In vitro toxicity of camalexin derivatives in human cancer and non-cancer cells

Toxicology in Vitro 27 (2013) 939–944

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In vitro toxicity of camalexin derivatives in human cancer and non-cancer cells Martina Pilatova a,1, Lenka Ivanova a,1, Peter Kutschy b, Lenka Varinska a, Lydia Saxunova a, Maria Repovska b, Marek Sarissky a, Robert Seliga a, Ladislav Mirossay a, Jan Mojzis a,⇑ a b

Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, Kosice, Slovak Republic Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, Pavol Jozef Safarik University, Kosice, Slovak Republic

a r t i c l e

i n f o

Article history: Received 13 April 2012 Accepted 2 January 2013 Available online 20 January 2013 Dedicated to Assoc. Prof. Peter Kutschy Keywords: Camalexin Benzocamalexin Indole phytoalexins Cytotoxicity

a b s t r a c t The aim of the study was to investigate the cytotoxic activity of camalexin and its five synthetic derivatives in cancer and non-cancer cells. In cancer cells the benzocamalexin (BC) displayed the most potent activity with an IC50 value of 23.3– 30.1 lmol/L. On the other hand, minimal toxicity (IC50 > 100.0 lmol/L) in non-cancer cells was observed. Based on these results, BC was selected for further studies. Flow cytometric analysis revealed a BC-induced arrest of the cell cycle in the G2 phase associated with downregulation of a-tubulin, a1-tubulin, b5-tubulin expression. These findings suggest that the inhibitory effect of BC is mediated via inhibition of microtubule formation. Moreover, BC downregulated the expression of microtubule-related protein indicating the effect of this compound on microtubule assembly. After treatment with BC increase of the sub-G1 DNA content fraction was noted which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by DNA fragmentation assay. Moreover, quantitative real-time PCR showed that BC downregulated the expression of antiapoptotic genes Bcl-2 and Bcl-xL and upregulated the expression of proapoptotic Bax. Taken together, our study suggests that the blockade of cell cycle progression and initiation of apoptosis may play an important role in the antiproliferative activity of BC in human cancer cells. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction In response to pathogen attack many plants elaborate a vast array of low molecular weight secondary metabolites called phytoalexins. Phytoalexins include a chemically diverse group of compounds biosynthesized de novo by plants as a defense response to fungal and bacterial pathogens (Pedras et al., 2011). The concept of phytoalexin was introduced by Müller and Borger in 1940, and since then these compounds have been extensively studied not only with respect to their role in defense against pathogens and pests, but also with respect to their health-promoting effects (Bishayee, 2009; Chakraborty et al., 2010; Holland and O’Keefe, 2010; Müller and Borger, 1940; Patel et al., 2011; Wu et al., 2011). Indole phytoalexins represent a specific group of phytoalexins synthesized by plants of the family Brassicaceae (syn. Cruciferae). They are indole alkaloids, most of which contain a side chain or another heterocycle, containing a nitrogen atom and one or two sulfur atoms. ⇑ Corresponding author. Address: Department of Pharmacology, Faculty of Medicine, Pavol Jozef Safarik University, Trieda SNP 1, 04011 Kosice, Slovak Republic. Tel./fax: +421 55 6428524. E-mail address: [email protected] (J. Mojzis). 1 These authors contributed equally to this work. 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.01.006

Until now, 44 cruciferous phytoalexins, i.e. metabolites produced de novo have been reported (Pedras and Yaya, 2010). The chemical diversity of cruciferous phytoalexins suggest substantially different biological activities as was confirmed experimentally. Principally, they display antimicrobial activity against bacterial and fungal plant pathogens (for rev. see Pedras et al., 2011). However, some indole phytoalexins have also been shown to exhibit significant antiproliferative/anticancer activity (Mehta et al., 1995; Monde et al., 2005; Mezencev et al., 2009; Pilatova et al., 2005). Camalexin (2-(1H-indol-3-yl)thiazole) is an indole phytoalexin present in Arabidopsis and closely related plant species. It was first isolated from Arabidopsis thaliana and Camelina sativa leaves (Browne et al., 1991). Like many of the phytoalexins, camalexin is substituted with sulfur and nitrogen-containing side chain (Glawischnig, 2007). Although camalexin biosynthesis is induced by a great variety of plant pathogens, it is suggested that camalexin possesses primary antifungal action (Glawischnig, 2007). In addition to its antifungal action, camalexin also exerts antiproliferative effects in human cancer cell lines. As documented by Moody and co-workers (1997) camalexin significantly inhibits growth of SKBr3 breast cancer cell line with IC50 = 2.7 lmol. Recently, a proapoptotic effect of camalexin in Jurkat leukemia cells was observed

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(Mezencev et al., 2011). However, the precise mechanisms of antiproliferative activity of camalexin still remain unknown. The present study was conducted to evaluate the antiproliferative activity of camalexin and its synthetic derivates in different human cancer cell lines. In order to determine the ability of these compounds to selectively inhibit tumor cell proliferation, their antiproliferative activities in cancer cell lines were compared with their effects on normal human cells (human umbilical vein endothelial cells; HUVECs). 2. Materials and methods 2.1. Tested compounds Camalexin (C), benzocamalexin (BC, 2-(1H-indol-3yl)benzo[d]thiazole), 1-methylbenzocamalexin (MBC), 1-methyl60 -methoxybenzocamalexin (MMBC), 1-methyl-60 -fluorobenzocamalexin (MFBC), 1-methyl-60 -cyanobenzocamalexin (MCBC). Camalexin and BC (Fig. 1) were prepared according to previously reported procedures (Ayer et al., 1992; Dzurilla et al., 1999). 1-Methylbenzocamalexin was obtained by methylation of benzocamalexin with methyl iodide in the presence of sodium hydride, MMBC, MFBC and MCBC were synthesized by Jacobson cyclization of 1-methyl-N-(4-substituted phenyl) indol-3-carboxthioamides. 2.2. Chemicals 3-(Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was obtained from Sigma–Aldrich Chemie (Steinheim, Germany). Medium 199 (M199) supplemented with 20 mM HEPES and penicillin/streptomycin were obtained from Cambrex (Verviers, Belgium); newborn calf serum (NBCS) was obtained from Cambrex (Verviers, Belgium) and heat-inactivated before use. Other materials used in the methods described below have been specified in detail in related references or in the text. 2.3. Cell culture The following human cancer cell lines were used for this study: Jurkat (human acute T-lymphoblastic leukemia), CEM (acute Tlymphoblastic leukemia), HeLa (cervical carcinoma cells), MDAMB-231 and MCF-7 (breast cancer cells). Jurkat, CEM, and HeLa cells were maintained in RPMI 1640 medium (PAA Laboratories, Pasching, Austria). MDA-MB-231, MCF-7 and A-549 were maintained in growth medium consisting of high glucose Dulbecco’s Modified Eagle Medium. Both media were with Glutamax-supplemented with 10% foetal calf serum, penicilin (100 IU/mL), and streptomycin (100 mg/mL) (all from Invitrogen, Carlsbad, CA USA), in the atmosphere of 5% CO2 in humified air at 37 °C. Cell viability, estimated by trypan blue exclusion, was greater than 95% before each experiment.

S

S

N N H

camalexin

N N H

benzocamalexin

Fig. 1. Chemical structure of camalexin and benzocamalexin.

2.4. Primary endothelial cells isolation and culture Human umbilical vein endothelial cells (HUVECs) were isolated, cultured, and characterized as previously described (Defilippi et al., 1991). Cells were cultured on gelatine-coated dishes in cM199 (=M199 medium supplemented with 10% heat-inactivated human serum, 10% heat-inactivated NBCS, 3.75 lg/mL crude endothelial cell growth factor (ECGF), 5 U/mL heparin, 100 IU/mL penicillin, and 100 lg/mL streptomycin) at 37 °C under 5% CO2/95% air atmosphere. Twenty-four hours before the experiments were started, ECGF and heparin were withdrawn from the endothelial cell cultures. HUVEC were confirmed as endothelial cells by their ‘‘cobblestone’’ morphology and positive expression of CD31 markers, universal markers for endothelium, using monoclonal antibody to the human CD31 antigen (Caltag Laboratories, Burlingame, CA, USA) by flow cytometry (FACS Vantage SE, Becton Dickinson, USA). 2.5. Assessment of cytotoxicity by MTT assay The cytotoxic effect of the tested compounds was studied by using colorimetric microculture assay with the MTT end-point. The amount of MTT reduced to formazan was proportional to the number of viable cells (Mosmann, 1983). Briefly, 5  103 cells were plated per well in 96-well polystyrene microplates (SARSTEDT, Nümbrecht, Germany) in the culture medium containing the tested chemicals at final concentrations 104–106 mol/L. After 72 h, 10 ll of MTT (5 mg/ml) were added in each well. After an additional 4 h, during which insoluble formazan was produced, 100 lL of 10% sodium dodecylsulphate were added in each well and another 12 h were allowed for the formazan to be dissolved. The absorbance was measured at 540 nm using the automated uQuant™ Universal Microplate Spectrophotometer (Biotek). Absorbance of control wells was taken as 100%, and the results were expressed as a percent of control. 2.6. Cell cycle analysis Cell cycle distribution in cells treated with the tested agents was analyzed by propidium iodide (PI) DNA staining. Briefly, 5  105 Jurkat cells were treated with the most active tested compound BC at the concentration of 50 lmol/L for 24, 48 and72 h. HUVEC (2  105) were treated with the concentration of 50 lmol/L for 24 and 48 h. After treatment, cells were harvested, washed twice in PBS and fixed overnight in 70% ethanol at 4 °C. Then, cells were washed with PBS, incubated for 1 h with 1 mg/ mL RNase and 10 lg/mL PI at room temperature in the dark. After staining, samples were immediately acquired in a FACS Canto flow cytometer using FACSDiva software (Becton Dickinson, USA). Ten thousand cells were acquired per sample. Data were analyzed using ModFit LT 3.0 software and expressed in the form of histograms. Percentages of cells corresponding to G1, S and G2 phases of the cell cycle were calculated. The cells with a DNA content lower than that of G1-phase cells (hypoploid population) were considered to be apoptotic (sub-G1). Because HUVEC undergo spontaneous apoptosis after 72 h of incubation we used 24 and 48 h incubation time for further experiments. 2.7. DNA fragmentation assay Treated and untreated Jurkat cells (for 24, 48 and 72 h; 1  106) and HUVEC (for 24 and 48 h; 2  105) were washed twice with 1X PBS calcium and magnesium free. Cells were lysed in a lysis buffer containing 10 mmol/L tris (hydroxymethyl)aminomethane, 10 mmol/L EDTA, 0.5% Triton X-100. Proteinase K (1 mg/mL) was

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added and cells were incubated at 37 °C for 1 h followed by 10 min. incubation at 70 °C. Then RNAase (200 lg/mL) was added and cells were incubated for another 1 h at 37 °C. Samples were transferred to 2% agarose gel and run with 40 V for 3 h. DNA fragments were visualized by a UV illuminator. 2.8. RNA isolation and cDNA synthesis Total RNA was isolated from Jurkat cells and HUVEC using the TRI Reagent (Molecular Research Center, Inc.) according to the manufacturer‘s instruction. Total RNA quality was verified on an agarose gel. Total RNA (1 lg) was reverse transcribed into cDNA by the RevertAid™ H Minus First Strand cDNA synthesis kit (Fermentas GmbH) according to the manufacturer‘s instruction, and used for quantitavive real time PCR. 2.9. Quantitative real-time PCR Quantitative real time PCR analysis was performed in Light Cycler (Roche USA) using iQTM SYBR Green Supermix (Bio-Rad Laboratories, USA) to verify the alterations of a-tubulin, a1-tubulin, b5tubulin, microtubule-related protein, bcl-2, bcl-xL and bax gene expression. The PCR program was initiated by 5 min at 95 °C before 40 thermal cycles, each of 30 s at 95 °C and 45 s at 60 °C. Data were analyzed according to the comparative Ct method and were normalized by b-actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.

significantly decreased survival of all cancer cell lines (p < 0.001) with IC50 ranging from 23.3 to 30.0 lmol/L. Differences in the effects of these two compounds were observed also in non-cancer HUVEC. Camalexin possessed a weak but significant toxicity (IC50 = 74.0 lmol/L) after 72 h of incubation. In contrast, BC (more toxic in cancer cells) was non-toxic in HUVEC (IC50 > 100.0). The methylation of benzocamalexin resulted in decreased antiproliferative activity and neither addition of methoxy, fluoro or cyano group increased it. Among camalexin analogues, BC displayed highest antiproliferative activity and was selected for further mechanistic studies. 3.2. Cell cycle analysis To determine the effect of the most potent indole phytoalexin on cell cycle progression, FACS analysis was done in Jurkat cells treated for 24, 48 and 72 h and HUVEC treated for 24 and 48 h with BC at 50 lmol/L. Comparison of control cells and cells treated for 24 h with BC showed increase in G1 fraction and after 48 h revealed an increase in the proportion of cells in the G2 phase from 9.7% to 18.8% (p < 0.01) followed by an increase in the proportion of cells having sub-G1 DNA content which was accompanied by a proportional decrease in the percentage of G1 phase cells. The G2 arrest did not persist and after 72 h treatment more than 57.4% cells were found to have sub-G1 DNA content (Table 2 and Fig. 2). BC did not show any effect on cell cycle distribution of HUVEC (Table 3 and Fig. 2). 3.3. DNA fragmentation assay

2.10. Statistical analysis Statistical data are expressed as mean ± standard deviation (SD). Student’s t-test and analysis of variance were employed to determine statistical significance. Values of p < 0.05 were considered to be statistically significant. 3. Results 3.1. Cytotoxicity The aim of this study was to examine the cytotoxicity of six indole phytoalexins in several cancer cell lines and non-cancer primary endothelial cells. Firstly, we compared biological activity of C and BC. Our data showed that C possesses selective cytotoxicity in cancer cells (Table 1). In comparison with non-treated cells, it significantly reduced the growth of HeLa and Jurkat cells (p < 0.01) as well as MDA and CEM cells (p < 0.05) with IC50 = 50.0, 46.2, 77.7 and 67.6 lmol/L, respectively. On the other hand, we found no significant toxic effect of camalexin in A549 and MCF cells even at the highest concentration used (100.0 lmol/L). The fusion of benzene to thiazole ring of camalexin structure significantly enhanced its cytotoxicity. In comparison with C, BC

Analysis of DNA fragmentation by agarose gel electrophoresis is one of the most widely used biochemical markers for cell death. The detection of internucleosomal DNA cleavage (DNA laddering) is considered to be an indicator of apoptosis. In our experiment DNA fragmentation of Jurkat cells into nucleosomal units was seen after 24 h incubation with BC at a concentration of 50 lmol/L. This effect was markedly enhanced after 48 and persisted after 72 h of incubation also. DNA fragmentation in HUVEC cells did not appear (Fig. 3). 3.4. Quantitative real-time PCR The cell cycle analysis showed that BC induces the G2 arrest. In order to verify whether the alterations of cell cycle are result in the alterations at the level of gene transcription, we conducted quantitative real-time PCR analysis for selected genes. After 48 h incubation of cancer cell with BC, we found a downregulation of the expression of a-tubulin, a1-tubulin, b5-tubulin suggesting the inhibitory effect of BC on microtubules. Furthermore, BC also downregulated the expression of microtubule-related protein suggesting an effect of this compound on microtubule assembly. As for the expression of apoptosis genes, BC downregulated the expression of anti-apoptotic bcl-2, bcl-xL and upregulated the

Table 1 The IC50 (lmol/L) of tested compounds in different cell lines and HUVEC cells after a 72 h of incubation.

C BC MBC MMBC MFBC MCBC

HeLa

Jurkat

A-549

MCF

MDA

CEM

HUVEC

50.0 ± 5.2 23.3 ± 3.2 >100.0 67.6 ± 8.2 50.0 ± 4.9 >100.0

46.2 ± 3.3 27.4 ± 08 >100.0 43.0 ± 6.3 >100.0 >100.0

>100.0 30.0 ± 1.5 >100.0 >100.0 >100.0 >100.0

>100.0 30.0 ± 2.1 >100.0 >100.0 >100.0 >100.0

77.7 ± 6.8 25.9 ± 3.9 >100.0 >100.0 >100.0 >100.0

67.6 ± 2.9 30.1 ± 2.4 74.8 ± 4.8 43.7 ± 5.9 >100.0 >100.0

74.0 ± 7.7 >100.0 >100.0 >100.0 66.0 ± 8.7 >100.0

C – camalexin, BC – benzocamalexin, MBC – 1-methylbenzocamalexin, MMBC – 1-methyl-60 -methoxybenzocamalexin, MFBC – 1-methyl-60 fluorobenzocamalexin, MCBC – 1-methyl-60 -cyanobenzocamalexin. The results are presented from six independent experiments.

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M. Pilatova et al. / Toxicology in Vitro 27 (2013) 939–944 Table 2 Flow cytometric analysis of cell cycle distribution in Jurkat cells treated with benzocamalexine (BC) (in%). Treatment

Conc. (lmol/L)

Time (h)

sub-G1

G1

S

G2

50.0

24 48 72

1.6 ± 0.2 3.0 ± 1.4 2.9 ± 3.5 57.4 ± 1.8a

44.4 ± 3.8 53.1 ± 3.9 54.7 ± 2.3a 38.4 ± 2.1a

45.1 ± 1.8 37.3 ± 2.6 26.6 ± 3.4 54.4 ± 1.5

10.5 ± 1.1 9.7 ± 3.1 18.8 ± 1.4b 7.3 ± 0.3 b

Control BC

b

The results are presented from three independent experiments. a p < 0.001. b p < 0.01 versus untreated cells (control).

A CTRL

B

24h

CTRL

48h

24h

72h

48h

Fig. 2. Cell cycle distribution detected by flow cytometric analysis. Jurkat cells (A) and HUVEC (B) were treated with BC (50.0 lmol/L) for the indicated time. The cells were then stained with PI and their nuclei analyzed for their DNA content by flow cytometry using CellQuest software. Each histogram is representative of three experiments.

Table 3 Flow cytometric analysis of cell cycle distribution in HUVEC cells treated with benzocamalexine (BC) (in %). Treatment

Conc. (lmol/L)

Time (h)

sub-G1

G1

S

G2

Control BC

50.0

24 48

1.7 ± 0.5 1.1 ± 0.3 2.3 ± 0.4

47.1 ± 2.5 41.3 ± 2.2 53.2 ± 1.3

22.4 ± 2.1 24.1 ± 0.8 17.8 ± 1.8

30.5 ± 0.7 34.4 ± 0.8 28.7 ± 0.4

The results are presented from three independent experiments.

A

NC

PC

1

2

3

B

NC

PC

1

2

3

4

Fig. 3. DNA fragmentation of (A) Jurkat cells after 24 (1), 48 (2) and 72 h (3) incubation with benzocamalexin (BC) at concentration 50 lmol/L and (B) HUVEC after 24 (1, 3) and 48 (2, 4) h incubation with benzocamalexin (BC) at concentration 50 lmol/L (1, 2) and 100 lmol/L (3, 4). Apoptotic DNA fragmentation was qualitatively analyzed by DNA gel electrophoresis. The extracted DNA was loaded on 2% agarose gel and was stained with ethidium bromide. Lanes indicate different treatments: NC – negative control (untreated cells); PC – positive control (etoposide 50 ll/mL). The pictures shown are representative of three independent experiments.

M. Pilatova et al. / Toxicology in Vitro 27 (2013) 939–944 Table 4 Effect of benzocamalexin (c = 50 lmol/L) on specific genes expression in Jurkat and HUVEC cells after 24 and 48 h of incubation. b-actin gene was used as a housekeeping gene to normalize each sample. Genes

Normalized ratio HUVEC

a-Tubulin a1-Tubulin b5-Tubulin MTRP bcl-2 bcl-xL bax

Jurkat

24 h

48 h

24 h

48 h

1.46 1.20 0.74 1.40 1.46 1.00 0.98

1.41 1.29 0.74 1.21 1.00 1.42 1.25

2.27 2.20 1.70 0.05 1.63 1.21 1.48

0.66 0.04 0.04 0.04 6.62E5 0.12 3.3

MTRP – microtubule-related protein. The results are presented from three independent experiments.

expression of pro-apoptotic gene bax (Table 4). The overall ratio of bax/bcl-2 mRNA levels was significantly increased in Jurkat cells following the treatment with BC. These results are in direct correlation with the data obtained in cell cycle analysis. No significant influence on gene expression was exerted in HUVEC (Table 4).

4. Discussion Previous reports indicate that consumption of cruciferous vegetables (from the Brassicaceae plant family) is associated with a reduced risk of several cancers (Murillo and Mehta, 2001; Powolny et al., 2011; Steinbrecher et al., 2009; Tang et al., 2010). It is believed that cancer chemopreventive effect of cruciferous vegetables is associated with sulfur-containing compounds called glucosinolates, which are cleaved by the plant enzyme myrosinase to biologically active compounds, such as indoles and isothiocyanates. Few years ago, we documented the antiproliferative effect of several indole phytoalexins or their synthetic analogues in different cancer cells types (Kutschy et al., 2009; Kutschy et al., 2010; Mezencev et al., 2008; Mezencev et al., 2009; Monde et al., 2005; Pilatova et al., 2005). In present study, the antiproliferative effects of camalexin and its synthetic derivatives on cancer and non-cancer cells were investigated. There is only limited data about effect of camalexin on mammalian cells (Mezencev et al., 2011; Moody et al., 1997). Mezencev and co-workers (2011) found that camalexin induces apoptosis in human T-leukemia Jurkat cells at micromolar concentration as detected by cell cycle analysis and annexin V/PI staining. Furthermore, activation of caspase-8, caspase-9, caspases-3/7 in camalexin-induced apoptosis was also demonstrated. As authors hypothesized, the ability of camalexin to stimulate reactive oxygen species production might play important role in camalexin-induced cells death. Herein, we report that camalexin, a naturally occurring indole phytoalexin, displayed only moderate cytotoxic activities against different cancer cells. The fusion of benzene to camalexin structure significantly increased its cytotoxicity. However, subsequent methylation of BC resulted in a decreased antiproliferative activity in cancer cell lines and neither addition of methoxy, fluoro nor cyano group increased it. Moreover, the effect of BC appeared to be selective for cancer cells (IC50 = 23.3–30.1 lmol/L versus IC50 > 100.0 lmol/L in HUVEC). Flow cytometric analysis indicated that the decrease in cell viability after Jurkat treatment with BC is associated with cell cycle arrest in the G2 phase followed by an increase in the fraction of cells with sub-G1 DNA content. In order to delineate mechanisms

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that may be involved in the response of Jurkat cells to BC treatment, we analyzed the expression of genes involved in microtubules formation as well as in apoptosis. Microtubules, formed from a-tubulin and b-tubulin, play a critical role in many cellular processes, including cell division, cell motility, intracellular trafficking, and cell shape maintenance. Perhaps most importantly they comprise the mitotic spindle, which controls the alignment and segregation of chromosomes during mitosis (Li et al., 2004). Our findings indicate that BC induced a decrease in the expression of a-tubulin and b-tubulin as well as the expression of microtubule-related protein as demonstrated by quantitative real-time PCR. Downregulation of tubulins may lead to deficient mitotic spindle formation resulting in a cell cycle arrest. Microtubule-associated proteins are a family of proteins that are involved in the regulation of microtubule assembly. Downregulation of microtubuleassociated proteins may promote destabilization of microtubules followed by non-repairable damage to the cells (Bhat and Setaluri, 2007). Recent studies have suggested that various cellular organelles including mitochondria are involved in apoptotic cell death (Faitova et al., 2006; Mukherjee et al., 2007). Mitochondrial Bcl-2 family proteins play an important role in mitochondria-mediated cell death (Rovini et al., 2011; Zinkel et al., 2006). Bcl-2, Bcl-xL and Bax are critical determinants of whether or not cells undergo apoptosis under experimental conditions that promote cell death. Several chemopreventive agents have been shown to act via induction of apoptosis through modulation of Bax/Bcl-2 family proteins (Bacˇkorová et al., 2012; Cai et al., 2011; Ferenc et al., 2010; Pilatova et al., 2010). In the present study, we have observed that BC caused a downregulation of the anti-apoptotic genes bcl-2 and bcl-xL accompanied with an upregulation of the proapoptotic gene bax in Jurkat cells. A critical determinant of the cell’s fate to enter apoptosis is the ratio of pro-apoptotic to anti-apoptotic protein levels. Increased ratio of Bax/Bcl-2 mRNA in Jurkat cells after treatment with BC indicates that induction of apoptosis is a key event in the cytotoxicity of this compound. To conclude, the data obtained in the present study indicate that molecular mechanisms of BC-mediated cytotoxicity in Jurkat cells might involve (1) downregulation of tubulin expression, (2) cell cycle arrest, (3) modulation of Bcl-2 family proteins leading to (4) apoptosis.

Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Slovak Research and Development Agency under the contracts no. APVV-0325-07 and APVV0514-06, by SEPO-II (ITMS code: 26220120039), and by VEGA 1/ 0304/10 and 1/0302/10.

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