Báo cáo khoa học: Strong cytotoxic effect of the bradykinin antagonist BKM-570 in ovarian cancer cells – analysis of the molecular mechanisms of its antiproliferative action

Strong cytotoxic effect of the bradykinin antagonist BKM-570 in ovarian cancer cells – analysis of the molecular mechanisms of its antiproliferative action Stephanie Jutras1,2, Magdalena Bachvarova2, Mamadou Keita1,2, Jean-Loup Bascands3,4, Anne-Marie Mes-Masson5,6, John M. Stewart7 and Dimcho Bachvarov1,2

1 Department of Molecular Medicine, Laval University, Que´ bec, Canada 2 Cancer Research Centre, Hoˆ pital L’Hotel-Dieu de Que´ bec, Centre Hospitalier Universitaire de Que´ bec (CHUQ), Canada 3 Inserm, U858 ⁄ I2MR, Department of Cardiac and Renal Remodeling, Toulouse, France 4 Universite´ Toulouse III Paul Sabatier, Institut de Me´ decine Mole´ culaire de Rangueil, France 5 Department of Medicine, Universite´ de Montre´ al, Canada 6 Centre de Recherche du Centre Hospitalier de l’Universite´ de Montre´ al (CHUM), Institut du Cancer de Montre´ al, Canada 7 Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO, USA

Keywords BKM-570; bradykinin antagonist; bradykinin receptor; DNA microarrays; ovarian cancer therapy

Correspondence D. Bachvarov, CHUQ Centre de recherche, Hoˆ tel-Dieu de Que´ bec, 9 rue McMahon, Que´ bec (Que´ bec), Canada G1R 2J6 Fax: +1 418 6915439 Tel: +1 418 525 4444, ext. 15590 E-mail: dimtcho.batchvarov@crhdq.ulaval.ca

(Received 8 September 2010, revised 9 October 2010, accepted 20 October 2010)

doi:10.1111/j.1742-4658.2010.07928.x

The standard chemotherapy for epithelial ovarian cancer (EOC) patients is currently a combination of taxane and platinum. However, most EOC patients still suffer relapses, and there is an immediate need for the devel- opment of novel and more effective therapeutic modalities against this deadly disease. Recently, the nonpeptide bradykinin (BK) antagonist 2,3,4,5,6-pentaﬂuorocinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino- 2,2,6,6-tetramethyl-piperidyl) amide (BKM-570) was shown to cause impressive growth inhibition of lung and prostate tumors, displaying supe- rior in vivo inhibitory effects than convential chemotherapeutic drugs. Here, we investigated BKM-570 cytotoxic effects in two EOC cell lines, derived from different EOC histopathologies: a clear cell carcinoma (TOV-21), and an endometrioid carcinoma (TOV-112). We showed that BKM-570 effec- tively inhibited the growth of ovarian cancer cells, as its cytotoxic effects were comparable to those of cisplatin, and were independent of the func- status of BK receptors. Moreover, BKM-570 synergized with tional cisplatin in inhibiting EOC cell growth. To better understand the molecular mechanisms of the antiproliferative action of this BK antagonist in EOC cells, we performed gene expression proﬁling in TOV-21 and TOV-112 cells following treatment with 10 lM BKM-570 for 24 h. BKM-570 displayed similar cytotoxic effects in the two cell lines analyzed, as genes with previ- ously shown involvement in apoptosis ⁄ antiapoptosis and cell adhesion were proportionally upregulated and downregulated in both cell lines, whereas including cell growth and genes involved in basic cellular mechanisms, inﬂammatory and immune maintenance, metabolism, cell cycle control,

Abbreviations AVP, arginine vasopressin; BK, bradykinin; BKM-570, 2,3,4,5,6-pentaﬂuorocinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino-2,2,6,6- tetramethyl-piperidyl) amide; B1R, B1 receptor; B2R, B2 receptor; CI, combination index; des-Arg9-BK, des-Arg9-bradykinin; EGF, epidermal growth receptor; EOC, epithelial ovarian cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G-protein-coupled receptor; HLA-B, human leukocyte antigen-B; HNF4a, hepatic nuclear factor 4a; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide; NF-jB, nuclear factor-kappaB; sqRT-PCR, semiquantitative RT-PCR; TNFRSF1B, tumor necrosis factor receptor superfamily, member 1B; YWHAG, tyrosine 3-monooxygenase ⁄ tryptophan 5-monooxygenase activation protein, gamma polypeptide.

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response, signal transduction, protein biosynthesis, transcription regulation, and transport, were predominantly downregulated upon treatment. Our data are indicative of the therapeutic potential of BKM-570 and related compounds in EOC management.

Introduction

therapeutic modalities against

of peptide and nonpeptide BK antagonists showed remarkable anticancer activities in both in vitro and in vivo cancer models [13,14]. Among these, the non- peptide (mimetic) BK antagonist 2,3,4,5,6-pentaﬂuoro- cinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino- 2,2,6,6-tetramethyl-piperidyl) amide (BKM-570) caused impressive growth inhibition of lung and prostate tumors, displaying superior in vivo inhibitory effects than the vascular endothelial growth factor inhibitor SU5416 [15,16] (see also Table 1 for the BKM-570 pri- mary structure). The efﬁcacy of BKM-570 and other newly developed BK antagonists in cancer treatment points the way for the development of new drugs against aggressive tumors, for which no satisfactory therapy now exists. In this study, we investigated in detail the cytotoxic effects of BKM-570 in two EOC cell lines, derived from different EOC histopathologies: a clear cell carcinoma (TOV-21), and an endometrioid carcinoma (TOV-112). Surprisingly, BKM-570 strongly and comparatively inhibited the growth of both EOC lines, although TOV-21 cells displayed only B2R expression, whereas TOV-112 cells expressed no func- tional BK receptors. Thus, by using the DNA micro- array approach, we tried to better comprehend the molecular mechanisms of the antiproliferative action of BKM-570 in EOC cells.

Epithelial ovarian cancer (EOC) is a disease that will affect approximately 1.7% of North American women during their lifetime, and is responsible for more can- cer deaths among women in the Western world than all other gynecologic malignancies [1]. Because of its asymptomatic nature, EOC is characterized by presen- tation at an advanced stage, the fact that it is spread primarily by an intraperitoneal route, and its relative sensitivity surgical to chemotherapy. An initial approach is essential for proper staging of the disease process and for aggressive cytoreduction, which in turn improves the response to chemotherapy and survival [2]. Chemotherapy has had an increasingly important role in the effective treatment of EOC. Since the early 1990s, the reference standard for postsurgical EOC ﬁrst-line chemotherapy has been the use of a plati- num–taxane combination, usually carboplatin paired with paclitaxel [3]. Although the standard taxane–plati- num regimen results in a complete response rate of 40–60% in advanced EOC patients [4], most of these patients relapse after a median period of 18 months, owing to the emergence of tumor resistance to these conventional drugs [4]. Thus, there is an immediate need for the development and application of novel and more this effective deadly disease.

Results

Cytotoxic effect of BKM-570 in the EOC cell lines TOV-21 and TOV-112 – synergistic interaction between BKM-570 and cisplatin

Bradykinin (BK), a vasoactive peptide released dur- ing inﬂammation [5], has been considered as a multi- potential stimulant of cancer growth. BK and its active metabolite des-Arg9-BK (des-Arg9-BK) exert their cellular effects through two pharmacologically distinct G-protein-coupled receptors (GPCRs), the B1 receptor (B1R) and the B2 receptor (B2R) [6].

especially

the B2R subtype,

High BK concentrations were found in ascitic and pleural ﬂuids obtained from patients with ovarian, gastric, pulmonary and hepatic cancers, as well as in experimental murine ascetic tumors [7–10]. Moreover, are BK receptors, expressed in different human cancers [11], suggesting a potential role for BK in inducing pathologic signal transduction in cancer growth and progression, nitric oxide production and vascular permeability enhance- ment in tumors [12]. BK antagonists may thus have applications in the modulation of cancer growth and in paraneoplastic syndromes. Indeed, new generations

To determine BKM-570-induced cytotoxicity in EOC cells, TOV-21 and TOV-112 cells were cultured in the absence (vehicle control) or in the presence of increas- ing doses of BKM-570 (0.05–100 lm) for 72 h. As shown in Fig. 1, BKM-570 displayed very strong cyto- toxic activity against the two EOC cell lines that was comparable to that of cisplatin, a drug that is com- monly used for ﬁrst-line treatment of EOC (Fig. 1). Thus, in TOV-21 cells, the IC50 values for BKM-570 and cisplatin were 19.37 lm and 9.85 lm, respectively, and in TOV-112 cells, the IC50 values were 21.51 lm and 19.92 lm for BKM-570 and cisplatin, respectively.

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Table 1. Primary structure of the BK antagonist BKM-570. The structures of the natural B1R and B2R agonists are presented for comparison. F5c, 2,3,4,5,6-pentaﬂuorocinnamoyl; OC2Y, O-2,6-di- chlorobenzyl-tyrosine; Atmp, 4-amino-2,2,6,6-tetramethylpiperidine.

Table 2. CI of the combination of BKM-570 and cisplatin, indicating synergism according to CALCUSYN software in TOV-21 and TOV-112 cells. CI < 0.1 indicates very strong synergism; CI = 0.1–0.3 indi- cates strong synergism; and CI = 0.3–0.85 indicates synergism.

Name

Structure

Function

BKM-570 (lM)

Cisplatin (lM)

BK des-Arg9-BK Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe BKM-570

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg B2R agonist B1R agonist BK antagonist

F5c-OC2Y-Atmp.HCl

2.5 5.0 10.0

0.01 0.015 0.032

TOV-21

A

120.0

TOV-21 5.0 10.0 20.0 TOV-112 5.0 10.0 20.0

5.0 10.0 20.0

0.011 0.036 0.033

BKM Cisplatin

100.0

)

80.0

( y t i l i

60.0

b a v l l

40.0

e C

20.0

0.0

0.01

0.1

100

1 Drug concentration (µM)

B

TOV-112

140.0

BKM Cisplatin

120.0

100.0

)

80.0

( y t i l i

values for each drug were used to determine equipo- tency for performance of consecutive synergistic analy- the BKM-570–cisplatin combined treatment. ses of Two serial dilutions (0.5 : 0.5 and 0.25 : 0.25) of BKM-570 and cisplatin were generated while a con- stant 1 : 1 equipotent ratio based on the IC50 values was maintained. Table 2 shows the combination index (CI) observed after treatment with the combination of the two drugs. At doses ranging from 5 to 20 lm BKM-570, combined with 2.5–10 lm cisplatin in TOV- 21 cells and 5–20 lm cisplatin in TOV-112 cells, CI ranged from 0.01 to 0.032 in TOV-21 cells, and from 0.01 to 0.036 in TOV-112 cells, suggesting that this combination displays a very strong synergistic effect in both EOC cell lines.

60.0

b a v l l

e C

40.0

Evaluation of B1R and B2R expression levels and functional status in TOV-21 and TOV-112 cells

20.0

0.0

0.01

0.1

100

1 Drug concentration (µM)

Fig. 1. Dose–response cytotoxicity curves for the EOC cell lines TOV-21 and TOV-112 treated with BKM-570 (BKM) and cisplatin. The dose range for both drugs was 0.05–100 lM. (A) Continual 72 h exposure of TOV-21 to BKM-570 (IC50, 19.37 lM) and cis- platin (IC50, 9.85 lM). (B) Continual 72 h exposure of TOV-112 to BKM-570 (IC50, 21.51 lM) and cisplatin (IC50, 19.92 lM). All results are expressed as mean ± standard deviation of three independent experiments.

Next, we investigated the expression levels of B1R and B2R in the TOV-21 and TOV-112 cells, using a semi- quantitative (duplex) RT-PCR approach. Surprisingly, TOV-21 cells displayed mostly B2R gene expression, whereas TOV-112 cells showed no BK receptor expres- sion (Fig. 2A). These ﬁndings were further conﬁrmed by functional analyses, as we examined the ability of BK (a speciﬁc B2R agonist) and des-Arg9-BK (a speciﬁc B1R agonist) to activate their corresponding receptors by mobilizing intracellular Ca2+ levels. As seen in Fig. 2B, only BK was able to induce intra- cellular release in TOV-21 cells, as both agonists showed no activity in TOV-112 cells. Additional GPCR agonists [angiotensin and arginine vasopressin (AVP)] were used as positive controls in order to con- ﬁrm the validity of the functional assays. These results indicate that the cytotoxic effect of BKM-570 in EOC cells could be entirely independent of BK receptor activity.

We next investigated the cytotoxic effects of the BKM- 570–cisplatin combination. The interaction between BKM-570 and cisplatin was analyzed by 3-(4,5-dim- ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays, and calcusyn software was used to determine whether this combination had additive or synergistic effects on EOC cell growth. The IC50

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TOV-21

A

B1R

TOV-112 B2R

B2R

B1R

kinin Rs

GAPDH

TOV-112

TOV-21

B

2.5

n o

i t

2.5

2.0

a r e b

2.0

i l + 2 a 1.5C

K B

P V A

n s n e t o g n A

K B - 9 g r A - s e d

Fig. 2. Analysis of the gene expression and functional activity of B1R and B2R in the EOC cell lines TOV-21 and TOV-112. (A) Analysis of B1R and B2R gene expression by RT-PCR. GAPDH was used as a reporter gene. (B) Functional (intracellular Ca2+ release) assays for BK receptor activity following treatments with speciﬁc B2R (BK) and B1R (des-Arg9-BK) agonists. Additional speciﬁc GPCR agonists (angiotensin and AVP) were used as positive controls (see details in text).

TOV-21 and TOV-112 cells display common and some distinct gene expression signatures following BKM-570 treatment

the differentially expressed genes

(‡ 2-fold)

nantly downregulated upon treatment. Additionally, TOV-112 cells displayed altered expression of DNA repair genes, upregulation of genes involved in the defense response, and substantial downregulation of genes associated with chromatin assembly and organi- zation, protein turnover and modiﬁcation, and ubiqui- tination. Tables S1 and S2 show the complete list in of the BKM-570-treated TOV-21 and TOV-112 cells, respectively.

line against

To better understand the molecular mechanisms of BKM-570 action in EOC cells, we employed Agilent human oligonucleotide (v2) microarrays, containing to identify global gene expression (cid:2) 22 000 genes, changes in the two EOC cell lines TOV-21 and TOV- 112 following treatment with 10 lm BKM-570 for 24 h. All microarray experiments were performed in duplicate, as two hybridizations were carried out for every BKM-570-treated cell the corre- sponding control cell line with a ﬂuorescent dye rever- sal (dye-swap) technique. For each cell line, a subset of differentially expressed genes was selected displaying at least two-fold difference upon BKM-570 treatment in both duplicate microarray experiments. Using these selection criteria, we found 126 genes to be upregulat- ed and 452 genes to be downregulated in TOV-21 cells following the BKM-570 treatment, whereas 400 genes were upregulated and 866 were downregulated in the BKM-570-treated TOV-112 cells. Table 3 shows a list of selected functionally related groups of genes that were differentially expressed (‡ 2-fold) in TOV-21 cells (Table 3A,B) and TOV-112 cells (Table 3C,D). BKM- 570 displayed similar cytotoxic effects in the two cell lines analyzed, as genes with previously shown involve- in apoptosis ⁄ antiapoptosis and cell adhesion ment were proportionally upregulated and downregulated in both cell lines, whereas genes involved in basic cellular mechanisms, including cell growth and maintenance, cell cycle control, inﬂammatory and immune responses, metabolism, signal transduction, protein biosynthesis, transcription regulation, and transport, were predomi-

Venn diagram analysis based on the above gene lists showed a comparatively large number of genes (314 genes) that were commonly downregulated in TOV-21 and TOV-112 cells, whereas only six genes were com- monly upregulated in both cell lines upon BKM-570 treatment (Fig. 3A). This analysis prompted us to eval- uate whether BKM-570-treated TOV-21 and TOV-112 cells could be distinguished by their gene expression proﬁles. Following ﬁltering on two-fold signal inten- sity, we used a one-way ANOVA parametric test (Welch t-test; variances not assumed to be equal) to select discriminatory genes. Indeed, a t-test with a P-value cutoff of 0.05 selected 148 genes for which expression differed in TOV-21 and TOV-112 cells. Clustering analysis based on the 148-gene list was per- formed with the standard Condition Tree algorithm provided in genespring, and revealed the formation of two major cluster groups that clearly distinguish TOV-21 and TOV-112 cells upon BKM-570 treatment (Fig. 3B). Sixty-ﬁve genes from the 148-gene list were upregulated in TOV-21 cells as compared with TOV- 112 cells. Major classiﬁcations of these genes include signal transduction, transport, transcription regulation, metabolism, and the inﬂammatory ⁄ immune response. Genes that were upregulated in TOV-112 cells in

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Fig. 3. Comparison of common and distinct gene expression across the different two- fold differentially expressed gene groups in TOV-21 and TOV-112 cells. (A) Comparison of the differentially expressed genes in TOV-21 and TOV-112 cells following BKM- 570 treatment, using Venn diagram analysis. (B) Hierarchical clustering based on the 148-gene list (P-value cutoff of 0.05), which discriminates TOV-21 and TOV-112 cells according to their response to BKM-570 treatment. Red signiﬁes upregulation, and green signiﬁes downregulation (see legend bar).

comparison with TOV-21 cells (83 genes) are mainly involved in metabolism, signal transduction, protein biosynthesis and modiﬁcation, regulation of transcrip- tion, transport, cell proliferation, and cell adhesion. The 148-gene list is presented in Table S3.

mostly upregulated in TOV-112 cells (Fig. 4C). Accord- ingly, pathways associated with cellular function and maintenance and cell signaling were most strongly downregulated in TOV-21 cells (Fig. 4B), whereas TOV-112 cells displayed speciﬁc downregulation of pathways related to post-translational modiﬁcation, cel- lular movement and protein synthesis (Fig. 4D).

Network analysis

identiﬁed 22 highly signiﬁcant networks with scores ‡ 3 in TOV-21 cells (Table S4) and 44 highly signiﬁcant networks with scores ‡ 3 in TOV-112 cells (Table S5). In TOV-21 cells, the ﬁve top-scoring networks were functionally associated with cancer, cell death and cell cycle control, and other pathologies, including genetic, hepatic, cardiovascular, gastrointestinal and genetic disorders. In TOV-112 cells, the ﬁve top-scoring networks were predominantly related to metabolism, signal transduction, cell–cell sig- naling and interaction, cellular assembly and organiza- tion, cancer, and cellular function and maintenance.

synthesis

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Pathway and network analyses, generated through the use of ingenuity pathways analysis (IPA) soft- ware, conﬁrmed the common and distinct major func- tionally related gene groups found to be differentially expressed in BKM-570-treated TOV-21 and TOV-112 cells. Thus, similar pathways were both induced and suppressed in both EOC cell types (including cell–cell signaling and interaction, cell cycle, cell death, gene expression, and antigen presentation), as pathways linked to cellular function and maintenance, cell growth and proliferation, metabolism, protein biosynthesis, transcription regulation and transport displayed pre- dominant downregulation in TOV-21 and TOV-112 cells (Fig. 4). The two EOC cell lines also displayed some differences upon BKM-570 treatment; thus, path- ways associated with cellular assembly and organiza- tion, post-translational modiﬁcation were predominantly upregulated in TOV- 21 cells (Fig. 4A), whereas pathways functionally related to DNA replication, recombination and repair, drug metabolism and cellular growth and proliferation were

Common networks obtained upon merging the ﬁve top-scoring networks (Fig. 5) revealed important nodes that are speciﬁc for each cell line. Thus, major nodes and associated interaction partners that were predomi- nantly downregulated in TOV-21 cells upon BKM-570 factor treatment 4a hepatic (EGF) and its (HNF4a), epidermal growth factor

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Fig. 4. Functional analysis for a dataset of differentially expressed genes (‡ 2-fold) in EOC cells following BKM-570 treatment. (A) Functional analysis of upregulated genes in TOV-21 cells. (B) Functional analysis of downregulated genes in TOV-21 cells. (C) Functional analysis of up- regulated genes in TOV-112 cells. (D) Functional analysis of downregulated genes in TOV-112 cells. Top functions that meet a P-value cutoff of 0.05 are displayed.

the nuclear

line (TOV-21 and TOV-112) following BKM-570 treat- ment, and quantiﬁed their expression by sqRT-PCR in control and treated spheroids. Table 4 summarizes the gene expression measurements of all validated genes. We found that both methods (microarray analysis and sqRT-PCR) detected similar patterns for the upregulat- ed and downregulated genes selected for validation.

Discussion

network, and genes and complexes that are mostly involved in inﬂammation and the immune response, including members of factor-kappaB (NF-jB) network (NF-jB complex and NF-jB ⁄ p65, or RELA) and the major histocompatibility complex class IB [human leukocyte antigen-B (HLA-B)] gene (Fig. 5A). Major gene nodes that were downregulated in TOV-112 cells included numerous members of the mitogen-activated protein kinase (MAPK) gene family (including ERK, Mapk, Jnk, MAPK9, MAPK12, MAPK13, Sapk, MAP4K5 and MATK). Other impor- tant gene nodes in BKM-570-treated TOV-112 cells also included the tumor necrosis factor receptor super- family, member 1B (TNFRSF1B) gene and the gene for the tyrosine 3-monooxygenase ⁄ tryptophan 5-mono- oxygenase activation protein, gamma polypeptide (YWHAG) (Fig. 5B).

In xenotransplants,

Validation of microarray ﬁndings with semiquan- titative RT-PCR (sqRT-PCR)

To validate the microarray results, we arbitrarily selected eight differentially expressed genes in each cell

Neuropeptides play a prominent role in the autocrine growth of EOC and a variety of other carcinomas [17,18]. Kinins and their receptors have been shown promote tumor-associated angiogenesis and tumor growth in vivo [11,19]. Speciﬁc peptide and nonpeptide BK antagonists, including BKM-570, displayed remark- able anticancer activities in both in vitro and in vivo cancer models, especially of lung and prostate cancers [13–16]. some BK antagonists showed higher potency than conventional anticancer drugs, including cisplatin, without evident toxicity to the hosts [15,20,21]. In certain combinations, they acted synergistically with standard anticancer drugs

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B

A

Fig. 5. Network analysis of dynamic gene expression in EOC cells based on the two-fold common gene expression list obtained following treatment with BKM-570. (A) Network analysis of BKM-570-treated TOV-21 cells. (B) Network analysis of BKM-570-treated TOV-112 cells. The ﬁve top-scoring networks for each cell line were merged, and are displayed graphically as node (genes ⁄ gene product) and edges (the biological relationships between the nodes). Intensity of the node color indicates the degree of upregulation (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product (square, cytokine; vertical oval, transmem- brane receptor; rectangle, nuclear receptor; diamond, enzyme; rhomboid, transporter; hexagon, translation factor; horizontal oval, transcrip- tion factor; circle, other). Edges are displayed with various labels that describe the nature of the relationship between the nodes: ____, binding only; ﬁ , acts on. The length of an edge reﬂects the evidence supporting that node-to-node relationship, in that edges supported by articles from the literature are shorter. Dotted edges represent indirect interactions.

[16,20]. Indeed, BKM-570 displayed very potent inhibi- tion of small cell lung cancer growth in nude mice that was stronger than that of cisplatin [22]. BKM-570 also showed activity against various other cancer cell lines in vitro, including small cell lung cancer, non-small cell lung cancer, and lung, prostate, pancreas, colon and cervix cancer [14].

receptor activity). These ﬁndings prompted us to use the microarray-based mRNA expression proﬁling approach to better understand the molecular determi- nants of the response of EOC cell lines to BKM-570. To our knowledge, the present work represents the ﬁrst effort to deﬁne global changes in gene expression in BKM-570-treated cancer cells by the use of high- density microarrays.

effects were

transcriptomics

We analyzed both functionally related genes that were commonly differentially expressed in the two EOC cell lines, as well as alterations in gene expression patterns that were speciﬁc for each cell line. In general, BKM-570 relatively similar to those displayed by some conventional EOC chemotherapeutic drugs (cisplatin, paclitaxel, and topotecan) that were previously analyzed in our group. Indeed, signiﬁcantly more genes were downregulated than induced following BKM-570 treatment in both EOC cell lines, and this was quite similar to the effect of these chemotherapeutic drugs [23]. The main goal of any cytotoxic (chemotherapy) treatment is the induc- tion of cell death (apoptosis), and we noticed a

In this study, we investigated the cytotoxic effect of BKM-570 in ovarian cancer cells. BKM-570 displayed potent anticancer activity that was comparable to that of cisplatin, which is used as a standard chemotherapeutic agent in EOC treatment. BKM-570 also induced very strong synergistic growth inhibition when combined with cisplatin. More importantly, we showed for the ﬁrst time that BKM-570 can equally suppress the growth of EOC cell lines irrespective of the expression of functional BK receptors, which is indicative of possible direct (BK receptor-independent) inhibitory effects of this mimetic compound on cancer cell growth. Indeed, BKM-570 displayed equipotent antitumor activity in TOV-21 cells (expressing a func- tional B2R) and TOV-112 cells (displaying no BK

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Table 4. Semiquantitative RT-PCR validation of microarray data.

Fold expression

Cell line

Gene

Common name

Microarray

sqRT-PCR

TOV-21 TOV-21 TOV-21 TOV-21 TOV-21 TOV-21 TOV-21 TOV-112 TOV-112 TOV-112 TOV-112 TOV-112 TOV-112 TOV-112 TOV-112

SENP7 APOE CLDN18 TSSK6 CTAG1A GRM4 CCL19 PLA1A CDC26 HOXA1 MARS TPD52L2 TNFAIP2 TXK INSR

SUMO1 ⁄ sentrin-speciﬁc protease 7 Apolipoprotein E Claudin 18 Testis-speciﬁc serine kinase 6 Cancer ⁄ testis antigen 1A Glutamate receptor, metabotropic 4 Chemokine (C-C motif) ligand 19 Phospholipase A1 member A Cell division cycle 26 Homeobox A1 Methionine-tRNA synthetase Tumor protein D52-like 2 Tumor necrosis factor, alpha-induced protein 2 TXK tyrosine kinase Insulin receptor

+4.13 )6.21 )5.08 )4.37 )3.85 )3.27 )2.86 +3.69 +3.04 +2.85 )7.25 )6.13 )4.95 )3.82 )2.89

+2.57 )7.23 )3.18 )4.55 )2.19 )2.43 )1.89 +2.12 +1.77 +3.01 )4.18 )3.97 )3.14 )1.66 )2.14

regulation of genes associated with chromatin assembly and organization, protein turnover and modiﬁcation, and ubiquitination. It cannot be assumed that these differences are attributable to the absence of functional BK receptors in the TOV-112 cell line as compared with TOV-21 cells expressing a functional B2R. More probably, the observed discrepancy is associated with the distinct histopathologic origins of the cell lines: clear cell carcinoma for TOV-21, and endometrioid carcinoma for TOV-112.

signal

comparative number of apoptosis-related genes that were upregulated and downregulated in both cell lines following BKM-570 treatment (Table 3); again, this was comparable to the effect obtained in EOC with the standard chemotherapeutic drugs [23]. Although induction of proapoptotic genes is expected upon treat- ment with chemotherapeutics, the downregulation of genes involved in apoptosis could be indicative of some compensatory mechanisms linked to BKM-570- induced cell death [24]. Likewise, different genes that are functionally involved in cell adhesion were compar- atively induced or suppressed following BKM-570 exposure. Indeed, destabilization of cellular adhesion in cancer cells following treatment was shown to be causal for apoptosis induction [25]; however, common overexpression of cell adhesion genes was also observed in both treated EOC cell lines, probably rep- resenting an adaptive response to BKM-570 treatment. As seen previously with conventional chemothera- peutic drugs, BKM-570 treatment triggered in both EOC cell lines similar downregulation of numerous genes that are functionally associated with cell growth and proliferation, metabolism, transduction, gene expression, protein biosynthesis and modiﬁcation, and molecular transport. In fact, suppressed transcrip- tion and lower metabolic rates have been previously linked with chemotherapeutic action [26].

The two cell

Although identiﬁcation of a list of individual genes that show expression changes is important, there is an increasing need to move beyond this level of analysis. Instead of simply enumerating a list of genes, we wanted to know how they interact as parts of complexes, path- ways, and biological networks. For this purpose, the microarray data were imported into the IPA software to identify relevant biological pathways and networks. Pathway and network analyses were highly conﬁrma- tory of the gene expression data obtained. Indeed, a number of functional pathways displayed similar expres- sion patterns in both EOC cell lines upon BKM-570 treatment (Fig. 4), although the cell lines displayed some speciﬁc pathway alterations, probably linked to their different histolopathologic origins. The observed upreg- ulation in TOV-112 cells of pathways functionally related to DNA replication, recombination and repair, drug metabolism and cellular growth and proliferation could be associated with some protective mechanisms against BKM-570 action in this cell line.

IPA network investigation was also indicative of lines in in TOV-21 the

some differences between the two EOC cell response to BKM-570 treatment. Thus, cells, network analysis was

suggestive

for

lines also displayed some differences upon BKM-570 exposure, conﬁrmed by hierarchical clustering analysis; the upregulation of DNA repair genes and genes implicated in the defense response in TOV-112 cells could be considered to represent a com- pensatory mechanism against BKM-570 action in these EOC cells. TOV-112 cells also displayed speciﬁc down-

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BKM-570

BKM-570 also exhibited a certain speciﬁcity of cyto- toxic action. This is shown by direct comparison of its transcriptomics effects in EOC cells with those of cis- platin and topotecan (analyses based on previously published data [23]; see also Fig. S1). This speciﬁcity is also substantiated by comparison of the most signiﬁ- cant functional pathways affected by BKM-570 in TOV-21 and TOV-112 cells (i.e. upregulation of cell– cell signaling and interaction pathways and predomi- nant downregulation of a number of metabolic path- ways in both cell lines) with those affected by cisplatin and topotecan (i.e. predominant upregulation of path- ways involved in cell cycle arrest and DNA replication, recombination and repair, and downregulation of path- ways mainly linked to cell growth and proliferation, cell cycle control, and cell–cell signaling and interac- tion; see Fig. 4 and [23]). These data indicate that the molecular mechanisms of BKM-570 cytotoxic action in EOC cells could be predominantly associated with sup- pression of EOC cell metabolism, and corroborate the hypothesis that the metabolic phenotype of EOC cells can be targeted for therapeutic intervention [37,38].

important roles of speciﬁc gene nodes [HNF4a, EGF, NF-jB complex, NF-jB ⁄ p65 (RELA) and HLA-B] in the cytotoxic action of BKM-570. All of these genes and their related networks could potentiate cancero- including ovarian cancerogenesis, and were genesis, mostly treatment upon suppressed (Fig. 5A). Indeed, HNF4a is a potent transcription factor that regulates the expression of multiple compo- nents within all three key compartments of the cell–cell junction, namely the adherens junction, the tight junc- tion, and the desmosome [27]. A role for HNF4a was suggested in tumor genesis, aggressiveness and metas- tasis in various tumors, including lung, colorectal and mucinous ovarian tumors [28–30], and suppression of HNF4a activity by natural or xenobiotic HNF4a ligand antagonists or by HNF4a small interfering RNA was proposed as a novel anticancer treatment strategy [29]. EGF and its receptors play etiologic roles in the molecular pathogenesis of cancer, and represent key therapeutic targets in many types of cancer, including ovarian cancer [31]. Similarly, it has been shown that the NF-jB pathway is frequently overactivated in aggressive ovarian cancers, and its modulation improved outcome in the subset of women who exhibit this pathway activity [32]. Finally, HLA-B and other members of the major histocompatibility class I com- plex have shown prognostic signiﬁcance in EOC, and could represent potential therapeutic targets in EOC management [33].

In conclusion, our studies show that the mimetic BK antagonist BKM-570 can effectively inhibit the growth of ovarian cancer cells, as BKM-570 displayed quite similar cytotoxic effects in EOC cells as cisplatin, a standard chemotherapeutic drug that is currently used for EOC therapy. Moreover, BKM-570 exhibited very strong synergism with cisplatin in inhibiting EOC cell growth. We have also demonstrated, for the ﬁrst time, that the anticancer activity of this BK antagonist could be independent of the functional status of the BK receptors.

BKM-570 displayed the same pattern of cytotoxic action in EOC cells as some standard chemotherapeu- tic drugs (cisplatin and topotecan) that are currently used for EOC therapy, as a number of genes and gene pathways involved in basic cellular mechanisms were similarly downregulated upon BKM-570 treatment. However, BKM-570 also demonstrated some apparent inhibitory effects, predominantly linked to its spe- ciﬁc suppression of major metabolic pathways in EOC cells.

Gene expression proﬁling and consecutive network and pathway analyses were also indicative of some dif- ferences between the two EOC cell lines in response to BKM-570 treatment, which are probably related to the different histolopathologic origins of each cell line.

In TOV-112 cells, network analysis demonstrated predominant suppression of the MAPK signaling path- way (Fig. 5B). Most of these genes are members of oncogenic pathways, which have been shown to be implicated in EOC initiation and ⁄ or progression [34]. Other important downregulated gene nodes in BKM- included TNFRSF1B 570-treated TOV-112 cells (TNFR2) and YWHAG. TNFR2 is one of the recep- tors for tumor necrosis factor (TNF)-a, and represents a potent antigenic factor; TNFR2 was found to be overexpressed in EOC tumors, and its expression cor- related with the highest risk of EOC progression [35]. YWHAG, or 14-3-3c, is a member of the 14-3-3 gene family, and is involved in the regulation of a variety of important cellular processes, such as the cell cycle, apoptosis, and mitogenic signaling. Recent evidence indicates that the expression of some of the 14-3-3 family members is elevated in human cancers, suggest- ing that they may play a role in tumorigenesis. More speciﬁcally, 14-3-3c was shown to induce oncogenic transformation by stimulating MAPK and phospho- inositide 3-kinase signaling [36].

The results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of BKM-570 actions in EOC cells. Although not directly clinically relevant, our data the therapeutic potential of could be indicative of

Despite the delineated similarities of BKM-570 action to that of conventional EOC chemotherapeutics,

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BKM-570 and related compounds in EOC manage- ment.

Experimental procedures

Cell lines, BKM-570 synthesis, and stock solution

2.5 mm KH2PO4, 1 mm CaCl2, 1 mm MgSO4, 10 mm glu- cose, pH 7.4, containing BSA (0.1%)] before loading with fura 2 acetoxymethyl ester (1 lm in Hepes buffer) for 45 min at 37 (cid:2)C. After two washes, the coverslips were mounted into the experimental chamber of the spectroﬂuo- rimeter and perfused at a rate of 6 mLÆmin)1 with Hepes buffer equilibrated at 37 (cid:2)C and containing various test substances: 100 lm neomycin, a nonspeciﬁc phospholi- pase C inhibitor, 2 mm EGTA, a Ca2+ chelator, and 1.5 lm thapsigargin, an inhibitor of the endoplasmic reticu- lum Ca2+ ATPases. Fluorescence measurements were made using a Spex Fluorilog spectroﬂuorometer (Spex Industry, Edison, NY, USA), set for alternative dual wavelength exci- tation at 340 and 380 nm. The light emitted at 520 nm was collected by a photomultiplier, and passed to a Spex system microcomputer, which averaged the emission collected over a 0.50 s period at each excitation wavelength.

The human EOC cell lines TOV-21 and TOV-112 were propagated in mixed (1 : 1 ratio) MCDB 105 Medium (Sigma, St-Louis, MS, USA) and 119 Medium (Invitrogen, CA, USA), containing 10% fetal bovine serum, 5 lgÆmL)1 fungizone (Bristol-Myers Squibb, Montreal, PQ, Canada) and 50 lgÆmL)1 gentamicin (Sandoz, Boucherville, PQ, Canada), as previously described [23]. The nonpeptide mimetic BK antagonist BKM-570 was synthesized as previ- ously described [15]. For cell treatment, BKM-570 stock solution was prepared in MCDB 105 ⁄ 119 medium without serum, containing 20% dimethylsulfoxide.

Gene expression proﬁling and data analysis

MTT assay

reagent

(v2) microarrays,

The MTT cell proliferation assay (Sigma) was used to mea- sure the cell growth inhibition effects of BKM-570 and cis- platin in EOC cell lines, as previously described [23]. Brieﬂy, cell suspensions (at 2 · 104 cellsÆmL)1) were trans- ferred to 96-well plates in triplicate, and incubated for 72 h with the indicated drug concentrations. Then, 38 lL of MTT (5 mgÆmL)1) was added to each well 4 h before the end of the incubation. After incubation, 200 lL of dimeth- ylsulfoxide was added to resolve the crystals, and the absor- bance was measured with a microplate reader at 595 nm. Cell viability in the presence of the medium alone or the dimethylsulfoxide vehicle was equivalent. Synergy between BKM-570 and cisplatin was determined with calcusyn software (Biosoft, Cambridge, UK), which analyzes the data from proliferation assays to determine the interaction between equipotent drug combinations [39].

IPA software

(see

Gene expression analysis was carried out as previously described [23,41]. Brieﬂy, total RNA was isolated from BKM-570-treated or control (vehicle-treated) EOC cell cul- tures with Trizol (Invitrogen, Burlington ON, Canada). The quality of all RNA samples was examined by capillary electrophoresis with an Agilent 2100 Bioana- lyzer (Agilent, Palo Alto, CA, USA). Fluorescently labeled cDNAs were generated from 10 lg of total RNA in each reaction, with an Agilent Fluorescent Direct Label Kit and 1.0 mm cyanine 3-labeled or cyanine 5-labeled dCTP following the user’s (PerkinElmer, Boston, MA, USA), manual. Cyanine-labeled cDNA from BKM-570-treated EOC cells was mixed with the same amount of reverse- color cyanine-labeled cDNA from the corresponding con- trol cells, and hybridized on the Agilent human oligonu- containing (cid:2) 22 000 genes. cleotide Array hybridization, washing, scanning, data extraction and analysis were performed as previously described the microarray data was [20,21]. Network analysis of http://www. with completed ingenuity.com).

Functional (intracellular Ca2+ release) assays for BK receptor activity

Semiquantitative duplex RT-PCR

TOV-21 and TOV-112 cells were treated for 5 min with 0.1 lm BK (a speciﬁc B2R agonist) or with 0.1 lm des- Arg9-BK (a speciﬁc B1R agonist). For positive controls, TOV-21 and TOV-112 cells were also treated with 0.1 lm angiotensin and 0.1 lm AVP, representing agonists for the corresponding GPCRs.

Changes in intracellular Ca2+ were measured with the ﬂuorescent Ca2+ indicator fura 2 acetoxymethyl ester, as previously described [40]. Brieﬂy, TOV-21 and TOV-112 cells (105 cellsÆmL)1) suspended in DMEM containing 10% fetal bovine serum were subcultured on 20 · 7 mm glass coverslips for 24 h. Conﬂuent cell-coated coverslips were rinsed with Hepes buffer [10 mm Hepes, 145 mm NaCl,

Analysis of BK receptor gene expression in TOV-21 and TOV-112 cells, as well as validation of microarray data for selected differentially expressed genes, was performed by sqRT-PCR as previously described [23,41]. The glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard. Comparative signal intensity was evaluated with imagej software (http://rsb.info.nih.gov/ij/). Primers were designed for these loci with the sequences freely available from the Entrez Nucleotide database and the primer3 algorithm for primer design (http://www- genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi).

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Acknowledgements

14 Stewart JM, Gera L, Chan DC, Bunn PA Jr, York EJ, Simkeviciene V & Helfrich B (2002) Bradykinin-related compounds as new drugs for cancer and inﬂammation. Can J Physiol Pharmacol 80, 275–280.

This study was supported by the Cancer Research Network of the ‘Fonds de la Recherche en Sante´ du Que´ bec’ (FRSQ), Que´ bec, Canada.

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The following supplementary material is available: Fig. 1. Comparison of the common and distinct differ- entially expressed genes in TOV-21 and TOV-112 cells following BKM-570 (BKM), cisplatin (CIS) and topo- tecan (TPT) treatment, using Venn diagram analysis. Table S1. List of common differentially expressed genes (‡ 2-fold) in BKM-570-treated TOV-21 cells. Table S2. List of common differentially expressed genes (‡ 2-fold) in BKM-570-treated TOV-112 cells. Table S3. List of 148 genes used for cluster analysis. Table S4. Networks with signiﬁcant scores that are commonly affected following BKM-570 treatment of TOV-21 cells. Table S5. Networks with signiﬁcant scores that are commonly affected following BKM-570 treatment of TOV-112 cells.

This supplementary material can be found in the

online version of this article.

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