Molecular mechanisms of antiproliferative effects ...

Fitoterapia 98 (2014) 241–247

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Molecular mechanisms of antiproliferative effects induced by Schisandra-derived dibenzocyclooctadiene lignans (+)deoxyschisandrin and (−)-gomisin N in human tumour cell lines Elisabetta Casarin a, Stefano Dall'Acqua a,⁎, Karel Šmejkal b, Tereza Šlapetová b, Gabbriella Innocenti a, Maria Carrara a a b

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, I-35131 Padova, Italy Department of Natural Drugs, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, CZ-612 42 Brno, Czech Republic

a r t i c l e

i n f o

Article history: Received 30 May 2014 Accepted in revised form 29 July 2014 Available online 7 August 2014 Keywords: Dibenzocyclooctadiene lignan (+)-Deoxyschisandrin (−)-Gomisin N Schisandra chinensis Antiproliferative activity Apoptosis

a b s t r a c t A different behavior of the two dibenzocyclooctadiene lignans (+)-deoxyschisandrin (1) and (−)-gomisin N (2), from Schisandra chinensis fruits, was observed against two human tumour cell lines, (2008 and LoVo). These lignans inhibited cell growth in a dose-dependent manner on both cell lines, but inducing different types of cell death. In particular, (+)deoxyschisandrin (1) caused apoptosis in colon adenocarcinoma cells (LoVo) but not in ovarian adenocarcinoma cells (2008), while (−)-gomisin N (2) induced apoptosis on both the cell lines used. Mitochondrial-mediated pathway was not involved in apoptotic stimuli. Both compounds caused G2/M phase cell growth arrest correlated with tubulin polymerization. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Natural products have played a crucial role especially on the field of anticancer compounds in last decades [1]. The induction of apoptotic cell death is considered to be an important mechanism of action of possible anti-cancer drugs. Many tumors are able to suppress or completely block apoptotic processes, allowing them to survive despite to the undergoing genetic and morphologic transformations [2]. Therefore, substances targeting Bcl2 and p53 proteins or corresponding genes, compounds affecting caspases or interacting with inhibitor of apoptosis (IAP) proteins, could bring advance into anticancer therapy [2,3]. Several lignan derivatives, such as the semisynthetic podophyllotoxin-related compounds etoposide or teniposide, are well known for their antitumor activity [1,4–6], but, despite several papers dealing with antiproliferative ⁎ Corresponding author. Tel.: +39 0498275344; fax: +39 0498275366. E-mail address: [email protected] (S. Dall'Acqua).

http://dx.doi.org/10.1016/j.ﬁtote.2014.08.001 0367-326X/© 2014 Elsevier B.V. All rights reserved.

activities of Schisandra lignans [5,7], only relatively few studies have evaluated the possible mechanisms of action and the effects on apoptosis of dibenzocyclooctadiene derivatives [8]. Dibenzocyclooctadiene lignans are the main group of bioactive compounds contained in the fruit of the wellknown medicinal plant Schisandra chinensis (Turcz.) Baill. (Schisandraceae) and up to date, more than 40 derivatives have been isolated from this source [9]. These lignans possess a unique structure of dibenzocyclooctadiene and are categorized into two series on the basis of their stereochemistry: with S- or R-biphenyl configuration. In addition, their cyclooctene ring exhibits a twist-boat-chair (TBC) or a twist-boat (TB) conformation. The structures of these lignans are complex because of substitution patterns, chiral centers and stereoisomery [7]. In previously published papers, some Schisandra lignans were studied for their potential effects against tumor cell lines. In particular schizandrin B was able to enhance doxorubicin induced apoptosis in hepatic carcinoma and breast tumor cell lines [10]. Lignans isolated from S. sphenathera were studied for

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their antiproliferative and estrogenic effects [11], derivatives from S. neglecta were evaluated for their cytotoxic properties against tumor cell lines [12,13]. The various biological activities of the dibenzocyclooctadiene lignans have been extensively investigated and recently, gomisin A, gomisin G, schisandrin, and schisanhenol were reported to possess activity by inhibiting Epstein-Barr virus early antigen (EBV-EA) activation, reversing P-glycoprotein-mediated multidrug resistance (Pgp-MDR) in cancer cells, and enhancing doxorubicin-induced apoptosis in human hepatic cancer cells [7]. A different mechanism of action was identified for two of these derivatives, gomisin N and gomisin A, against different cell lines [10], and some structure-activity relationships of several dibenzocyclooctadiene lignans able to overcome the MDR in a lung cancer cell line were also reported [14]. In our previous work, we assessed the antiproliferative activity of two Schisandra lignan derivatives, (+)-deoxyschisandrin (1) and (−)-gomisin N (2) isolated from S. chinensis, in different in vitro models [5]. (+)-deoxyschisandrin (1) and (−)-gomisin N (2) present similar chemical structure (with the exception of methylenedioxy substituted 1 and two free phenolic groups substituted 2, and +/− optical rotation) and in this paper we report results showing the different behavior in assays on two human tumor cell lines. In particular, the ability to induce apoptosis, the capability to modify cell cycle progression and the interaction with tubulin were evaluated. Significant differences between 1 and 2 were observed, showing that small changes in the chemical structure of dibenzocyclooctadiene lignans may induce modification of the antiproliferative activity.

HAM's F-12 medium [31]. All culture media were supplemented with 10% heat-inactivated FBS and 1% antibiotics (Biochrom KG Seromed). All cells were maintained at 37 °C in humidified atmosphere containing 5% CO2. 2.1.3. Antiproliferative activity assay Cells were seeded (1 × 104 cells/mL) in 96-well tissue plates (Falcon Plymouth, England) and, following overnight incubation, exposed for 24, 48 and 72 h to different concentrations of the lignans 1 and 2 (from 25 to 250 μM) and to podophyllotoxin (from 1 to 20 μM), used as a positive control. Cell viability was assessed by MTT assay [32]: 20 μL of MTT solution (5 mg/mL in PBS) was added to each well 4 h before the end of the treatment, and plates were incubated at 37 °C. Then, the culture medium was discarded and the pigment produced was dissolved in DMSO (150 μL/well). Absorbance was measured on a microculture plate reader (Titertek Multiscan) using 570 nm and 630 nm as test and reference wavelength, respectively.

2.1. General experimental procedures

2.1.4. Apoptosis assay Cells were seeded (1 × 105 cells/mL) in 25 mL flasks (Falcon) and after 24 h treated with various concentrations of lignans 1 and 2 or podophyllotoxin for 72 h. Then, cells and supernatants were collected and centrifuged. Pellets were re-suspended in Hepes buffer with Annexin V (2.5 μL; Invitrogen Molecular Probes, Oregon, USA) and Propidium Iodide (PI; 3.5 μL; Sigma-Aldrich, St. Louis USA). Stained cells were incubated for 15 min in dark and on ice and then analyzed by flow cytometry (Epics XL, Beckmann Coulter). Cells negative for both Annexin V and PI are viable, cells Annexin V positive /PI negative are in early stage of apoptosis, and cells Annexin V positive /PI positive stained are necrotic or in late stage of apoptosis.

2.1.1. Chemicals (+)-deoxyschisandrin (1) and (−)-gomisin N (2) were isolated from fruits of S. chinensis as previously described [5]. Information on the structure was obtained on the basis of both 1D and 2D NMR experiments; the stereospecifity was determined using NMR and CD analysis (data not shown), purity of isolated compounds was N 98% on the basis of NMR and HPLC assays. Compounds, dissolved in DMSO at 10 mg/mL and stored at 4 °C, were diluted with growth medium before each experiment and used immediately.

2.1.5. Mitochondrial membrane potential assay Cells were seeded (1 × 105 cells/mL) in 6-well plates, after 24 h incubation treated with different concentrations of lignans for 72 h, then washed and incubated with Rhodamine 123 (10 μM; Sigma-Aldrich) for 15 min in dark at room temperature. The samples were analyzed by flow cytometry. Rhodamine 123, a cell-permeable cationic probe, is able to highlight the depolarization of mitochondrial membrane potential (ΔΨm). A depolarization of ΔΨm, resulting from apoptotic signals, triggers a loss of Rhodamine 123 from mitochondria.

2.1.2. Cell lines Two human cell lines were used: 2008 cells derived from ovary carcinoma and maintained in RPMI 1640 medium; LoVo cells derived from colon-rectal adenocarcinoma and grown in

2.1.6. Cell cycle analysis Cells were seeded (1 × 105 cells/mL) in 6-well plates and, after 24 h incubation, treated for 72 h with different concentrations of lignans. Cells were detached, centrifuged, permeated

2. Experimental

Table 1 Growth inhibition effect of (+)-deoxyschisandrin, (−)-gomisin N and podophyllotoxin on two human cancer cell lines. Each value represents means ± standard deviation (SD) of three independent experiments. IC50 (μM) LoVo

(+)-Deoxyschisandrin(1) (−)-Gomisin N(2) Podophyllotoxin

2008

24 h

48 h

72 h

24 h

48 h

72 h

198.5 ± 36.7 173.2 ± 52.1 21.3 ± 1.6

79.6 ± 3.6 97.5 ± 17.1 13.6 ± 1.8

54.3 ± 13.6 68.5 ± 10.0 4.1 ± 1.3

195.4 ± 28.9 169.7 ± 15.9 14.3 ± 1.6

156.3 ± 18.2 129.2 ± 10.9 11.8 ± 1.3

72.6 ± 3.7 118.0 ± 13.2 3.2 ± 0.6


with 70% ethanol and then the RNAase (Sigma-Aldrich) was added. The cells were labelled with Propidium Iodide (PI), left in dark at 37 °C for 30 minutes and then analyzed by flow cytometry.

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2.1.7. Western blot analysis Cells were seeded (1 × 105 cells/mL) in 25 mL flasks, and after 24 h of incubation treated with different concentrations of lignans for 72 h. Then, cells were washed and lysed by lysis

Fig. 1. Induction of cell death by dibenzocyclooctadiene lignans in A) Lovo and B) 2008 cells after 72 h treatment. The data are presented as the mean ± SD of the results for three independent experiments.

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rabbit monoclonal anti-β-actin, 1:1000 dilution (Cell Signaling Technology). Then, the membranes were incubated for 1 h with HPR-conjugated secondary antibody (Cell Signaling Technology). All blots were developed by ECL PLUS Detection Kit Reagent (Amersham Biosciences, GE healthcare).

Chart 1. Structures of (+) deoxyschisandrin (1) and (−) gomisin N (2).

buffer (25 mM Tris–HCl pH 7.4; 150 mM NaCl; 1% Igepal; 1% sodium deoxycholate; 0.1% SDS; 1 mM EDTA pH 8), harvested and centrifuged. Protein concentration was determined using Lowry method [33]. For western blot analysis, 30 μg of proteins were loaded onto 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, GE healthcare, UK). The membranes were blocked by incubation in 1% TBS-T containing 10% semi-skimmed milk, and incubated overnight at 4 °C with primary antibodies: rabbit monoclonal anti-cyclin B1, 1:1000 dilution (Cell Signaling Technology, Danvers USA), mouse monoclonal anti-acetylated tubulin, 1:2000 dilution (Santa Cruz Biotechnology, USA),

2.1.8. Mitotic mechanisms study After 72 h of treatment, cells were fixed with cold methanol, permeated with 0.1% triton and then labeled with a mouse monoclonal antibody against acetylated tubulin diluted 1:250 (Santa Cruz Biotechnology) and secondary anti mouse antibody (Sigma-Aldrich), that allows to underline the polymerization of microtubules. PI staining was used to mark cell nuclei and the cell cycle distribution was studied by the confocal microscopy. 2.1.9. Statistical analysis Three independent experiments were performed in triplicate for each assay. The results were evaluated by Student's ttest. The IC50 95% confidence limits were estimated using the software GraphPad Prism 3.0 (GraphPad software, Inc., San Diego, CA). 3. Results and discussion Many cancer chemotherapeutic agents are natural products or natural product-derived compounds. Etoposide and teniposide are clinically used and belong to the family of

Fig. 2. Cell cycle progression after 72 h of dibenzocyclooctadiene lignans treatment in A) Lovo and B) 2008 cells after 72 h treatment. Each value is the mean of three independent experiments.


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Table 2A Cell cycle progression [%] after 72 h dibenzocyclooctadiene lignans treatment in Lovo cells after 72 h treatment. LoVo

G0/G1

Control Podophyllotoxin 2.5 μM (+)-deoxyschisandrin 24 μM (+)-deoxyschisandrin 48 μM (−)-gomisin N 25 μM (−)-gomisin N 50 μM

40.3 11.9 39.5 27.0 39.6 28.0

± ± ± ± ± ±

S 6.2 1.4 6.9 7.5 7.1 3.6

26.7 9.8 26.5 18.2 25.3 24.4

G2/M ± ± ± ± ± ±

0.3 0.5 0.6 2.2 1.9 4.9

33.0 78.3 34.0 54.8 35.1 47.6

± ± ± ± ± ±

1.1 1.4 1.8 2.9 2.2 3.4

podophyllotoxin-related lignans [15]. Compounds 1 and 2, sharing the basic skeleton of the dibenzocyclooctadiene-type lignans, but differing in the pattern substitution at one of the aromatic rings and different optical activity, were evaluated in this paper as model compounds in order to assess potential differences in the mechanism of action against tumor cell lines. As summarized in Table 1 and Chart 1, neither treatment with 1 or 2 was able to induce a cell growth reduction similar to that of podophyllotoxin, used as reference compound. The observed antiproliferative activity of the two compound was low, compared to the reference lignan podophyllotoxin, but we deeply investigated their mode of action in order to highlight possible different mechanisms. Despite the moderate antiproliferative effects only, the compounds 1 and 2 were able to induce different types of death on tumor cells treated (Fig. 1A and B), in particular on LoVo cells, 1 mainly inducing apoptosis in a dose-dependent manner, while 2 caused a mixed apoptotic and necrotic effect. Conversely, on 2008 cells compound 1 induced necrosis especially at the highest dose used, while 2 induced apoptosis in a dose-dependent manner (Fig. 1A and B). Apoptosis, a programmed cell death, can be initiated in two ways: by an extrinsic (death receptor-mediated) or by an intrinsic (mitochondrial-mediated) pathway [9,16,17,18]. In the first, plasma membrane death receptors are involved and the apoptosis signal is provided by the interaction between these receptors and their ligands. Whereas in the second, changes in the mitochondrial integrity in response to a broad range of physical and chemical stimuli can trigger apoptosis. Mitochondria have recently emerged as intriguing targets for some anticancer agents which would destabilize them and cause apoptosis, if possible selectively in cancer cells [19,20]. Mitochondrial dysfunction, including the loss of mitochondrial membrane potential (ΔΨm), permeability transition, and release of cytochrome c, is associated with apoptosis induced

Fig. 3. Effects of dibenzocyclooctadiene lignans on cyclin B1 expression in Lovo cells. A typical immunoblot of cell cycle regulation related protein is reported (A). Lines: a) control, b) podophyllotoxin 1 μM, c) podophyllotoxin 2.5 μM, d) (+)-deoxyschisandrin 24 μM, e) (+)-deoxyschisandrin 48 μM, f) (−)gomisin N 25 μM, g) (−)-gomisin N 50 μM. The data are expressed as the mean ± SD of three independent experiments and are reported in the graph (B) as cyclin B1 % versus control.

by chemotherapeutic drugs [21]. We observed on both cell lines, that neither compound 1 or 2, like podophyllotoxin, can induce remarkable loss of ΔΨm (data not shown), suggesting that the apoptosis induced by these three lignans is not related to changes in the potential of the mitochondria membrane. If these results could be expected on 2008 cells treated by compound 1, which induced predominantly necrotic cells, on

Table 2B Cell cycle progression [%] after 72 h dibenzocyclooctadiene lignans treatment in 2008 cells after 72 h treatment. 2008

G0/G1

Control Podophyllotoxin 2.5 μM (+)-Deoxyschisandrin 24 μM (+)-Deoxyschisandrin 48 μM (−)-Gomisin N 50 μM (−)-Gomisin N 100 μM

60.6 11.3 62.7 61.5 59.2 68.0

± ± ± ± ± ±

S 4.4 1.2 4.9 4.8 2.9 5.1

19.7 10.8 18.7 18.7 23.7 15.4

G2/M ± ± ± ± ± ±

2.3 1.7 3.6 2.5 3.5 2.7

19.7 77.9 18.6 19.8 17.1 16.6

± ± ± ± ± ±

4.3 1.4 2.6 4.4 3.2 2.4

Fig. 4. Effects of dibenzocyclooctadiene lignans on tubulin expression in Lovo cells. A typical immunoblot of cell cycle regulation related protein is reported (A). Lines: a) control, b) podophyllotoxin 1 μM, c) podophyllotoxin 2.5 μM, d) (+)-deoxyschisandrin 24 μM, e) (+)-deoxyschisandrin 48 μM, f) (−)gomisin N 25 μM, g) (−)-gomisin N 50 μM. The data are expressed as the mean ± SD of three independent experiments and are reported in the graph (B) as acetylated tubulin % versus control.

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the other hand our data demonstrated that compound 2 and podophyllotoxin induce apoptosis in LoVo and 2008 cells via mitochondrial dysfunction-independent pathway. Other authors report a apoptosis mitochondrial damage-dependent pathways for 1 [22] and 2 [23], but these data were obtained on leukemic cells (HL-60 and U937), suggesting a different response in dependence on the cell type. Some lignans have recently been reported to induce cell cycle arrest in human cancer cells [23,33], therefore we examined whether 1 or 2 affected cell cycle progression in our cell models. Tests carried out on 2008 cells showed, that neither 1 nor 2 were able to change cell number in the different cell cycle phases. Lignans 1 and 2 caused a dose dependent G2/M phase arrest when assayed on LoVo cells only, unlike podophyllotoxin, which caused a remarkable G2/M phase arrest on both cell lines used for assays (Fig. 2A and B, Tables 2A and 2B). It is known that cell cycle progression is mediated by the activation of a highly conserved family of protein kinases, the cyclin-dependent kinases (Cdks) which activation requires a binding of specific regulatory proteins, cyclins [24,25]. The

entry into mitosis is under the control of B-type cyclins associated with Cdk1. The cyclin B/Cdk1 complex was originally defined as the maturation-promoting factor or M phasepromoting factor [26,27]. Regulation of cyclin B/Cdk1 complex at multiple levels ensures the tight control of the timing of mitotic entry and cell division. Without synthesis of cyclin B prior to the G2/M transition, Cdk1 remains inactive, the cells cannot enter mitosis and the cell cycle will arrest at G2 phase. We observed a decrease in the level of cyclin B1 (42%) after LoVo treatment with (−)-gomisin N (2) at 50 μM only. This decrease was comparable to that obtained with podophyllotoxin (but at 2.5 μM) (Fig. 3). These results indicated that the down-regulation of cyclin B1 expression might contribute to LoVo cell cycle arrest caused by 2, while further investigations are needed to clarify the molecular changes observed in (+)-deoxyschisandrin (1) mediated G2/M cell cycle arrest. Microtubule dynamics play a central role in the process of mitosis. During the majority of process of the cell cycle, microtubules form an intracellular lattice-like structure. However, when cells enter mitosis, this microtubule net-

Fig. 5. Effects of podophyllotoxin at 2.5 μM, (+)-deoxyschisandrin at 48 μM and (−)-gomisin N at 50 μM on acetylated tubulin expression in Lovo cells. Cells were viewed under confocal microscopy at 60x magnification.


work is reorganized into the mitotic spindle. The critical role, which microtubules play in cells division, makes them a very suitable target for the development of chemotherapeutic drugs against the rapidly dividing cancer cells. The effectiveness of microtubules-targeting drugs has been validated by the successful use of several Vinca alkaloids and taxanes [28]. Then, to clarify the molecular events involved in G2/M phase arrest induced by deoxyschizandrin and gomisin N on LoVo cells we investigated the effect of both lignans on the expression of acetylated tubuline and on the capability to interfere with microtubule dynamics. Our results, reported in Fig. 4, showed that 1 caused a dose-dependent decrease in the level of acetylated tubulin, namely 52 and 81% in respect to untreated cells at concentration of 24 and 48 μM, respectively. 2 also caused a remarkable decrease in the level of acetylated tubulin which was not dose-dependent and was the 78% respect to untreated cells, for both doses used. A significant reduction was observed after treatment with podophyllotoxin, which caused a strong decrease of tubulin expression (85 and 95 % at 1 and 2.5 μM, respectively). These results were confirmed by confocal microscopy, where the cells treated with 1 and 2 showed microtubule depolymerization similar to that induced by podophyllotoxin (Fig. 5). Then, both lignans effectively target microtubules, when exert their inhibitory effects on cells proliferation via blocking mitosis by microtubule depolymerization. The results obtained are in agreement with literature only as regards the different responses related to the type of tumor cells treated. Gomisin J and N (2) were reported to inhibit Wnt/β-catenin signaling in HCT116 cells, by disrupting the interaction between β-catenin and its specific target DNA sequences and not altering the expression of the β-catenin protein [29]. The same paper reported that gomisins J and N (2) inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase thus showing different behavior compared to our results in LoVo and 2008 cells. In HeLa cells, gomisin N (2) was able to overcome TRAIL resistance through ROS-mediated upregulation of DR4 and DR5 expression [30], showing the ability to act also via different mechanism of action. In the same cell line, gomisin N (2) enhanced TNF-α-induced apoptosis by suppressing of NF-κB and EGFR signaling pathways [7]. In conclusion, we showed that small change in structure of dibenzocyclooctadiene lignans can modify the mode of action against different tumor cell lines. Acknowledgment The authors are grateful to MIUR for financial support. References [1] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461–77. [2] Wong RSY, Journal of Experimental and, Cancer Research Clinical. Apoptosis in cancer: From pathogenesis to treatment. 2011;30. [3] Zhang JY. Apoptosis-based anticancer drugs. Nat Rev Drug Discov 2002;1: 101–2. [4] Saleem M, Hyoung JK, Ali MS, Yong SL. An update on bioactive plant lignans. Nat Prod Rep 2005;22:696–716. [5] Šmejkal K, Šlapetová T, Krmenčík P, Babula P, Dall'Acqua S, Innocenti G, et al. Evaluation of cytotoxic activity of schisandra chinensis lignans. Planta Med 2010;76:1672–7.

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