prostate cancer cells was determined using four different concentrations. The compound 6MPTOX at a lower concentration of 0.001 μM did not show any effect on cell death. When the concentration was increased to 0.002 μM some apoptotic bodies were observed (Figure 6(e)); with 0.005 μM, few cells were in late apoptosis and others were necrotic (orange or red color, Figure 6(f)); and at the highest concentration of 0.011 μM the effect caused cell death mainly by necrosis (Figure 6(g)). Three controls were used: boiling water for necrosis, where most of the cells stained in red (Figure 6(c)); H₂O₂ for apoptosis, where some of the cells stained in green or orange with apoptotic bodies (Figure 6(b)), and a negative control with most of the cells stained in green (cells without treatment, Figure 6(a)). The effect of 6MPTOX on prostate cancer cells was dose-response, inducing apoptosis followed by necrosis when the concentration increased.

4. Conclusion

For the first time, the metabolic content of the Mexican species L. scabrellum was established. Eight compounds including lignans, terpenes, and fatty acids were isolated and identified. This study demonstrated the cytotoxic activity of 6MPTOX toward four selected human carcinomas and against a normal fibroblast cell line. Biochemical and biological experiments showed that 6MPTOX specifically arrested PC3 cells in their G2/M phase as well as a higher mitotic index. Moreover, at its lowest concentration it induces apoptosis as shown by an increase in its subG1 cell population and in stained apoptotic cells. Necrotic cell death was observed at higher concentrations. Data presented in this study show that 6MPTOX binds at the colchicine binding site of tubulin with a K_b = 7.26 × 10⁶ M⁻¹, causing disruption of tubulin polymerization. These results showed that 6MPTOX inhibit tubulin polymerization and arrest cells in G2/M as a mechanism of cytotoxic activity in PC3 cells. This is the first report describing the mechanism of action of 6MPTOX.

Supplementary Material

Supplementary Material provides all the spectroscopic data of isolated compounds (NMR and MS spectra).

298463.f1.pdf

Figure 1

Chemical structure of Linum scabrellum secondary metabolites.

Figure 2

Effect of 6MPTOX on cell cycle in the prostate PC3 cell line. (a) Negative control with DMSO 0.02%; (b) PC3 cells exposed to 0.002 μM PTOX as positive control; (c–f) PC3 cells exposed to different concentrations of 6MPTOX; (c) 0.011 μM; (d) 0.005 μM; (e) 0.002 μM; (f) 0.001 μM.

Figure 3

Effect of 6MPTOX on mitosis of the prostate cancer cell line PC3.

Figure 4

Effect of microtubules polymerization. (a) DMSO 0.02%, (b) 6MPTOX, and (c) PTOX.

Figure 5

Displacement of MTC by 6MPTOX measured with fluorescence, (a) MTC with tubulin, (b) 6MPTOX, (c) PTOX, and (d) MTC.

Figure 6

Effect of 6MPTOX on the prostate cancer cells (PC3). (a) Negative control; (b) apoptosis control; (c) necrosis control; (d–g) cells exposed to different concentrations of 6MPTOX; (d) 0.001 μM; (e) 0.002 μM; (f) 0.005 μM; (6) 0.011 μM. The arrow points out the cells that were amplified (40x). (b) Apoptotic cells; (c) necrotic cells; (e) apoptotic cells (0.002 μM); (f) late apoptosis (0.005 μM); (g) necrotic cells (0.011 μM).

Table 1

IC₅₀ values (μg/mL) of dichloromethane/methanol extract and 6MPTOX isolated from Linum scabrellum.

Extract/compound	Cell lines
Extract/compound	PC3	MCF7	HF6	SiHa	HFS-30
CH₂Cl₂:MeOH extract	1.60 ± 0.07	5.7 × 10⁻¹ ± 0.07	5.7 × 10⁻¹ ± 0.07	1.54 ± 0.07	1.02 ± 0.07
6MPTOX	1.7 × 10⁻¹ ± 0.07	6.6 × 10⁻² ± 0.07	7.9 × 10⁻² ± 0.07	2.74 ± 0.07	6.0 × 10⁻² ± 0.07
PTOX	3 × 10⁻³ ± 0.07	1 × 10⁻⁴ ± 0.07	1.4 × 10⁻³ ± 0.07	1.23 ± 0.07	1 × 10⁻⁴ ± 0.07
Taxol	6.2 × 10⁻¹ ± 0.07	1.3 × 10⁻¹ ± 0.07	2.8 × 10⁻¹ ± 0.07	2.30 ± 0.07	1.2 × 10⁻¹ ± 0.07

PC3: prostate cancer, MCF7: breast cancer, HF6: colon cancer, SiHa: cervical cancer, and HFS-30: fibroblasts.

Acknowledgments

Ivonne Alejandre-García acknowledges fellowship 226354 from CONACYT. The authors thank Dr. Alfonso Lejia from Centro de Ciencias Genómicas, UNAM, for technical assistance. The authors are indebted to Dr. T Fitzgerald from Florida A&M University for his kind gift of the colchicine analog 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). Partial support from CONACYT (Grants CB 156276 and 222714) is acknowledged. The authors thank Laboratorio Nacional de Estructura de Macromoléculas (Conacyt 251613) for the spectroscopic and mass analyses and support given to J. F. D. from BIPPED2 and BIO2013-42984R from the Ministry of Economy of Spain.

Conflict of Interests

The authors declare no conflict of interests.

References

1. VasilevN.EbelR.EdradaR.Metabolic profiling of lignan variability in Linum species of section Syllinum native to BulgariaPlanta Medica2008743273280[PubMed][Google Scholar]
2. EichE.PertzH.KalogaM.(−)-Arctigenin as a lead structure for inhibitors of human immunodeficiency virus type-1 integraseJournal of Medicinal Chemistry19963918695[PubMed][Google Scholar]
3. LiuK. C. S.LeeS.-S.LinM.-T.Lignans and tannins as inhibitors of viral reverse transcriptase and human DNA polymerase-α: QSAR analysis and molecular modelingMedicinal Chemistry Research199771168179[PubMed][Google Scholar]
4. HaraH.FujihashiT.SakataT.KajiA.KajiH.Tetrahydronaphthalene lignan compounds as potent anti-HIV type 1 agentsAIDS Research and Human Retroviruses1997138695705[PubMed][Google Scholar]
5. GhisalbertiE. L.Cardiovascular activity of naturally occurring lignansPhytomedicine199742151166[PubMed][Google Scholar]
6. CostaM. C. A.TakahataY.Conformational analysis of synthetic neolignans active against leishmaniasis, using the molecular mechanics method (MM2)Journal of Computational Chemistry1997185712721[Google Scholar]
7. IwasakiT.KondoK.NishitaniT.Arylnaphthalene lignans as novel series of hypolipidemic agents raising high-density lipoprotein levelChemical and Pharmaceutical Bulletin1995431017011705[PubMed][Google Scholar]
8. KurodaT.KondoK.IwasakiT.OhtaniA.TakashimaK.Synthesis and hypolipidemic activity of diesters of arylnaphthalene lignan and their heteroaromatic analogsChemical and Pharmaceutical Bulletin1997454678684[PubMed][Google Scholar]
9. DelormeD.DucharmeY.BrideauC.Dioxabicyclooctanyl naphthalenenitriles as nonredox 5-lipoxygenase inhibitors: structure-activity relationship study directed toward the improvement of metabolic stabilityJournal of Medicinal Chemistry1996392039513970[PubMed][Google Scholar]
10. ZacchinoS.RodríguezG.PezzenatiG.OrellanaG.EnrizR.SierraM. G.In vitro evaluation of antifungal properties of 8.O.4′-NeolignansJournal of Natural Products1997607659662[PubMed][Google Scholar]
11. Rantapaa-DahlqvistS.LandbergG.RoosG.NorbergB.Cell cycle effects of the anti-rheumatic agent CPH82British Journal of Rheumatology1994334327331[PubMed][Google Scholar]
12. LerndalT.SvenssonB.A clinical study of CPH 82 vs. methotrexate in early rheumatoid arthritisRheumatology2000393316320[PubMed][Google Scholar]
13. LeanderB.RosenK.Medicinal used for podophyllotoxinUS patent: 4, pp. 788, 1988
14. IwasakiT.KondoK.KurodaT.Novel selective PDE IV inhibitors as antiasthmatic agents. Synthesis and biological activities of a series of 1-aryl-2,3-bis(hydroxymethyl)naphthalene lignansJournal of Medicinal Chemistry1996391426962704[PubMed][Google Scholar]
15. DamayanthiY.LownJ. W.Podophyllotoxins: current status and recent developmentsCurrent Medicinal Chemistry199853205252[PubMed][Google Scholar]
16. HandeK. R.Etoposide: four decades of development of a topoisomerase II inhibitorEuropean Journal of Cancer1998341015141521[PubMed][Google Scholar]
17. HemmatiS.MohagheghzadehA.AlfermannA. W.Quantification of aryltetralin lignans in Linum album organs and in vitro culturesIranian Journal of Pharmaceutical Sciences2006214756[Google Scholar]
18. KonuklugilB.SchmidtT. J.AlfermannA. W.Accumulation of lignans in suspension cultures of Linum mucronatum ssp. Armenum (Bordz.) DavisZeitschrift fur Naturforschung—Section C: Journal of Biosciences2001561111641165[PubMed][Google Scholar]
19. DumontetC.JordanM. A.Microtubule-binding agents: a dynamic field of cancer therapeuticsNature Reviews Drug Discovery2010910790803[PubMed][Google Scholar]
20. AyresD. C.LoikeJ. D.Lignans, Chemical, Biological and Clinical Properties19901stCambridge, UKCambridge University Press
21. BussA. D.WaighR. D.WolffM. E.Natural Products as Leads for New Pharmaceuticals19951stNew York, NY, USAJohn Wiley & Sons
22. GordalizaM.CastroM. A.del CorralJ. M. M.San FelicianoA.Antitumor properties of podophyllotoxin and related compoundsCurrent Pharmaceutical Design200061818111839[PubMed][Google Scholar]
23. van UdenW.HomanB.WoerdenbagH. J.Isolation, purification, and cytotoxicity of 5-methoxypodophyllotoxin, a lignan from a root culture of Linum flavumJournal of Natural Products1992551102110[PubMed][Google Scholar]
24. RojasA. M.MendietaM. A.AntúnezM. Y.Cytotoxic podophyllotoxin type-lignans from the steam bark of Bursera fagaroides var. FagaroidesMolecules201217895069519[PubMed][Google Scholar]
25. CastroM. Á.del CorralJ. M. M.GarcíaP. A.Synthesis and biological evaluation of new podophyllic aldehyde derivatives with cytotoxic and apoptosis-inducing activitiesJournal of Medicinal Chemistry2010533983993[PubMed][Google Scholar]
26. NepomucenoA.IshikiM.Las plantas empleadas para el tratamiento de las infecciones respiratorias en los altos de Chiapas (México)Etnobiología20108111130[Google Scholar]
27. ZollaC.ArguetaA.Atlas de las Plantas de la Medicina Tradicional Mexicana19941stMéxico, MexicoBiblioteca de Medicina Tradicional Mexicana, Instituto Nacional Indigenista
28. RzedowskiJ.RzedowskyG.Flora del Bajío y Regiones Adyacentes19926Instituto de Ecología
29. MuirA.WestcotN.Flax: The Genus Linum20033rdTaylor & Francis
30. LautiéE.QuinteroR.FliniauxM.-A.VillarrealM.-L.Selection methodology with scoring system: application to Mexican plants producing podophyllotoxin related lignansJournal of Ethnopharmacology20081203402412[PubMed][Google Scholar]
31. SkehanP.StorengR.ScudieroD.New colorimetric cytotoxicity assay for anticancer-drug screeningJournal of the National Cancer Institute1990821311071112[PubMed][Google Scholar]
32. SuffnessM.PezzutoJ. M.HostettmannK.Assays related to cancer drug discoveryMethods in Plant Biochemistry: Assays for Bioactivity, 61990London, UKAcademic Press71133[Google Scholar]
33. la ReginaG.EdlerM. C.BrancaleA.Arylthioindole inhibitors of tubulin polymerization. 3. Biological evaluation, structure−activity relationships and molecular modeling studiesJournal of Medicinal Chemistry2007501228652874[PubMed][Google Scholar]
34. BueyR. M.BarasoainI.JacksonE.Microtubule interactions with chemically diverse stabilizing agents: thermodynamics of binding to the paclitaxel site predicts cytotoxicityChemistry and Biology2005121212691279[PubMed][Google Scholar]
35. YangG.-Y.LiaoJ.KimK.YurkowE. J.YangC. S.Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenolsCarcinogenesis1998194611616[PubMed][Google Scholar]
36. KasibhatlaS.Amarante-MendesG.FinucaneD.BrunnerT.Bossy-WetzelE.Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosisCold Spring Harbor Protocols200620063p. 4493[PubMed][Google Scholar]
37. MohanS.BustamamA.IbrahimS.In vitro ultramorphological assessment of apoptosis on CEMss induced by linoleic acid-rich fraction from Typhonium flagelliforme tuberEvidence-Based Complementary and Alternative Medicine2011201112[PubMed][Google Scholar]
38. FiniL.HotchkissE.FoglianoV.Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of the ATM-p53 cascade in colon cancer cell linesCarcinogenesis2008291139146[PubMed][Google Scholar]
39. San FelicianoA.del CorralJ. M. M.GordalizaM.CastroA.Lignans from Juniperus sabinaPhytochemistry199029413351338[PubMed][Google Scholar]
40. BroomheadA. J.DewickP. M.Aryltetralin lignans from Linum flavum and Linum capitatumPhytochemistry1990291238393844[PubMed][Google Scholar]
41. van MinhC.NhiemN. X.YenH. T.Chemical constituents of Trichosanthes kirilowii and their cytotoxic activitiesArchives of Pharmacal Research2015[PubMed][Google Scholar]
42. CorreaG.Da Costa AbreuV.MartinsD.Anti-inflamatory and antimicrobial activities of steroids and triterpenes isolated from aerial parts of Justicia acuminatissima (Acanthaceae)International Journal of Pharmacy and Pharmaceutical Sciences2014667581[Google Scholar]
43. SchmidtT. J.KlaesM.SendkerJ.Lignans in seeds of Linum speciesPhytochemistry2012828999[PubMed][Google Scholar]
44. MohagheghzadehA.GholamiA.SoltaniM.HemmatiS.AlfermannA. W.Linum mucronatum: organ to organ lignan variationsZeitschrift für Naturforschung C2005605-6508510[PubMed][Google Scholar]
45. KonuklugilB.IonkovaI.VasilevN.Lignans from Linum species of sections Syllinum and LinumNatural Product Research200721116[PubMed][Google Scholar]
46. KumarS.YouF. M.DuguidS.BookerH.RowlandG.CloutierS.QTL for fatty acid composition and yield in linseed (Linum usitatissimum L.)Theoretical and Applied Genetics20151285965984[PubMed][Google Scholar]
47. HemmatiS.von HeimendahlC. B. I.KlaesM.AlfermannA. W.SchmidtT. J.FussE.Pinoresinol-lariciresinol reductases with opposite enantiospecificity determine the enantiomeric composition of lignans in the different organs of Linum usitatissimum L.Planta Medica2010769928934[PubMed][Google Scholar]
48. HendzelM. J.WeiY.ManciniM. A.Mitosis-specific phosphorylation of histone H₃ initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensationChromosoma19971066348360[PubMed][Google Scholar]
49. KasibhatlaS.GourdeauH.MeerovitchK.Discovery and mechanism of action of a novel series of apoptosis inducers with potential vascular targeting activityMolecular Cancer Therapeutics200431113651373[PubMed][Google Scholar]
50. HeldmanA. W.ChengL.JenkinsG. M.Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosisCirculation20011031822892295[PubMed][Google Scholar]
51. GordalizaM.GarcíaP. A.del CorralJ. M. M.CastroM. A.Gómez-ZuritaM. A.Podophyllotoxin: distribution, sources, applications and new cytotoxic derivativesToxicon2004444441459[PubMed][Google Scholar]
52. KamalA.Srinivasa ReddyT.PolepalliS.Synthesis and biological evaluation of podophyllotoxin congeners as tubulin polymerization inhibitorsBioorganic & Medicinal Chemistry2014221954665475[PubMed][Google Scholar]
53. HastieS. B.Interactions of colchicine with tubulinPharmacology & Therapeutics1991513377401[PubMed][Google Scholar]
54. FltzgeraldT. J.Molecular features of colchicine associated with antimitotic activity and inhibition of tubulin polymerizationBiochemical Pharmacology1976251213831387[PubMed][Google Scholar]
55. MedranoF. J.AndreuJ. M.GorbunoffM. J.TimasheffS. N.Roles of ring C oxygens in the binding of colchicine to tubulinBiochemistry1991301537703777[PubMed][Google Scholar]
56. KroemerG.GalluzziL.VandenabeeleP.Classification of cell death: recommendations of the nomenclature committee on cell death 2009Cell Death & Differentiation2009161311[PubMed][Google Scholar]
57. KerrJ. F.WyllieA. H.CurrieA. R.Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kineticsBritish Journal of Cancer1972264239257[PubMed][Google Scholar]
58. DegterevA.YuanJ.Expansion and evolution of cell death programmesNature Reviews Molecular Cell Biology200895378390[PubMed][Google Scholar]