Nephroblastoma overexpressed gene (NOV) enhances cell motility and COX-2 upregulation of human osteosarcoma involves avb5 integrin, ILK and AP-1-dependent pathways

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Nephroblastoma overexpressed gene (NOV) enhances cell motility and COX-2

upregulation of human osteosarcoma involves

a

v

b

5 integrin, ILK and

AP-1-dependent pathways

Chun-Yin Huang

a,b

, Chun-Yi Lee

a,b

, Meng-Yi Chen

a

, Hsiao-Chi Tsai

c

, Horng-Chaung Hsu

d

,

Chih-Hsin Tang

e,f,

*

a

Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan

b

Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan

c_{Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan} d

Department of Orthopaedic Surgery, China Medical University Hospital, Taichung, Taiwan

e

Department of Pharmacology, School of Medicine, China Medical University and Hospital, Taichung, Taiwan

f

Graduate Institute of Basic Medical Science, China Medical University and Hospital, Taichung, Taiwan

1. Introduction

Osteosarcoma is a high-grade malignant bone neoplasm that occurs primarily in children and adolescents. The principles of treatment of osteosarcoma have undergone dramatic changes in the past 20 years. Until recently, 5-year survival of 20% with surgical treatment alone was considered acceptable. This outcome suggested that 80% of the patients had pulmonary metastasis at the time of presentation[1]. Hence, chemotherapy is usually employed in an adjuvant situation to improve the prognosis and long-term survival. Recurrence usually occurs as pulmonary metastases or, less frequently, metastases to distant bones or as a local recurrence

[2–4]. Thus, a novel strategy that would efﬁciently inhibit

metastasis, especially to the lung, from the primary osteosarcoma site is highly desirable.

Decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors[5]. The invasion of tumor cells is a complex, multistage process. To facilitate cell motility, invading cells need to change cell–cell adhesion properties, rearrange the extracellular matrix (ECM) environment, suppress anoikis and reorganize their cytoskeletons[6]. Cyclooxygenases (COXs) are the rate-limiting enzymes that catalyze the conversion of arachidonic acid to prostaglandins (PGs). COX-2 is an inducible enzyme and is activated by extracellular stimuli such as growth factors and pro-inﬂammatory cytokines[7]. Over-expression of COX-2 is frequent-ly found in many types of cancer, including colon, lung, breast, pancreas, head, and neck cancers[8–10]and is usually associated with poor prognosis and short survival. Therefore, COX-2 may play a critical role in tumorigenesis, and its disruption may prevent metastasis.

NOV is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins, with developmental

A R T I C L E I N F O Article history:

Received 22 October 2010 Accepted 1 December 2010 Available online 9 December 2010 Keywords: NOV Integrin Osteosarcoma Migration ILK A B S T R A C T

Osteosarcoma is characterized by a high malignant and metastatic potential. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Nephroblastoma overexpressed gene (NOV), also called CCN3, was regulated proliferation and differentiation of cancer cells. However, the effect of NOV on migration activity and COX-2 expression in human osteosarcoma cells is mostly unknown. Here we found that NOV increased the migration and expression of COX-2 in human osteosarcoma cells.avb5 monoclonal antibody (mAb), integrin-linked kinase (ILK) and Akt inhibitor reduced the NOV-enhanced the migration and COX-2 up-regulation of osteosarcoma cells. NOV stimulation increased the ILK kinase activity and phosphorylation of Akt. In addition, c-Jun siRNA also antagonized the NOV-mediated migration and COX-2 expression. Moreover, NOV enhanced the AP-1 binding activity and promoter activity. Taken together, these results suggest that the NOV acts throughavb5 integrin to activate ILK and Akt, which in turn activates c-Jun and AP-1, resulting in the activations of COX-2 and contributing the migration of human osteosarcoma cells.

* Corresponding author at: Department of Pharmacology, School of Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung, Taiwan.

Tel.: +886 4 22052121 7726; fax: +886 4 22053764. E-mail address:[email protected](C.-H. Tang).

Contents lists available atScienceDirect

Biochemical Pharmacology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m

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functions[11,12]. Recent studies have shown that the CCN protein family members also play important roles in tumorigenesis, including cancer cell proliferation, survival, adhesion, and invasion

[13,14]. CCN proteins are mostly secreted and extracellular matrix associated and have been proposed to connect signaling pathways and facilitate cross talks between epithelium and stroma[11].

NOV molecule is widely expressed, especially in the nervous and musculoskeletal systems as well as in the blood vessels. The functions of NOV protein among these different tissues might, however, be very different. Although NOV was originally described as antiproliferative [15] and its expression was associated with differentiation and growth arrest in Wilm’s tumor, chondrosarcomas and rhabdomyosarcomas[16,17], more recent data correlate NOV with increased proliferative index of 3T3 ﬁbroblast and tissue samples of prostate and renal carcino-mas [18,19]. In osteosarcoma, it is inversely associated with expression of liver/bone/kidney alkaline phosphatase isoform early marker of osteoblastic differentiation[20,21]. In Ewing’s sarcoma, expression of NOV was only sporadically observed, but it was associated with a signiﬁcantly higher risk of developing lung and bone metastasis[20].

Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture

The human osteosarcoma cell lines (U2OS, MG-63, HOS and G292) were purchased from the American Type Cell Culture Collection (Manassas, VA). The cells were maintained in RPMI-1640 medium which was supplemented with 20 mM HEPES and 10% heat-inactivated FCS, 2 mM-glutamine, penicillin (100 U/ml) and streptomycin (100

m

g/ml) at 37 8C with 5% CO2.

2.3. Migration assay

The migration assay was performed using Transwell (Costar, NY; pore size, 8-

m

m) in 24-well dishes. Before performing the migration assay, cells were pretreated for 30 min with different concentrations of inhibitors, including the KP392, Akt inhibitor or vehicle control (0.1% DMSO). Approximately 2 104_{cells in 100}

_m

_l of serum-free RPMI-1640 medium were placed in the upper chamber, and 300

m

l of the same medium containing NOV was placed in the lower chamber. The plates were incubated for 24 h at 37 8C in 5% CO2, then cells were fixed in methanol for 15 min and stained with 0.05% crystal violet in PBS for 15 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each clone was plated in triplicate in each experiment, and each experiment was repeated at least three times[24].

2.4. Western blot analysis

The cellular lysates were prepared as described previously[25]. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldiﬂuoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against Akt, p-Akt or p-c-Jun (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS ﬁlm (Eastman Kodak, Rochester, NY). Quantitative data were obtained using a comput-ing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

2.5. ILK kinase assay

ILK enzymatic activity was assayed in osteosarcoma cells lysed in Nonidet 40 buffer (0.5% sodiumdeoxycholate, 1% Nonidet P-40, 50 mM HEPES (pH 7.4), 150 mM NaCl) as previously reported

g of total RNA was reverse transcribed into cDNA by using oligo(dT) primer. The quantitative

real time PCR (qPCR) analysis was carried out using Taqman1 one-step PCR Master Mix (Applied Biosystems, CA). Two

m

l of cDNA were added per 25-

m

l reaction with sequence-speciﬁc primers and

Taqman1

probes. Sequences for all target gene primers and probes were purchased commercially (GAPDH was used as internal control) (Applied Biosystems, CA). qPCR assays were carried out in triplicate on an StepOnePlus sequence detection system. The cycling conditions were 10-min polymerase activation at 95 8C followed by 40 cycles at 95 8C for 15 s and 60 8C for 60 s. The

Fig. 1. NOV induced migration and COX-2 expression in human osteosarcoma cells. (A) Cells were incubated with various concentrations of NOV, and in vitro migration activities measured with the Transwell after 24 h (n = 5). (B) The NOV expression from osteosarcoma cell lines by using Western blot (n = 4). (C) Cells were incubated with NOV (100 ng/ml) for indicated time intervals, and COX-2 expression was examined by Western blot and qPCR (n = 4). (D) Cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 h followed by stimulation with IPTG (5 mM) for 24 h, the COX-2 expression and migration activity were determined by Western blot and Transwell (n = 4). (E) Cells were and pretreated with celebrex (10mM) or NS-398 (20mM) for 30 min followed by stimulation with NOV, and in vitro migration was measured with the Transwell after 24 h (n = 4). (F) Cells were transfected with COX-2 siRNA for 24 h followed by stimulation with NOV, and in vitro migration measured with the Transwell after 24 h (n = 4). Results are expressed as the mean S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with NOV-treated group.

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threshold was set above the non-template control background and within the linear phase of target gene ampliﬁcation to calculate the cycle number at which the transcript was detected (denoted CT).

2.8. Electrophoretic mobility shift assay (EMSA)

EMSA was performed by using EMSA ‘gel shift’ kit (Panomics, Fremont, CA) according to the manufacturer’s protocol. Oligonu-cleotides corresponding to canonical human COX-2 promoter AP-1 binding sequence (50_{- AGAAACAGTCATTTC-3}0_{) was used. Cells}

nuclear extract (3

m

g) was incubated with poly d(I–C) at room temperature for 5 min. The nuclear extract was then incubated with biotin-labeled probes and the incubated at RT for 30 min. After electrophoresis on a 6% polyacrylamide gel, the samples on gel were transferred onto a presoaked immobilon-Ny+ membrane (Millipore, Billerica, MA, USA). The membrane was baked at 80 8C for 1 h, crosslinked in an oven for 3 min, and then developed by adding the blocking buffer and streptavidin-HRP conjugate and then subjected to Western blot analysis.

2.9. Statistics

The values given are means S.E.M. The signiﬁcance of difference between the experimental groups and controls was assessed by Student’s t test. The difference is signiﬁcant if the p value is <0.05.

3. Results

3.1. NOV-directed osteosarcoma cells migration through COX-2 upregulation

Previous studies have shown that NOV regulates cell migration and invasion in human cancer cells[28,29]. However, the effect of NOV in migration of osteosarcoma is mostly unknown. NOV-triggered migration in osteosarcoma cells was examined by using the Transwell assay. NOV directed human osteosarcoma cells (U2OS, MG-63, HOS and G292 cells) migration (Fig. 1A). On the other hand, NOV did not induce proliferation and apoptosis of osteosarcoma cells (Supplementary Fig. S1). However, treatment of

Fig. 2. NOV induced osteosarcoma cell migration throughavb5 integrin. (A and B) Cells were and pretreated withavb3 andavb5 monoclonal antibody (10mg/ml) for 30 min followed by stimulation with NOV. The cell migration and COX-2 expression were measured with the Transwell and qPCR (n = 4). (C) MG63 cells were exposed to NOV for indicated time intervals, and cell lysates were immunoprecipitated with an antibody speciﬁc for ILK. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with anti-pGSK3bor GSK3b(n = 4). (D–F) Cells were and pretreated with KP-392 (10mM) for 30 min or transfected with ILK siRNA for 24 h followed by stimulation with NOV. The cell migration and COX-2 expression were measured with the Transwell, qPCR and Western blot (n = 4). Results are expressed as the mean S.E. *p < 0.05 compared with control;#

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osteoblastic cells (MC3T3-E1) with NOV (100 ng/ml) did not induce migration activity (data not shown). We next examined human osteosarcoma cell lines for expression of the NOV using Western blot. We found that human osteosarcoma cell lines expressed the protein level of NOV (Fig. 1B). Previous study has shown that COX-2 mediated cell motility in human cancer cells

[30,31]. We therefore, hypothesized that COX-2 may be involved in NOV-mediated osteosarcoma migration. Treatment of osteosarco-ma with NOV enhanced mRNA and protein expression of COX-2 (Fig. 1C). We used an IPTG-inducible COX-2 gene expression vector to examine the role of COX-2 in osteosarcoma cells. U2OS cells were transfected with IPTG-inducible COX-2 gene expression vector or a control vector, and then IPTG (5 mM) was added for 24 h. Using Western blot analysis, we found that IPTG induced

COX-2 expression (Fig. 1D; upper panel). Furthermore,

over-expression of COX-2 enhanced cell migration in osteosarcoma

cells (Fig. 1D; lower panel). To conﬁrm COX-2 mediated

5 integrin. 3.3. ILK and Akt signaling pathways are involved in the NOV-mediated COX-2 upregulation and migration of osteosarcoma

ILK has been shown to be capable of regulating integrin-mediated signaling [32]. We directly measured the ILK kinase activity in response to NOV stimulation by the immunoprecipita-tion of ILK from lysates. Fig. 2C shows that NOV exposure in osteosarcoma time-dependently increased ILK kinase activity, assessing the phosphorylation of the recombinant GSK3

b

on Ser9. To further explore whether ILK is involved in NOV-induced cell migration and COX-2 expression, ILK inhibitor KP-392 or ILK siRNA were used. As shown inFig. 2D–F, pretreatment of osteosarcoma with KP-392 inhibited NOV-induced cell migration and COX-2 expression. Transfection of osteosarcoma with ILK siRNA also antagonized the potentiating effect of NOV (Fig. 2D and E). Therefore, ILK plays an important role in NOV-induced cell migration and COX-2 expression. It has been reported that ILK is an upstream regulator of the phosphorylation of Akt on Ser473

[33], we then examined whether NOV stimulation also enhances the association of ILK with Akt. Stimulation of osteosarcoma cells with NOV increased Akt phosphorylation time dependently (Fig. 3A). Pretreatment of cells for 30 min with Akt inhibitor inhibited the NOV-induced cell migration and COX-2 expression (Fig. 3B–D). Transfection of cells with Akt mutant also reduced NOV-mediated cell migration and COX-2 expression (Fig. 3B and C). Taken together, these results indicate that the ILK and Akt pathways are involved in NOV-induced migration activity and COX-2 up-regulation in human osteosarcoma cells.

Fig. 3. Akt is involved in NOV-mediated migration and COX-2 expression in osteosarcoma. (A) MG63 cells were incubated with NOV for indicated time intervals, and p-Akt expression was determined by Western blot (n = 4). (B–D) Cells were and pretreated with Akt inhibitor (10mM) for 30 min or transfected with Akt mutant for 24 h followed by stimulation with NOV. The cell migration and COX-2 expression were measured with the Transwell, qPCR and Western blot (n = 4). Results are expressed as the mean S.E. *p < 0.05 compared with control;#

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3.4. Involvement of AP-1 in NOV-induced cell migration and COX-2 expression

AP-1 plays a critical role in COX-2 expression[34]. To examine the role of the AP-1 binding site in NOV-mediated migration and COX-2 expression, an AP-1 inhibitor (curcumin) was used. Pretreatment of cells with curcumin inhibited NOV-enhanced cell migration and COX-2 expression (Fig. 4A–C). AP-1 activation was further evaluated by analyzing the electrophoretic mobility shift assay, as well as by the phosphorylation of c-Jun. Stimulation of cells with NOV resulted activation of AP-1 specific DNA–protein complex formation (Fig. 4D). To identify the specific subunit involved in the formation of the AP-1 complex, supershift assay was performed using antibody specific for anti-c-Jun. Incubation of nuclear extracts with anti-c-Jun antibody increased supershift of AP-1 DNA–protein complex (Fig. 4E). Pretreatment of cells with KP392 and Akt inhibitor reduced NOV-mediated AP-1-specific DNA–protein complex formation (Fig. 4F). Transfection of cells with c-Jun siRNA suppressed the cell migration and COX-2

expression (Fig. 5A–C). Treatment of cells with NOV resulted in a marked phosphorylation of c-Jun (Fig. 5D). To directly determine AP-1 activation after NOV treatment, cells were transiently transfected with AP-1 driven luciferase as an indicator of AP-1 activation. As shown inFig. 5E, NOV treatment of cells for 24 h increased in AP-1 driven luciferase activity. In addition,

a

v

b

5 mAb, KP392 and Akt inhibitor antagonized the NOV-induced AP-1 driven luciferase activity (Fig. 5E). Co-transfection of cells with ILK siRNA, Akt mutant and c-Jun siRNA also reduced NOV-increased AP-1 driven luciferase activity (Fig. 5F). Taken together, these data suggest that activation of

a

v

b

5 integrin, ILK, Akt and c-Jun are required for NOV-induced AP-1 activation in human osteosarcoma cells.

4. Discussion

Osteosarcoma is a debilitating, though not always fatal, high-grade malignant bone neoplasm that targets children and adolescents. The chemotherapies are not fully effective, and as a

Fig. 4. AP-1 is involved in NOV-mediated migration in human osteosarcoma. (A–C) Cells were and pretreated with curcumin (10mM) for 30 min followed by stimulation with NOV. The cell migration and COX-2 expression were measured with the Transwell, qPCR and Western blot (n = 5). (D and F) MG63 cells were exposed to NOV for indicated time intervals or pretreated for 30 min with KP392 or Akt inhibitor followed by stimulation with NOV for 240 min, and electrophoretic mobility shift assay was performed as described in Section2(n = 4). (E) Nuclear extracts from MG63 cells exposed to NOV for 120 min, and c-Jun binding to AP-1 element was examined by EMSA analysis. Preincubation of c-Jun antibody (Ab, 5mg) with nuclear extracts isolated from cells exposed to NOV induced the supershift of AP-1 DNA complex. Results are expressed as the mean S.E. *p < 0.05 compared with control;#

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result, 20% of all patients die due to metastasis of osteosarcoma cells to the lungs[3]. Therefore, it is important to develop effective adjuvant therapy for preventing osteosarcoma metastasis. We hypothesized that NOV and its receptor would help to direct the migration of osteosarcoma cells. We found that NOV increased cell migration in human osteosarcoma cell lines. However, NOV induces only a 2.5-fold increases in migration of osteosarcoma cells. In our previous study, we found a maximum 3-fold increase in migration of chondrosarcoma cells induced by another chemokine (SDF-1) [35]. Therefore, the maximum increase in migration of osteosarcoma cells is 2- to 3-fold. One of the mechanisms underlying NOV directed migration was transcrip-tional upregulagtion of COX-2 and activation of

a

v

b

5 integrin, ILK, Akt and AP-1 pathways. Due to the presented data we recommend the COX-2 selective inhibitor may possible treatment regime for the prevention of osteosarcoma metastasis.

COX-2 is a pleiotropic enzyme that mediates many physiologi-cal functions such as inhibition of cell apoptosis, augmentation of angiogenesis, and increased cell motility. It has been reported that the expression of COX-2 is associated with a metastatic phenotype

of human cancer cells[30,31]. In this study, we found that over-expression COX-2 induced cell motility of osteosarcoma. Treat-ment of cells with NOV increased COX-2 expression in human osteosarcoma. On the other hand, COX-2 inhibitors antagonized reduced cell motility. In addition, the inhibition of NOV-induced COX-2 protein expression with siRNA signiﬁcantly reversed NOV-inhibited migration. Besides, NOV also increased PGD2, PGI2 and PGE2 production in osteosarcoma cells ( Supple-mentary Fig. S2). Therefore, COX-2-producted PGs may be the NOV-responsive mediator, and lead to enhance cancer migration and metastasis.

ILK, a potential candidate signaling molecule, has been shown to be capable of regulating integrin-mediated signaling[32]. ILK can interact with the cytoplasmic domain of

b

-integrin subunits and is activated by both integrin activation as well as growth factors and is an upstream regulator of Akt[36]. The current study showed that NOV stimulation increased kinase activity of ILK. Treatment with the ILK inhibitor of KP-392 inhibited NOV-induced cell migration and COX-2 expression. Furthermore, the ILK siRNA also antagonized the NOV-mediated potentiation of migration and

Fig. 5. Integrin/ILK/Akt pathway is mediated NOV-induced AP-1 activation. (A–C) Cells were transfected with c-Jun siRNA for 24 h followed by stimulation with NOV. The cell migration and COX-2 expression were measured with the Transwell, qPCR and Western blot (n = 5). (D) MG63 cells were incubated with NOV for indicated time intervals, and p-c-Jun expression was determined by Western blot (n = 4). (E and F) MG63 cells were pretreated withavb5 mAb, KP392 and Akt inhibitor for 30 min or co-transfected with ILK siRNA, Akt mutant and, c-Jun siRNA before exposure to NOV. The AP-1 driven luciferase activity was measured, and the results were normalized to theb-galactosidase activity and expressed as the mean S.E. for three independent experiments performed in triplicate. Results are expressed as the mean S.E. *p < 0.05 compared with control;

#

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COX-2 expression. Therefore, ILK activation is involved in NOV-induced cell migration and COX-2 expression in osteosarcoma. ILK possibly regulated the cell function by promoting the phosphory-lation of Akt on Ser473 [37]. Our results demonstrate that pretreatment of osteosarcoma with Akt inhibitor antagonized the increase of cell migration and COX-2 expression under NOV stimulation. This was further conﬁrmed by the result that the dominant negative mutant of Akt inhibited the enhancement of cell migration and COX-2 expression under NOV stimulation. Here we also found that the cytoplasmic kinase Akt was activated by NOV stimulation in osteosarcoma. These effects implicate the involvement of ILK and Akt activation in NOV-mediated induction of cell migration and COX-2 expression.

It has been reported that chemokines induced COX-2 expres-sion through AP-1 dependent pathway[34,38]. The results of this study show that AP-1 activation contributes to NOV-induced migration and COX-2 expression in osteosarcoma cells. The AP-1 sequence binds to members of the Jun and Fos families of transcription factors. These nuclear proteins interact with the AP-1 site as Jun homodimers or Jun-Fos heterodimers formed by protein dimerization through their leucine zipper motifs. The results of this study show that NOV induced c-Jun phosphorylation. In addition, c-Jun siRNA abolished the NOV-induced cell migration and COX-2 expression in osteosarcoma cells. Furthermore, NOV also increased the AP-1 speciﬁc DNA–protein complex formation. The AP-1 speciﬁc DNA–protein complex formation was attenuated by KP392 and Akt inhibitor. On the other hand, KP392, Akt inhibitor and curcumin or ILK siRNA and Akt mutant reduced NOV-mediated AP-1 promoter activity. These results indicate that NOV might act through the ILK, Akt, c-Jun, and AP-1 pathway to induce COX-2

activation in human osteosarcoma cells. NF-

k

B is reported to be the main transcriptional factor involved in COX-2 mRNA tran-scription[39]. We also found that stimulation of cells with NOV increased the accumulation of p65 in the nucleus (Supplementary Fig. S3). Therefore, NF-

k

B also participate the NOV-mediated migration and COX-2 expression in human osteosarcoma.

Due to the prognosis of patients with osteosarcoma distant metastasis is generally considered as very poor. Thus, preventing human osteosarcoma metastasis is an important issue nowadays. Our study presents that NOV increases the expression of COX-2 via

a

v

b

5 integrin, ILK, Akt, c-Jun/p65, and AP-1/NF

k

B-dependent pathway and increasing migration of human osteosarcoma cells (Fig. 6). Furthermore, the discovery of NOV-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and may help us to develop effective therapy in the future. Acknowledgments

This work was supported by grants from China Medical University (CMU99-S-17) and China Medical University Beigang Hospital (CMUBH R990001). We thank Dr. W.M. Fu for providing Akt dominant negative mutant; Dr. M.L. Kuo for providing COX-2 IPTG-induced expression plasmid.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.bcp.2010.12.005.

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