Cytotoxic and apoptotic effects of leptocarpin, a plant-derived sesquiterpene lactone, on human cancer cell lines

Accepted Manuscript Cytotoxic and Apoptotic effects of Leptocarpin, a plant-derived sesquiterpene lactone, on human cancer cell lines Claudia Bosio, Giacomo Tomasoni, Rolando Martínez, Andrés F. Olea, Héctor Carrasco, Joan Villena PII:

S0009-2797(15)30108-3

DOI:

10.1016/j.cbi.2015.11.006

Reference:

CBI 7514

To appear in:

Chemico-Biological Interactions

Received Date: 3 July 2015 Revised Date:

18 October 2015

Accepted Date: 4 November 2015

Please cite this article as: C. Bosio, G. Tomasoni, R. Martínez, A.F. Olea, H. Carrasco, J. Villena, Cytotoxic and Apoptotic effects of Leptocarpin, a plant-derived sesquiterpene lactone, on human cancer cell lines, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cytotoxic and Apoptotic effects of Leptocarpin, a plant-derived sesquiterpene lactone, on human cancer cell lines Claudia Bosio1, Giacomo Tomasoni1, Rolando Martínez1*, Andrés F. Olea2, Héctor Carrasco2 and Joan Villena3*. 1

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Departamento de Química, Facultad de Ciencias Exactas, Universidad Andrés Bello, Quillota 910, Viña del Mar 2520000, Chile; E-Mails: [email protected] (CB); [email protected] (GT); [email protected] (RM). 2 Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Llano Subercaseaux 2801, San Miguel, Santiago, Chile; E-Mails: [email protected] (AFO); [email protected] (HC). 3 Centro de Investigaciones Biomédicas (CIB), Escuela de Medicina, Universidad de Valparaíso, Av. Hontaneda 2664, Valparaíso 234000, Chile.

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* Authors to whom correspondence should be addressed; E-Mails: [email protected]; [email protected]; Tel.: +56-032-250-7354.

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ABSTRACT

Sesquiterpene lactones have attracted much attention in drug research because they present a series of biological activities such as anticancer, antifungal, anti-inflamatory, antimicrobial and antioxidant. Leptocarpin (LTC) is a sesquiterpene lactone isolated from a native Chilean plant, Leptocarpha rivularis, which has been widely used in traditional medicine by Mapuche people. Previous work has demonstrated that LTC decreases cell viability of cancer cell lines. In this

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contribution, we analyze the mechanism of LTC cytotoxicity on different cancer cell lines. The results show that in all cases LTC induces an apoptotic process and inhibition of NF-κB. Apoptosis has been confirmed by observing condensation of chromatin, nuclear fragmentation, release of cytochrome c into the cytosol, and increasing of caspase-3 activity. It has also been found that LTC

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is an effective inhibitor of NF-κB, which suggests that leptocarpin-induced cytotoxicity involves in some degree the inhibition of NF-κB signaling pathway. The concentration at which LTC inhibits

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NF-κB activity to the control level is similar or even lower than that found for parthenolide and others sesquiterpen lactones. These results indicate that leptocarpine is a very interesting molecule that could be considered as therapeutic agent for cancer treatment.

Keywords: Sesquiterpene lactones; Leptocarpin; Cytotoxicity; Apoptosis; Factor NF-κB; Cancer

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cell lines

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1. INTRODUCTION

The number of chemotherapeutics drugs that are natural products or chemical derivatives of them is increasing every day [1]. Thus, the search of new products with potential biological activity has been focused on medicinal plants that have been traditionally used for their various curative abilities. Plants synthesize many chemicals both as constitutive products and as secondary

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metabolites. In this context, the family of Asteraceas emerges as an interesting alternative, since they are considered as the most developed group of plants, and they have been used by many indigenous people in folk medicine. Members of this family are rich in bioactive compounds such as polyacetylenes, diterpenes and sesquiterpene lactones (SQL). The latter are highly interesting since they have demonstrated a number of clinical activities, such as antitumor, antiulcer, anti-

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inflammatory, neurocytotoxic and cardiotonic activities [2]. This wide range of biological activities are known to be due to inactivation of nuclear factor kappa B (NF-κB) via a α-methylene γ-

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butyrolactone group which is chemically reactive [3,4]. Thus, SQL may be able to interfere with key biological processes such as: cell signaling, cell proliferation, apoptosis and mitochondrial respiration [5-7]. Apoptosis is a particularly interesting process because it has been proposed as a new approach for the treatment of cancer. Apoptosis is a programmed, physiological mode of cell death that occurs naturally in cells and is characterized by morphological changes such as chromatin condensation and nuclear fragmentation with the formation of apoptotic bodies followed by ordered

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cell demise through phagocytosis. Apoptosis is triggered by signals involving the mitochondria (intrinsic) or death receptors (extrinsic), which initiate activation of Caspases, a family of intracellular proteases, that are responsible of the morphological and biochemical events that

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characterize classical apoptosis. The mitochondrial pathway involves release of cytochrome c and others pro-apoptotic factors into the cytoplasm, through pores formed in the mitochondrial membrane, leading to activation of Caspase-9. This process is produced by change of mitochondrial

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membrane permeability (MMP), which leads to loss of mitochondrial membrane integrity. The changes in MMP are considered a determinant factor in the execution of death cell. The ability of cancer cells of resisting cell death has been considered a hallmark in cancer [8]. The signal that triggers the apoptosis process is product of a delicate balance between apoptotic and antiapoptotic proteins. On the other hand, anticancer studies have shown that constitutive activation of NF-κB is involved in the tumor initiation, progression, and therapeutic resistance in most of the major forms of human cancer [9,10]. Thus, a hallmark-targeting cancer drug could be any natural or synthetic compound, which by inhibiting NF-κB increases effectively apoptosis in cancer cells. In other words, the inhibition of this transcription factor can increase the efficiency of cancer therapy by killing cancer cells directly or render them more vulnerable to chemotherapeutic agents [11-14]. 3

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Therefore, the concept in this new approach to cancer treatment is to use naturally occurring chemical compounds that can trigger or enhance cell death through the regulation of the above factors. For example, it has been proposed that compounds able of inhibiting NF-κB can be used in a combinatory therapy with chemoagents for which cancer cells have developed chemoresistance. Escin a natural mixture of triterpene saponins, augments the efficacy of gemcitabine through downregulation of nuclear factor-κB and nuclear factor-κB-regulated gene products in pancreatic cancer

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both in vitro and in vivo [15]. In the case of sesquiterpene lactones there have been a number of studies where their anticancer activity has been associated to some of these factors. Some examples of active SQL are parthenolide obtained from the herb feverfew, which has shown potent antitumor activity against cancer stem cells [16,17] and synergistic effect on treatment of pancreatic

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adenocarcinoma and a xenograft model of breast cancer when used in combination with docetaxel or sulindac, respectively [18,19]; cynaropicrin that induces cytotoxicity of leukocyte cancer cell

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lines such as U937 and Jurkat T cells [20]. In both cases, it has been suggested that these molecules triggers apoptosis in cancer cells by inhibition of NF-κB.

On the other hand, our research group has been engaged for some time in the study of an autochthonous plant that has been widely used as folk remedy by Mapuche people. This plant, called "palo negro" and whose scientific name is Leptocarpha rivularis, belongs to the family of Asteraceae, and it has been shown that contains a high percentage of SQL, with leptocarpin (LTC)

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as the major component [21-24] (Figure 1). In a previous study, conducted on cell lines SK-Hep-1 (adenocarcinoma of the liver), and epithelial cell line HeLa, we have shown that LTC exhibits a significant cytotoxic effect [23]. In this work, we have studied the effect of leptocarpin on the

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acting on the NF-κB.

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apoptosis mechanism. The results show that this compound triggers programmed cell death by

CH3 O O

H O 9

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10

CH3

3 4

H3C

8

13

6

5

7

HO

11

O

12

H3C O

Figure 1. Chemical structure of leptocarpin, extracted from Leptocarpha rivularis.

2. MATERIAL AND METHODS 2.1 Materials

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LTC (Fig 1, MW 362 g/mol) was obtained from Leptocarpha rivularis as previously described [24]. Structural identity of leptocarpin was determined by using spectroscopic techniques (1H and

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NMR, IR, MS). Staurosporin, 5-fluorouracil 5-FU, sulforhodamine B (SRB), rhodamine 123, Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Fetal bovine serum, penicillin, streptomycin were obtained from Hyclone (Santiago, Chile) and used as received.

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HT-29 cells (colon cancer cell line), PC-3 and DU-145 (prostate cancer cell lines), MDA-MB-231 and MCF7 (breast cancer cell lines), CCD 841 CoN (human colon epithelial cells) and HDF (human dermal fibroblasts) were obtained from the American Type Culture Collection (Rockville, MD,

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USA).

2.2 Cell culture

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All tested cell lines were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium, containing 10% heat-inactivated fetal bovine serum (FBS), amphotericin (2.5 µg/mL), penicillin (100 U/mL) and streptomycin (100 µg/mL).

2.3 In Vitro Cytotoxicity Screening by using Sulforhodamine B Assay [25,26] Stock cells were incubated at 37 °C under humid atmosphere with 5% CO2 for 24 h before the test.

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The cell suspension was set up at 3,000 cells per well of a 96-flat-bottomed 200 µL well microplate. Leptocarpin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mg/mL and diluted with the growth medium to the desired concentrations (0-25 µM). Negative control cultures were prepared by adding just 0.1% DMSO. All culture microplates were incubated at 37 °C in a CO2

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incubator with humidified 5% CO2 for 72 h. At the end of drug exposure, cells were fixed with 50% trichloroacetic acid at 4 °C. After washing with water, cells were stained with 0.1% sulforhodamine

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B (SRB) dissolved in 1% acetic acid (50 µL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain. Protein-bound stain was solubilized with 100 µL of 10 mM unbuffered Tris base, and the cell density was determined using an ELISA fluorescence plate reader at an emission wavelength of 540 nm using the program Gen5 1.07. The obtained data were expressed as percentages of viability cells versus solvent control, whose viability was considered 100%. Values shown are the mean ±SD of three independent experiments in triplicate. The software used to calculate the IC50 values was Prism 6® version 6.0d. 2.4 Morphological Assessment of Cell Apoptosis

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Cell and nuclear morphology were analyzed, after 48 hours of treatment with LTC (0, 7 and 25 µM), using phase contrast microscopy and immune fluorescence microscopy, respectively. As a positive control of apoptosis was used 5-FU at 25 µM. Morphological changes in the nuclear chromatin of cells undergoing apoptosis were revealed by a nuclear fluorescent dye, Hoechst 33342. Briefly, CCD 841 CoN, HT-29, PC-3 and MCF7 (1 × 104 cells/mL) were cultured on 24-well chamber slides, and exposed to LTC or 5-FU (positive control)

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for 48 h. The control group was exposed to 0.1% DMSO. The cells were washed twice with phosphate buffer solution, fixed with 3.7% formaldehyde and washed again with phosphate buffer solution. Following the addition of Hoechst 33342 solution (1 µM diluted with PBS) cells were incubated in a dark room at room temperature for 30 min. After washing, they were examined under

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an immunofluorescence microscope at 465 nm (Olympus IX 81 model inverted microscope).

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2.5 Determination of mitochondrial potential by flow cytometry

Rhodamine 123, a cationic voltage-sensitive probe that reversibly accumulates in mitochondria, was used to detect changes in mitochondrial membrane potential [27,28]. Cells were incubated with leptocarpin (0, 7 and 25 µM) for 48 h, and subsequently were stained with rhodamine 123 (1 µM) and incubated in darkness for 1 h at 37 °C. Then, the medium was removed and cells were washed twice with PBS. Later the cells were trypsinized and collected by centrifugation (10 min at 1500 g).

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The supernatant was discarded and the cells pellets were resuspended in PBS and analyzed by flow cytometry using the filter FL1. Results are expressed as percentage of cells stained with rhodamine 123.

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2.6 Evaluation of Cytochrome c release.

The release of cytochrome c from the mitochondria into the cytoplasm was assessed by using the

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FlowCellect™ cytochrome c kit (Millipore Corp.). Briefly, cells were plated in 5 mL of culture medium into 60 mm Petri dishes. After treatment with LTC (0, 7, 25 µM for 24 h), cells were collected and centrifuged at 300 g for 5 min at 4°C. Subsequently, cells were submitted to permeabilization,

fixation

and

blocking

buffers

according

to

supplier´s

instructions.

Immunolabeling for cytochrome c is achieved by incubating permeabilized cells with anticytochrome c-FITC antibody (10 µL) for 30 min at room temperature. Cells were centrifuged and the pellets were resuspended in blocking buffer and analyzed by flow cytometry. Results are expressed as percentage of cells with reduced fluorescence of FITC. This percentage is a measure of the number of apoptotic cells that have already released their cytochrome c from the mitochondria to the cytoplasm.

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2.7 Determination of Caspases activation

Analysis of Caspase-3 Activity. The activity of Caspases was determined by using a fluorescent inhibitor of caspases tagged with fluorescein isothiocyanate, FITC-VAD-FMK. The CaspACE™ FITC-VAD-FMK In Situ Marker was obtained from Promega. Briefly, cells were treated with leptocarpine (0, and 25 µM) for 48 h. The cells were incubated with CaspACE™ FITC-VAD-FMK in darkness for 20 min at room temperature. Then, the medium was removed and cells were washed

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twice with PBS. Exposed cells were collected by tripsinization and centrifugation (10 min at 1500 g). The supernatant was discarded and the cells were re-suspended in PBS and analyzed by flow cytometry using the filter FL3. Results are expressed as percentage of cells stained with

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CaspACE™ FITC-VAD-FMK [29].

2.8 NF-κB Analysis

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2.8.1 Detection of the DNA-binding activity of NF-κB factor

Cells previously stimulated with TNF-α (10 ng/mL, 1 hour) [30,31] were submitted to different treatments: LTC (7 µM) for 48 h; 0.1% DMSO (for 48 h); and caffeic acid phenethyl ester (CAPE) (25 µg/mL, 24 h), an inhibitor of NF-κB that was used as negative control [32]. After these treatments, cells were collected and centrifuged at 300 g for 5 min at 4°C. Nuclear fractions were isolated and subsequently analyzed for NF-κB activity following manufacturer’s instructions for

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detecting specific transcription factor DNA binding assay, NF-κB (p65) transcription factor assay kit (Cat# 10011223, Cayman).

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2.9 Statistical analysis

Data are reported as mean values ± SD. Experiments were repeated three times, with triplicate samples for each. Data were analyzed by Prism 6® version 6.0d. Statistical significance was

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defined as p < 0.05.

To analyze the normality in the distribution of the data, the test "Shapiro-Wilk” was used. While for the statistical analysis of data with no normal distribution, the non-parametric test of “Wilcoxon” with designed range was utilized.

3. RESULTS

The cytotoxicity of leptocarpin was evaluated in vitro against different cancer cell lines: HT-29 colon cancer; PC-3 and DU-145 human prostate cancer; MCF7 and MDA-MB-231 breast cancer and two non tumoral cell lines; CCD 841 CoN human colon epithelial cells (CoN) and human dermal fibroblasts (HDF). The sulforhodamine B colorimetric assay was set up to estimate the IC50 7

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values; nine serial dilutions (from 1 to 25 µM) for each sample were evaluated in triplicate. The results obtained from these assays are shown in Table 1. The IC50 values obtained for cancer cell lines are in the range of 2.0 − 6.4 µM, being the lowest value (highest cytotoxicity) that observed in cancer cell line DU-145. On the other hand, LTC is much less cytotoxic for fibroblasts with an IC50 value of 18.7 µM. This result indicates that LTC acts selectively on cancer cell lines. The inhibitory

[20,33,34].

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effect of LTC on cancer cells viability is similar to that found for other sesquiterpene lactones

Table 1. IC50 values (µM) of LTC for different cancer cell lines and human dermal fibroblasts PC-3

HT-29

MCF7

MDA

2.0 ± 0.8

4.5 ± 0.3

3.8 ± 0.2

3.1 ± 0.3

6.4 ± 0.5

CoN

HDF

5.2 ± 0.3

18.7 ± 3.5

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DU-145

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Data are reported as mean values ± SD of three different experiments with samples in triplicate

In order to determine whether the effect of LTC on the reduction in cell viability involves cell death by triggering apoptosis, changes in cell and nuclear morphology were assessed with phase contrast microscopy and by Hoechst 33342 staining using fluorescence microscopy, respectively. In Figure

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2 are shown some representative images obtained for MCF-7.

Figure 2. Effect of leptocarpin on cellular morphology, chromatin condensation and fragmentation, of MCF-7. Ctrl: cells exposed to 0.1% DMSO; Lepto: cells treated with LTC (7µM) for 48 h. Arrow 8

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heads and arrows indicate not condensed nuclei and condensed and/or fragmented nuclei, respectively.

Images obtained with phase contrast microscope of control cells (0.1% DMSO) (Ctrl) and cells treated with LTC (7 µM) for 48 h are given in the left side. These images show clearly that MCF-7 cells treated with LTC are smaller in size and they lose their characteristic shape appearing as round

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mass. In addition, the number of cells is also reduced indicating a progression to cell death. On the other hand, the nuclear morphology changes of MFC-7 cells were examined on images obtained by fluorescence microscopy after Hoechst 33342 staining (Figure 2, right side). It can be seen that cells treated with LTC (7µM) for 48h show significant chromatin condensation and fragmentation,

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compared to control cells.

The results obtained for different cells lines were quantified by measuring the percentages of

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condensed and/or fragmented nuclei observed after treatment with leptocarpin (7 and 25 µM for 48 h). Cells exposed to 0.1% DMSO were used as control (Ctrl), and 5-fluorouracil (5-FU), an apoptosis inductor compound, was used as positive control. The obtained results (* p < 0.01) are summarized in Table 2.

Table 2. Percentages of condensed and/or fragmented nuclei of different cell lines treated with LTC (7 and 25 µM) or with 5-FU (25 µM) for 48 h. Cells exposed to 0.1% DMSO were used as control.

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Leptocarpin 7 µM

25 µM

Control

5-FU, 25 µM

MCF7

33.1±5.3*

52.1±6.3*

5.1±0.6

30.4±4.5*

PC-3

28.2±3.6*

47.3±6.5*

6.2±2.4

36.9±5.1*

HT29

21.5±5.7*

43.4±2.9*

7.3±1.9

33.0±4.9*

8.0±1.2

13.6±0.3*

5.5±1.0

21.5±0.6*

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CoN

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Cell Lines

As shown in Table 2, in cancer cell lines the number of cells with condensed and/or fragmented nuclei increases significantly by treatment with LTC, reaching percentages values even higher than those obtained with 5-FU. Interestingly, almost no effect is observed for CoN cells exposed to LTC 7 µM. These results suggest the occurrence of a LTC-induced apoptosis that acts selectively on cancer cells [35]. Mitochondria play a crucial role in the apoptotic cascade by serving as a convergent center of apoptotic signals originating from both the extrinsic and intrinsic pathways [36]. Changes induced in the mitochondrial membrane permeability (MMP) have been reported previously to represent a determinant effect in the execution of cell death [37]. Thus, the effect of leptocarpin on MMP has been analyzed using flow cytometry with rhodamine 123 stain [27]. The results, given as percentage 9

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of rhodamine 123 stained-cells, are collected in Table 3, and show that these values are significantly reduced by treatment with LTC (7-25 µM for 24 h) as compared to control cells (0.1% DMSO). FCCP (10 µM) was used as positive control. The effect of LTC on mitochondrial membrane permeability is higher in cancer cells than in non-tumor cells. Actually, LTC (25 µM) has almost no effect on HDF cells when compared to cancer cells. Thus, LTC-induced loss of mitochondrial

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membrane potential correlates quite well with decreased cell viability (see Table 1).

Table 3. Changes induced by treatment with leptocarpin, in mitochondrial membrane permeability of HDF, CoN, MCF7, HT-29 and PC-3 cells. Cells were stained with rhodamine 123, and then analyzed by flow cytometry. Permeability changes are measured as percentage of

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rhodamine 123 stained cells found in cell lines treated with leptocarpin (7-25 µM) against

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control cells (0.1% DMSO). As positive control, FCCP (10 µM) was used.

Leptocarpin 7 µM

25 µM

Control

FCCP, 10 µM

HDF

99.9±1.1

73.4±1.1

98.9±6.5

8.7±1.4**

MCF7

53.8±2.3*

21.5±0.4*

97.1±3.9

10.4±0.9**

PC-3

57.3±6.7*

46.5±7.2*

96.8±6.4

6.9±1.0**

HT29

61.5±5.7*

53.4±2.9*

101.3±4.9

8.0±1.7**

CoN

100.1±2.7

100.5±6.5

7.5±0.6**

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Cell Lines

23.1±0.2*

* p < 0.05, ** p < 0.001.

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It has been proposed that depletion of mitochondrial membrane potential leads to release of apoptogenic factors, such as cytochrome c in the apoptotic cascade and activation of caspases [38].

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To confirm that LTC-induced apoptosis involves the mitochondria-dependent pathway both the release of cytochrome c and caspases activity was evaluated next. The release of cytochrome c was assessed by flow cytometry analysis of permeabilized-cells labeled with anti-cytochrome c-FITC antibody. Cells that have not released their cytochrome c are highly fluorescent, whereas apoptotic cells that have already released their cytochrome c exhibit a reduced fluorescence. Thus, from flow cytometry data the percentage of cells that have released their cytochrome c can be determined. The results obtained for PC3, HT29 and HDF cells treated with LTC (0, 7 and 25 µM) for 24 h are shown in Figure 3. These results indicate that cytochrome c is released from all cell lines treated with LTC, and that this effect is higher in cancer cell lines than in normal cells. These results follow the same trend found for the LTC-induced decrease of MMP.

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Figure 3. Percentage of cells which have released their cytochrome c from the mitochondria into the cytosol: PC-3 (black bars), HT-29 (white bars) and HDF (grey bars). Cell lines were treated with leptocarpin (0, 7, 25 µM) for 24 h. The percentage of cells that releases cytochrome c was obtained by measuring FITC fluorescence by flow cytometry. Results are presented as means +/- SD of three independent experiments. (*) Values statistically significant in comparison to control cells, p <

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0.05; (#) Statistically significant differences observed between the cancer cell lines and non-tumoral cell HDF, p < 0,05.

Regarding caspases activity, the focus was put on Caspase-3 that is a main executor of apoptosis

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playing a central role in its biological processing. The effect of treatment with LTC (7 µM) on

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Caspase-3 activation on normal and cancer cells is shown in Table 4.

Table 4. Activity of Caspase-3 in HDF, MCF7, PC-3, HT-29 and CoN cells treated with LTC (7µM). Data is reported as ratio of activities of treated cells and control cells (0.1% DMSO), which were arbitrarily assigned a unitary value.

HDF

MCF7

PC-3

HT29

CoN

LTC

1.10±0.13

2.51±0.31**

5.5±0.45**

3.45±0.23**

1.32±0.30

Control

1.12±0.16

0.98±0.15

0.91±0.16

1.11±0.10

1.07±0.10

* p < 0.05 or ** p < 0.001

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As shown in Table 4, the activation of Caspase-3 is higher in cells exposed to LTC than in control cells (0.1% DMSO), except for HDF cells. In other words, LTC increases the activity of Caspase-3 in cancer cells but has no effect on normal cells. It is worth to mention that these results were obtained by using a caspases inhibitor tagged with fluorescein isothiocyanate FTC-VAD-FMK, and it was assumed that binding to apoptotic cells is due mainly to activation of caspase-3, even though other caspases could be contributing to this binding [29].

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To determine if the observed apoptotic effect induced by leptocarpin is consequence of inhibition of NF-κB, we have analyzed the effect of LTC on the NF-κB activity using a DNA binding assay. All studied cells were previously treated with TNF-α, a tumor necrosis factor that actives NF-κB. The results given in Table 5 indicate that stimulation of different cancer cells with TNF-α increases

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significantly the activity of NF-κB, and this activity is almost 2-3 times higher than in control cells. On the other hand, cells pretreated with TNF-α that are exposed to LTC show a significant

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reduction of NF-κB activation. The magnitude of this inhibition is similar to that observed in cells treated with CAPE (25 µg/mL), a specific and potent inhibitor of NF-κB [32]. The treatment with LTC or CAPE alone has no effect on NF-κB activation.

Table 5. Inhibitory effect on NF-κB activity of LTC (7 µM, 24 h), in MCF-7, CoN and PC-3 cells, which were previously exposed to TNF-α. Values are mean ± S.D. (n = 3). All data values are

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reported as percentage in comparison with control cells (0.1% DMSO), which were arbitrarily assigned

MCF7

HT29

CoN

101.9±4.3

101.5±6.7

102.1±5.7

100.1±5.7

Leptocarpin

71.4±4.9

97.1±5.5

79.8±5.2

97.7±7.2

CAPE

89.2±7.7

104.1±3.8

99.7±5.4

92.9±6,3

TNF-α

323.1±8.9*

301.3±11.1* 279.8±15.3* 231.7±24.0*

TNF-α +Lepto

81.4±10.9#

93.7±7.2#

86.0±4.1#

105.1±8.5#

TNF-α +CAPE

112.1±10.4# 85.9±6.6#

98.3±5.4#

81.4±5.3#

Ctrl

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PC-3

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a 100% value.

*p < 0.05, significantly different from control; #p < 0.05, significantly different from cells treated with TNF-α

The present data demonstrates that LTC is an effective inhibitor of NF-κB in different cancer cell lines, suggesting that the mechanism of cell death induced by LTC involves in some degree the inhibition of NF-κB signaling pathway. 12

ACCEPTED MANUSCRIPT 4. DISCUSSION

Leptocarpin is a sesquiterpene lactone that has been isolated as one of the major components of “palo negro” a member of the Asteracea family [24]. In a previous work we have shown that LTC exhibit high cytotoxicity on human liver adenocarcinoma (SK-Hep-1) and HeLa cells. It has also

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been shown that acid hydrolysis of the oxirane ring reduces significantly the activity against these cell lines [21,23]. There are other sesquiterpene lactones such as costunolide, cynaropicrin, parthenolide, with chemical structures that are very similar to LTC, which have anticancer and antitumoral effects [20,34,39]. For example, parthenolide has shown potent antitumor activity

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against cancer stem cells [16,17], breast [19], gastric [40], and renal cancer lines [41]. Therefore, in this study we have explored more deeply the molecular mechanism by which LTC affects the

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viability of cancer cells. The results indicate that LTC has inhibitory and proapoptotic effects on cell viability in a dosage and time dependent manner.

Cytotoxicity results show that, under the same conditions, LTC is more cytotoxic for cancer cells than for fibroblasts. The IC50 values obtained for cancer cell lines are in the range of 2.0 − 6.4 µM, whereas an IC50 value of 18.7 µM was measured for fibroplast cells. Cho et al. have also shown that cynaropicrin inhibited efficiently proliferation of leukocyte cancer cells lines, whereas human

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fibroblast or Chang cells were slightly affected [20]. These results indicate that LTC and other similar SQL inhibit selectively the proliferation of cancer cell lines. The cytotoxic property of SQL has been attributed to the presence of a α-methylene-γ-lactone moiety in the molecule regardless of other differences in structure [3,34]. The mechanism of action associated to the activity of these

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molecules involves a covalent bond with sulfhydryl groups present in the cysteine aminoacid of proteins or enzymes through a Michael type reaction [33,42], which would alter the functions of

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these macromolecules.

SQL are known to induce cytotoxicity by triggering apoptosis, and therefore changes in cell and nuclear morphology were used to demonstrate the occurrence of apoptosis. The results shown in Figure 2 and Table 3 indicate that after treatment with LTC, cells change their morphology becoming rounded, and besides chromatin condensation and nuclear fragmentation are observed. These results are clear evidence that LTC induces an apoptosis process [35]. Similar effects have been reported in the apoptosis induced by parthenolide in multiple myeloma and prostate cancer [43]. Mitochondria play a crucial role on apoptosis initiation, and therefore induction of mitochondrial apoptotic activity is considered a potential therapeutic approach. Proapoptotic drugs that act on mitochondrial pores can affect the mitochondrial membrane permeability enhancing the release of 13

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cytochrome c into the cytoplasm, where it forms a complex polymer with apoptotic enzyme activators [36,38]. This complex activates and starts a caspase cascade reaction, in which the downstream caspase 3 and other members of the caspase family are activated. Caspase 3 is the terminal factor in the enzymatic cascade reaction in mammalian cells and it is also an effector leading to apoptosis [38]. This process is the mitochondrial pathway and is one of the caspase cascade activation pathways. Our results show that treatment with LTC increases MMP, induces

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release of cytochrome c, and activation of caspase 3. These effects indicate that LTC induces apoptosis through the mitochondrial pathway, and its effect is higher in cancer cells than in HDF cells. The activity of downstream effector caspase 3 is also upregulated in cancer cells treated with parthenolide [44].

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On the other hand, the extrinsic pathway involves death receptors and is activated by TNF. However, the apoptotic effect of TNF is counter-balanced by simultaneous activation of NF-κB, an anti-apoptotic factor that is over expressed in most cancer types. It has been demonstrated that NF-

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κB inhibits the induction of apoptosis by stimulating the production of antiapoptotic proteins as Bcl-2 and Bcl-XL [15,45]. Thus, the inhibition of this transcription factor can increase the efficiency of cancer therapy by killing cancer cells directly or render them more vulnerable to chemotherapeutic agents [46-49]. Some SQL, such as parthenolide, helenalin, costunolide and cynaropicrin, that are important inhibitors of NF-κB [4,50-53] have been evaluated as potential

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anticancer drugs. Thus, we have determined the effect of LTC on NF-κB activation in different cancer cells, where apoptosis was induced with TNF-α. The results indicate that cancer cells treated with TNF-α increase their NF-κB activity in almost 2-3 times over the level measured in control cells. However, cells pretreated with TNF-α that have been exposed to LTC show a significant

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reduction of NF-κB activation. The magnitude of this inhibition is similar to that observed in cells treated with CAPE (25 µg/mL), a specific and potent inhibitor of NF-κB [32]. The treatment with

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LTC or CAPE alone has no effect on NF-κB activation. It is interesting to mention that the concentration at which leptocarpin reduces NF-κB activity to the control level (7 µM) is similar or even lower than that found for parthenolide and others sesquiterpene lactones [54,55]. It has also been shown that parthenolide inhibit completely NF-κB activation mainly by alkylation of p65, independent of the cell type used, at concentrations ranging between 10 to 20 µM [54]. Our results suggest that the effect of LTC on NF-κB activity is associated to a decreased DNA binding capacity of this transcription factor; however the specific mechanism is still unknown. Thus, in this study it has been proved that LTC can effectively induce apoptosis in different cancer cell lines by activating the mitochondrial pathway, and by inhibiting NF-κB. These results suggest that leptocarpin has a great potential for application in treating cancer cells. 14

ACCEPTED MANUSCRIPT 5. CONCLUSIONS Leptocarpin significantly decreases cell viability of cancer cell lines, such as HT-29, PC-3, DU145, MCF7 and MDA MB-231. This inhibitory effect is dose-dependent with IC50 in the range of 2.0 – 6.4 µM. However, the effect of LTC on human dermal fibroblasts is much lower with IC50 of 18.7 µM. This effect has being linked to apoptosis by showing that LTC induces morphological

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changes, significant chromatin condensation and fragmentation in cancer cells as compared to control cells. As further evidence of apoptosis, it has been shown by flow cytometry that LTC induces depletion of the mitochondrial membrane potential and, consequently release of cytochrome c and an increase of caspase-3 activity has also been observed. Finally, the effect of

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LTC on NF-κB activation has been assessed. The results indicate that cells treated with LTC exhibit an important reduction of NF-κB activity at concentrations as low as 7 µM.

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In summary, our results suggest that leptocarpin decreases cell viability of cancer cell lines by inducing caspase-dependent apoptosis and by inhibition of the NF-κB factor.

ACKNOWLEDGMENTS

The authors are grateful to DIUV of Universidad de Valparaíso (J.V.) and FONDECYT for

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financial support of this work under Grants 50/2011 and1130742, respectively.

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Figure 2

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HIGHLIGHTS

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Leptocarpin exhibits citotoxicity against different cancer cell lines The IC50 values are similar to those reported for other sesquiterpene lactones Leptocarpin induces apoptosis by activating the mitochondrial pathway It is shown that leptocarpin is also a potent inhibitor of NF-κB activation These results indicate that leptocarpin could be used as anticancer agent

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