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2 and p38 pathways are required for P2Y receptor-mediated prostate cancer invasion
Cancer Letters 215 (2004) 239–247 www.elsevier.com/locate/canlet
ERK1/2 and p38 pathways are required for P2Y receptor-mediated prostate cancer invasion Ling Chen1, Hui-ying He1, Hong-mei Li, Jie Zheng, Wan-jie Heng, Jiang-feng You, Wei-gang Fang* Department of Pathology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100083, China Received 16 December 2003; received in revised form 4 May 2004; accepted 19 May 2004
Abstract The G protein-coupled P2Y purinoceptors have wide physiological functions, but their role(s) in tumor progression remain unclear. Here, we report that stimulation of P2Y receptors enhances prostate cancer cell invasion in two human prostate carcinoma cell lines, which is mediated by ERK1/2 and p38 signaling pathways. P2Y agonists stimulated prostate cancer cell invasion, and increased the activities of ERK1/2 and p38 protein kinases. The stimulated cancer cell invasion was inhibited by the presence of MEK1 inhibitor PD98059 or p38 inhibitor SB203580. Expression of dominant-negative mutant of MEK1 (KA-MEK1), or up-regulation of MKP-5 (a dual-specificity phosphatase of p38), both reduced the invasion of cultured prostate cancer cells. These results suggest that P2Y receptors and their down-stream ERK1/2 and p38 protein kinases are important regulators promoting prostate cancer invasion. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: P2Y receptor; ERK1/2; p38; Prostate cancer; Invasion
1. Introduction Prostate cancer is the leading diagnosed malignancy in men in western countries. Due to lack of effective treatments, development of hormone-independence and metastases remains the most lethal aspect of this disease. Loss of cell–cell contact caused by dysfunction of E-cadherin [1], and increased proteolysis and * Corresponding author. Tel.: C86-10-82802599; fax: C86-1062015547. E-mail address: [email protected] (W.- Fang). 1 These authors contributed equally to this work.
cell motility [2,3] have been shown to be critical steps in invasive and metastatic process. Several growth factors and their receptors have been implicated in this process through activation of their down-stream signaling pathways, and mitogen-activated protein kinase (MAPK) pathway has been extensively investigated. In a study of 50 cases of high-grade prostatic intraepithelial neoplasia (PIN) using immunohistochemistry and in situ hybridization, increased expressions of MAPK family have been demonstrated in all cases of high-grade PIN compared with normal prostate [4]. More recently, interleukin 6 (IL-6) induces an androgen response in androgen dependent
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.05.023
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LNCaP cells by mechanisms dependent on androgen receptor, ERK1/2 and p38 activities [5]. The ERK1/2 MAPK pathway has been shown to be involved in the invasive and metastatic development of various types of cancers including rat prostatic adenocarcinoma [6], melanoma cancer [7] and vascular smooth muscle cells [8]. Modulated by the androgen responsive product STE20/SPS-related kinase in LNCaP cells [9], the p38 MAPK is an important mediator for cell motility or invasive phenotype in cancers including human breast epithelial cells [10] and endothelial cells [11]. Nucleotides, such as ATP and UTP, can function as potent extracellular stimuli by exerting their effects via activation of a distinct family of cell surface receptors, the P2 purinoceptor family. P2 receptors have two groups: the P2X ligand-gated ionotropic channel family and the P2Y metabotropic GPCR family [12]. Our previous study demonstrated that the ATP receptors on PC-3 prostate cancer cells belong to P2Y subtype [13]. Expression of P2Y receptor messenger RNA is also detected in PC-3 cells [14]. P2Y receptor activation induces arterial smooth muscle cell migration [15] and regulates cell growth of different cell types [13,16]. However, little is known of connection between P2Y receptor and tumor pro-gression. We reported that ATP activates p38 as well as ERK1/2 cascades, and stimulates prostate cancer cell invasion [17]. To address whether ERK1/2 and p38 pathways mediate P2Y receptor-induced prostate cancer progression, in the present study we demonstrate that P2Y receptor-induced prostate cancer cell invasion is regulated through ERK1/2 and p38 pathways.
2. Materials and methods 2.1. Tumor cell lines and cell culture Two subclones from the PC-3 human prostate carcinoma cell line (American Type Culture Collection, Rockville, MD) were derived. The 1E8 cells are highly metastatic (100% metastasis to lungs and lymph nodes of nude mice 5 weeks after subcutaneous inoculation), whereas the 2B4 cells are non-metastatic [18]. All cells were routinely grown in RPMI 1640 supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 8C and used when they reach 80% confluency.
2.2. Plasmid construction and cell transfection Plasmid encoding dominant-negative human MEK1 (pcDNA3-KA-MEK1) was kindly provided by Dr Rong Xu (Weizmann Institute, Israel). Lysine 97 of MEK1 was mutated to alanine (K97A) to produce an inactive enzyme. The K97A served as dominant-negative mutant by suppressing MAPKK, MAPK and p90rsk activation in vivo [19]. Plasmid pcDL-SRa 296-Myc containing wild type human MKP-5 (-wt) was kindly provided by Dr Eisuke Nishida (Kyoto University, Japan). Transfection was performed using the LipofectAMINEe reagent (Gibco BRL, Gaitherburg, MD), and the transfectants were maintained in the presence of 400 mg/ml G418. 2.3. Immunoblotting analysis of activated kinases Activated MAPKs (ERK1/2, p38 and JNK/SAPK) were detected using phospho-specific antibodies directed against the dually phosphorylated, active forms of these kinases. Cells were serum-deprived for 24 h before treatment with reagents at the indicated time. Cells were lysed in buffer containing 62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), and 0.1% (w/v) bromphenol blue. Equal amounts of protein samples were separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes, which were blotted with appropriate antibodies to determine MAPK activities, and the signals were detected by enhanced chemiluminescence (ECL analysis kit, Amersham Corp). The membranes were stripped in buffer (62.5 mM Tris–HCL, 2% SDS, and 100 mM b-mercaptoethanol, pH 6.7, at 50 8C), and re-probed with anti-ERK1/2, -p38 or -JNK antibodies to determine the total amounts of each kinase. 2.4. In vitro invasion assay The invasive ability of carcinoma cells was evaluated using Boyden Chamber assay as previously described by Albini et al. [20]. Briefly, the upper and lower compartments of the chamber were separated with a PVP-free polycarbonate filter (8-mm pore size, Neuro Probe, CA) coated with 50 mg reconstituted basement membrane Matrigel (Collaborative Res, Bradford, MA). Cells in 400 ml medium were placed
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in the upper compartment, and the lower compartment was filled with 200 ml NIH3T3-conditioned medium. After incubation for 12 h at 37 8C, the filters were removed. Cells on the upper side of the filter were removed with cotton-tipped swabs. Filters were fixed in methanol and stained with hematoxylin and eosin. Cells on the underside of the filters were counted by microscopic inspection. Each sample was assayed in triplicate. 2.5. Statistical analysis Comparison of multiple means was performed with nonparametric ANOVA. Data are expressed as meanGs.d.m. P values are considered to be significant at !0.05.
3. Results 3.1. Stimulation of P2Y receptor led to activation of ERK1/2 and p38 kinases in prostate cancer cells Treatment of both 1E8 and 2B4 cells with P2Y receptor agonist ATP resulted in a significant activation of ERK1/2 and p38, but not JNK (data not shown) kinases. These responses were time- and dose-dependent (Fig. 1A–D), with peak activation at 100 mM in 15 min. ERK1/2 activation in response to ATP was significantly inhibited by pretreatment with MEK1 inhibitor PD98059 (25 mM) (Fig. 1E), and p38 phosphorylation was also inhibited significantly in the presence of SB203580 (10 mM) (Fig. 1F), confirming that both ERK1/2 and p38 are implicated in downstream signal transduction of P2Y receptor. To verify that it is P2Y receptors that mediate ATP activation of the kinases, we tested another hydrolysis-resistant P2Y receptor agonist AMP-PNP (b, g, -imido ATP)
Fig. 1. ATP activates ERK1/2 and p38 kinases in 1E8 (A and C) and 2B4 (B and D) cells in a time- and dose-dependent manner.
PD98059 and SB203580 inhibit ATP-induced ERK1/2 (E) and p38 (F) activation. AMP-PNP (G) activates ERK1/2 and p38 kinases in 1E8 cells in a time-dependent manner. Cells were serum-deprived for 24 h and then pretreated without or with 25 mM PD98059 for 1 h or 10 mM SB203580 for 20 min before treatment with stimulators at the indicated time. Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane and probed with antiphospho-ERK1/2 or anti-phospho-p38 antibody. The blots were stripped and re-probed with anti-ERK1/2 or anti-p38 antibody to assess equal loading of samples.
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and found that it stimulated ERK1/2 and p38 activation to the same extent as ATP (Fig. 1G). To confirm the specific role of P2Y receptor in p38 and ERK1/2 activation, cells were pretreated
with P2 receptor antagonist suramin (300 mM), and then stimulated with ATP for 15 min. As shown in Fig. 2A, ERK1/2 phosphorylation by ATP was significantly reduced approximately 82.4%
Fig. 2. P2 receptor antagonist suramin blocks ATP-induced ERK1/2 (A) and p38 (B) activation in 1E8 and 2B4 cells. Serum-starved cells were pretreated with 300 mM suramin for 30 min prior to stimulation with 100 mM ATP for 15 min. (C) Different levels of ERK1/2 and p38 phosphorylation by ATP in 1E8 and 2B4 cells. (D) KA-MEK1 transfection inhibits ERK1/2 activation. (E) MKP-5 transfection inhibits p38 phosphorylation.
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(P!0.001) and 80.7% (P!0.001) in 1E8 and 2B4 cells, respectively. ATP-induced p38 activation was also inhibited by suramin (1E8 cell: 71.6%, P! 0.01; 2B4 cell: 69.2%, P!0.01) (Fig. 2B). These results confirmed our finding that P2Y receptors were coupled to ERK1/2 and p38 pathways in prostate cancer cells. 3.2. Activation of P2Y receptors stimulated invasion of prostate cancer cells in vitro Using the in vitro invasion assay, incubation with 100 mM ATP for 12 h enhanced the invasion of metastatic 1E8 cells (63.4% increases) through Matrigel when compared with control (Fig. 3A). The invasion of non-metastatic 2B4 cells was also stimulated by ATP (46.4% increase). In a parallel experiment, AMP-PNP showed similar effect to that of ATP (data not shown). Since P2Y receptor stimulates activation of ERK1/2 and p38, we examined the effects of blocking ERK1/2 and p38 pathways on ATP-induced invasion. Pretreatment of the cells with p38 inhibitor SB203580 significantly attenuated the stimulatory effect of ATP on cell invasion (69.9% decreases in 1E8, 54% decreases in 2B4). Blocking the ERK1/2 pathway with PD98059 showed the same effect (79.8% decrease in 1E8, 61% decrease in 2B4) (Fig. 3A). These data suggest that both the ERK1/2 and p38 cascades were involved in mediating the invasive phenotype seen in human prostate carcinoma cells. 3.3. ERK1/2 and p38 pathways were involved in P2Y receptor-mediated prostate cancer invasion To further confirm the involvement of the ERK1/2 and p38 pathways in mediating the P2Y receptorinduced cancer invasion, we further tested individual contribution of each pathway to this effect by modulating gene expression. Transfection with the dominant-negative mutant of MEK1 cDNA (KA-MEK1) inhibited ERK1/2 phosphorylation significantly (Fig. 2D). KA-MEK1 transfectants showed a 40.7% decrease in the number of cells passing through Matrigel-coated membranes as compared with vector control (P!0.01), and a 45.2% decrease as compared with ATP-treated control (P!0.01) (Fig. 3B). Together with the result
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obtained in PD98059 pretreatment as shown above (Fig. 3A), the data suggest that ERK1/2 pathway promotes prostate cancer cell invasion. Treatment of the KA-MEK1-transfected cells with SB203580 further inhibited ATP-stimulated cell invasion by 76.2% as compared with ATP-treated control (a further 21.4% decrease as compared with KA-MEK1-transfected control) (Fig. 3B). In order to confirm that p38 is also involved in P2Y receptormediated cancer cell invasion, we overexpressed MKP-5, a dual-specificity phosphatase specific for p38, by transfection of MKP-5 cDNA into 1E8 cells. Phosphorylation of p38 was inhibited significantly in MKP-5 transfectants (Fig. 2E). A 66.6% decrease in invasive cells was seen in MKP-5 transfected cells as compared with vector control, and a 65.9% decrease as compared with ATP-treated control (Fig. 3C). Pretreatment of the MKP-5-transfected cells with PD98059 could further inhibit cell invasion as compared with ATP-treated control, though not so significantly. These results further confirm that both the ERK1/2 and p38 pathways are required for the P2Y receptor-mediated 1E8 cell invasion. 3.4. Activation levels of ERK1/2 and p38 pathways were correlated with invasive potential of prostate cancer cells 1E8 and 2B4 cells derived from PC-3 differ in their metastatic potentials in nude mice as well as invasive ability in vitro. In order to correlate the difference in their invasive abilities to the levels of ERK1/2 and p38 activation, we compared the levels of ERK1/2 and p38 activation by ATP between the two subclones. As shown in Fig. 2C, the phosphorylation levels of ERK1/2 and p38 stimulated by ATP were significantly higher in 1E8 cells than those in 2B4 cells (2.85 and 2.57 folds, respectively). Since, there was no difference in ERK1/2 and p38 constitutive expression between the two subclones, and 1E8 cells have greater metastatic potentials, the findings suggest that ERK1/2 and p38 signaling pathways are involved in prostate cancer progression and other mechanisms such as coupling of P2Y receptor to the MAPK cascades are required in prostate cancer invasion.
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Fig. 3. Effects of different treatments on ATP-induced in vitro invasion of 1E8 or 2B4 cells in Boyden chamber invasion assay. (A) Effects of PD98059 and SB203580 on in vitro invasion of 1E8 and 2B4 cells. (B) Effect of KA-MEK1 transfection on in vitro invasion of 1E8 cells. (C) Effect of MKP-5 transfection on in vitro invasion of 1E8 cells. (D) 1E8 transfectants migrate through Matrigel coated filters (!200). Cells were plated on filters pre-coated with Matrigel, incubated with or without PD98059 (25 mM for 1 h) or SB203580 (10 mM for 20 min), and then stimulated with 100 mM ATP for 12 h and assayed in vitro invasion. The data are expressed as percent of the number of cells passing through filters in control. Fifteen fields were counted on each filter. Values are the meanGs.d.m. (nZ3).
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4. Discussion Intracellular signaling pathways mediating malignant progression of human prostate cancers are yet to be fully determined. In the present study, we investigated roles of ERK1/2 and p38 in cancer cell invasion using two human prostate carcinoma cell lines with different metastatic potentials. We demonstrate for the first time that P2Y purinoceptor agonists stimulate in vitro invasion of prostate cancer cells, and the stimulated invasion is mediated by ERK1/2 and p38 kinases. Inhibition of ERK1/2 and p38 kinases, either singly or in combination, inhibits the invasive ability of prostate cancer cells, providing a novel baseline of research for applied study in human prostate cancer invasion and metastasis. Although the mechanisms underlying tumor invasion and metastasis are very complex, considerable evidence has indicated that Ras oncogenes and Rasassociated signaling pathways contribute significantly to tumor invasion and metastasis [10,21]. Our findings of the participation of ERK1/2 and p38 in mediating prostate cancer cell invasion are consistent with the current notion that Ras-MAPK signaling pathway plays an important role in tumor invasion and metastasis. Consistently, previous studies have also shown that ERK1/2 is necessary for acquisition of the metastatic phenotype of NIH3T3 cells [22] and required for cells with increased RasGEF to express matrix-degrading activity in vitro and tissue invasiveness in vivo [23]. Furthermore, our data show that there is significant difference in the levels of ERK1/2 and p38 activity between the two human prostate cancer cell lines with or without metastatic capacity, although both kinases can be activated by P2Y receptor agonists. This difference in ERK1/2 and p38 MAP kinase signaling pathways may provide a useful index for prediction of not only the molecular mechanism underlying prostate cancer but also the potential of prostate cancer metastasis. The signaling pathways in mediating the tumorigenic and metastatic activities of Ras have been suggested to be segregated and activation of the p38MAPK cascade plays a part in the process. The p38 pathway is activated in many cell types in response to cell stresses including hyperosmotic shock, UV
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irradiation and exposure to pro-inflammatory cytokines, and plays an important role in cell proliferation, apoptosis, motility and invasive phenotype [10,11,24]. To date, however, limited data are available in the potential roles of p38 kinase in prostate cancer progression. We show here that the p38-signaling cascade is activated by ATP treatment and responsible for P2Y receptor-stimulated in vitro invasion in androgen-independent prostate cancer cells. In addition, previous studies have shown that IL-6 induced an androgen response in LNCaP cells through ERK1/2 and p38 pathways [5]. An androgen responsive product, the STE20/SPS-related kinase, modulated p38 MAP kinase activity in androgen-dependent LNCaP cells [9]. Based on these findings, we propose that the p38 pathway may play an important role in mediating the progression of prostate carcinoma to a more advanced stage. Extracellular purines and pyrimidines are important signaling molecules that mediate diverse biological effects via cell surface receptors. The G protein-coupled P2Y receptor subfamilies have extensive pathophysiological functions in cell growth and differentiation, but their roles in tumor progression have not been adequately investigated. Our results that P2Y receptor stimulated prostate cancer invasion via ERK1/2 and p38 pathways provide insight into the potential role of P2Y receptor in prostate cancer progression. Since there are eight P2Y receptors [25] and such multiple P2Y receptors as P2Y2, P2Y6 and P2Y11 have also been shown to coexist on prostate cancer PC-3 cells [14], it can not be ruled out that multiple P2Y receptor subtypes contribute to the observed invasion of prostate cancer cells. Further studies are required to dissect the functions of different P2Y receptors. Furthermore, ERK1/2 and p38 are only part of the effector pathways down-stream of P2Y receptors, contributions of other pathway(s) such as PI-3K/Akt/PTEN system may also need to be explored. Convincing evidence exists that PI-3K/Akt/PTEN pathway is involved in tumor invasion and metastasis as well as prostate cancer progression [26,27]. In summary, ERK1/2 and p38 kinases are important regulators in P2Y receptorinduced prostate cancer invasion, and may provide diagnostic and therapeutic targets for advanced stage prostate carcinoma.
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Acknowledgements The authors wish to thank Dr Jun-ping Liu for his careful reading of the manuscript. This work was supported by grants to WGF from National Natural Science Foundation of China (30070293 and 30270518) and Key Research Project of Beijing (H020920030390).
[12]
[13]
[14]
References [1] S. Hirohashi, Inactivation of the E-cadherin-mediated cell adhesion system in human cancers, Am. J. Pathol. 153 (1998) 333–339. [2] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [3] T. Genda, M. Sakamoto, T. Ichida, H. Asakura, M. Kojiro, S. Narumiya, S. Hirohashi, Cell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma, Hepatology 30 (1999) 1027–1036. [4] C. Magi-Galluzzi, R. Montironi, M.G. Cangi, K. Wishnow, M. Loda, Mitogen-activated protein kinases and apoptosis in PIN, Virchows Arch. 432 (1998) 407–413. [5] H.L. Kim, D.J. Griend, X. Yang, D.A. Benson, Z. Dubauskas, B.A. Yoshida, et al., Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers, Cancer Res. 61 (2001) 2833–2837. [6] T. Suthiphongchai, P. Promyart, S. Virochrut, R. Tohtong, P. Wilairat, Involvement of ERK1/2 in invasiveness and metastatic development of rat prostatic adenocarcinoma, Oncol. Res. 13 (2003) 253–259. [7] X. Ge, Y. Fu, G.G. Meadows, U0126, a mitogen-activated protein kinase kinase inhibitor, inhibits the invasion of human A375 melanoma cells, Cancer Lett. 179 (2002) 133–140. [8] K. Graf, X.P. Xi, D. Yang, E. Fleck, W.A. Hsueh, R.E. Law, Mitogen-activated protein kinase activation is involved in platelet-derived growth factor directed migration by vascular smooth muscle cells, Hypertension 29 (1997) 334–339. [9] H. Qi, Y. Labrie, J. Grenier, A. Fournier, C. Fillion, C. Labrie, Androgens induce expression of SPAK, a STE20/SPS1related kinase, in LNCaP human prostate cancer cells, Mol. Cell Endocrinol. 182 (2001) 181–192. [10] M.S. Kim, E.J. Lee, H.R. Choi Kim, A. Moon, p38 kinase is a key signaling molecule for H-ras-induced cell motility and invasive phenotype in human breast epithelial cells, Cancer Res. 63 (2003) 5454–5461. [11] T. Matsumoto, K. Yokote, K. Tamura, M. Takemoto, H. Ueno, Y. Saito, S. Mori, Platelet-derived growth factor activates p38
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration, J. Biol. Chem. 274 (1999) 13954–13960. G. Burnstock, M. Williams, P2 purinergic receptors: modulation of cell function and therapeutic potential, J. Pharmacol. Exp. Ther. 295 (2000) 862–869. W.G. Fang, F. Pirnia, Y.J. Bang, C.E. Myers, J.B. Trepel, P2purinergic receptor agonists inhibit the growth of androgenindependent prostate carcinoma cells, J. Clin. Invest. 89 (1992) 191–196. R. Janssens, J.M. Boeynaems, Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells, Br. J. Pharmacol. 132 (2001) 536–546. H. Chaulet, C. Desgranges, M.A. Renault, F. Dupuch, G. Ezan, F. Peiretti, et al., Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin, Circ. Res. 89 (2001) 772–778. M.T. Tu, S.F. Luo, C.C. Wang, C.S. Chien, C.T. Chiu, C.C. Lin, C.M. Yang, P2Y2 receptor-mediated proliferation of C6 glioma cell via activation of Ras/Raf/MEK/MAPK pathway, Br. J. Pharmacol. 129 (2000) 1481–1489. H.Y. He, W.G. Fang, J. Zheng, J.F. You, W.J. Heng, Y. Li, Mechanism of the mitogen-activated protein kinase phosphatase-5 regulating the growth and invasion of a human prostate cancer cell line, Natl Med. J. China 83 (2003) 1812–1817. Y.X. Liu, J. Zheng, W.G. Fang, J.F. You, J.L. Wang, X.L. Cui, B.Q. Wu, Identification of metastasis associated gene G3BP by differential display in human cancer cell sublines with different metastatic potentials—G3BP as highly expressed in non-metastatic cells, Chin. Med. J. 114 (2001) 35–38. R. Seger, D. Seger, A.A. Reszka, E.S. Munar, H. EldarFinkelman, G. Dobrowolska, et al., Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells, J. Biol. Chem. 269 (1994) 25699– 25709. A. Albini, Y. Iwamoto, H.K. Kleinman, G.R. Martin, S.A. Aaronson, J.M. Kozlowski, R.N. McEwan, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (1987) 3239–3245. P.M. Campbell, C.J. Der, Oncogenic Ras and its role in tumor cell invasion and metastasis, Semin. Cancer Biol. 14 (2004) 105–114. D.R. Welch, T. Sakamaki, R. Pioquinto, T.O. Leonard, S.F. Goldberg, Q. Hon, Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells, Cancer Res. 60 (2000) 1552–1556. Y. Ward, W. Wang, E. Woodhouse, I. Linnoila, L. Liotta, K. Kelly, Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and ral- specific guanine exchange factor pathways, Mol. Cell Biol. 21 (2001) 5958–5969. J.M. Kyriakis, J. Avruch, Sounding the alarm: protein kinase cascades activated by stress and inflammation, J. Biol. Chem. 271 (1996) 24313–24316.
L. Chen et al. / Cancer Letters 215 (2004) 239–247 [25] M.P. Abbracchio, J.M. Boeynaems, E.A. Barnard, J.L. Boyer, C. Kennedy, M.T. Miras-Portugal, et al., Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family, Trends Pharmacol. Sci. 124 (2003) 52–55. [26] H. Kobayashi, M. Suzuki, N. Kanayama, T. Terao, Genetic down-regulation of phosphoinositide 3-kinase by bikunin
247
correlates with suppression of invasion and metastasis in human ova-rian cancer HRA cells, J. Biol. Chem. 279 (2004) 6371–6379. [27] M.A. Davies, S.J. Kim, N.U. Parikh, Z. Dong, C.D. Bucana, G.E. Gallick, Adenoviral-mediated expression of MMAC/PTEN inhibits proliferation and metastasis of human prostate cancer cell, Clin. Cancer Res. 8 (2002) 1904–1914.
ERK1/2 and p38 pathways are required for P2Y receptor-mediated prostate cancer invasion Ling Chen1, Hui-ying He1, Hong-mei Li, Jie Zheng, Wan-jie Heng, Jiang-feng You, Wei-gang Fang* Department of Pathology, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100083, China Received 16 December 2003; received in revised form 4 May 2004; accepted 19 May 2004
Abstract The G protein-coupled P2Y purinoceptors have wide physiological functions, but their role(s) in tumor progression remain unclear. Here, we report that stimulation of P2Y receptors enhances prostate cancer cell invasion in two human prostate carcinoma cell lines, which is mediated by ERK1/2 and p38 signaling pathways. P2Y agonists stimulated prostate cancer cell invasion, and increased the activities of ERK1/2 and p38 protein kinases. The stimulated cancer cell invasion was inhibited by the presence of MEK1 inhibitor PD98059 or p38 inhibitor SB203580. Expression of dominant-negative mutant of MEK1 (KA-MEK1), or up-regulation of MKP-5 (a dual-specificity phosphatase of p38), both reduced the invasion of cultured prostate cancer cells. These results suggest that P2Y receptors and their down-stream ERK1/2 and p38 protein kinases are important regulators promoting prostate cancer invasion. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: P2Y receptor; ERK1/2; p38; Prostate cancer; Invasion
1. Introduction Prostate cancer is the leading diagnosed malignancy in men in western countries. Due to lack of effective treatments, development of hormone-independence and metastases remains the most lethal aspect of this disease. Loss of cell–cell contact caused by dysfunction of E-cadherin [1], and increased proteolysis and * Corresponding author. Tel.: C86-10-82802599; fax: C86-1062015547. E-mail address: [email protected] (W.- Fang). 1 These authors contributed equally to this work.
cell motility [2,3] have been shown to be critical steps in invasive and metastatic process. Several growth factors and their receptors have been implicated in this process through activation of their down-stream signaling pathways, and mitogen-activated protein kinase (MAPK) pathway has been extensively investigated. In a study of 50 cases of high-grade prostatic intraepithelial neoplasia (PIN) using immunohistochemistry and in situ hybridization, increased expressions of MAPK family have been demonstrated in all cases of high-grade PIN compared with normal prostate [4]. More recently, interleukin 6 (IL-6) induces an androgen response in androgen dependent
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.05.023
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LNCaP cells by mechanisms dependent on androgen receptor, ERK1/2 and p38 activities [5]. The ERK1/2 MAPK pathway has been shown to be involved in the invasive and metastatic development of various types of cancers including rat prostatic adenocarcinoma [6], melanoma cancer [7] and vascular smooth muscle cells [8]. Modulated by the androgen responsive product STE20/SPS-related kinase in LNCaP cells [9], the p38 MAPK is an important mediator for cell motility or invasive phenotype in cancers including human breast epithelial cells [10] and endothelial cells [11]. Nucleotides, such as ATP and UTP, can function as potent extracellular stimuli by exerting their effects via activation of a distinct family of cell surface receptors, the P2 purinoceptor family. P2 receptors have two groups: the P2X ligand-gated ionotropic channel family and the P2Y metabotropic GPCR family [12]. Our previous study demonstrated that the ATP receptors on PC-3 prostate cancer cells belong to P2Y subtype [13]. Expression of P2Y receptor messenger RNA is also detected in PC-3 cells [14]. P2Y receptor activation induces arterial smooth muscle cell migration [15] and regulates cell growth of different cell types [13,16]. However, little is known of connection between P2Y receptor and tumor pro-gression. We reported that ATP activates p38 as well as ERK1/2 cascades, and stimulates prostate cancer cell invasion [17]. To address whether ERK1/2 and p38 pathways mediate P2Y receptor-induced prostate cancer progression, in the present study we demonstrate that P2Y receptor-induced prostate cancer cell invasion is regulated through ERK1/2 and p38 pathways.
2. Materials and methods 2.1. Tumor cell lines and cell culture Two subclones from the PC-3 human prostate carcinoma cell line (American Type Culture Collection, Rockville, MD) were derived. The 1E8 cells are highly metastatic (100% metastasis to lungs and lymph nodes of nude mice 5 weeks after subcutaneous inoculation), whereas the 2B4 cells are non-metastatic [18]. All cells were routinely grown in RPMI 1640 supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 8C and used when they reach 80% confluency.
2.2. Plasmid construction and cell transfection Plasmid encoding dominant-negative human MEK1 (pcDNA3-KA-MEK1) was kindly provided by Dr Rong Xu (Weizmann Institute, Israel). Lysine 97 of MEK1 was mutated to alanine (K97A) to produce an inactive enzyme. The K97A served as dominant-negative mutant by suppressing MAPKK, MAPK and p90rsk activation in vivo [19]. Plasmid pcDL-SRa 296-Myc containing wild type human MKP-5 (-wt) was kindly provided by Dr Eisuke Nishida (Kyoto University, Japan). Transfection was performed using the LipofectAMINEe reagent (Gibco BRL, Gaitherburg, MD), and the transfectants were maintained in the presence of 400 mg/ml G418. 2.3. Immunoblotting analysis of activated kinases Activated MAPKs (ERK1/2, p38 and JNK/SAPK) were detected using phospho-specific antibodies directed against the dually phosphorylated, active forms of these kinases. Cells were serum-deprived for 24 h before treatment with reagents at the indicated time. Cells were lysed in buffer containing 62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), and 0.1% (w/v) bromphenol blue. Equal amounts of protein samples were separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes, which were blotted with appropriate antibodies to determine MAPK activities, and the signals were detected by enhanced chemiluminescence (ECL analysis kit, Amersham Corp). The membranes were stripped in buffer (62.5 mM Tris–HCL, 2% SDS, and 100 mM b-mercaptoethanol, pH 6.7, at 50 8C), and re-probed with anti-ERK1/2, -p38 or -JNK antibodies to determine the total amounts of each kinase. 2.4. In vitro invasion assay The invasive ability of carcinoma cells was evaluated using Boyden Chamber assay as previously described by Albini et al. [20]. Briefly, the upper and lower compartments of the chamber were separated with a PVP-free polycarbonate filter (8-mm pore size, Neuro Probe, CA) coated with 50 mg reconstituted basement membrane Matrigel (Collaborative Res, Bradford, MA). Cells in 400 ml medium were placed
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in the upper compartment, and the lower compartment was filled with 200 ml NIH3T3-conditioned medium. After incubation for 12 h at 37 8C, the filters were removed. Cells on the upper side of the filter were removed with cotton-tipped swabs. Filters were fixed in methanol and stained with hematoxylin and eosin. Cells on the underside of the filters were counted by microscopic inspection. Each sample was assayed in triplicate. 2.5. Statistical analysis Comparison of multiple means was performed with nonparametric ANOVA. Data are expressed as meanGs.d.m. P values are considered to be significant at !0.05.
3. Results 3.1. Stimulation of P2Y receptor led to activation of ERK1/2 and p38 kinases in prostate cancer cells Treatment of both 1E8 and 2B4 cells with P2Y receptor agonist ATP resulted in a significant activation of ERK1/2 and p38, but not JNK (data not shown) kinases. These responses were time- and dose-dependent (Fig. 1A–D), with peak activation at 100 mM in 15 min. ERK1/2 activation in response to ATP was significantly inhibited by pretreatment with MEK1 inhibitor PD98059 (25 mM) (Fig. 1E), and p38 phosphorylation was also inhibited significantly in the presence of SB203580 (10 mM) (Fig. 1F), confirming that both ERK1/2 and p38 are implicated in downstream signal transduction of P2Y receptor. To verify that it is P2Y receptors that mediate ATP activation of the kinases, we tested another hydrolysis-resistant P2Y receptor agonist AMP-PNP (b, g, -imido ATP)
Fig. 1. ATP activates ERK1/2 and p38 kinases in 1E8 (A and C) and 2B4 (B and D) cells in a time- and dose-dependent manner.
PD98059 and SB203580 inhibit ATP-induced ERK1/2 (E) and p38 (F) activation. AMP-PNP (G) activates ERK1/2 and p38 kinases in 1E8 cells in a time-dependent manner. Cells were serum-deprived for 24 h and then pretreated without or with 25 mM PD98059 for 1 h or 10 mM SB203580 for 20 min before treatment with stimulators at the indicated time. Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane and probed with antiphospho-ERK1/2 or anti-phospho-p38 antibody. The blots were stripped and re-probed with anti-ERK1/2 or anti-p38 antibody to assess equal loading of samples.
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and found that it stimulated ERK1/2 and p38 activation to the same extent as ATP (Fig. 1G). To confirm the specific role of P2Y receptor in p38 and ERK1/2 activation, cells were pretreated
with P2 receptor antagonist suramin (300 mM), and then stimulated with ATP for 15 min. As shown in Fig. 2A, ERK1/2 phosphorylation by ATP was significantly reduced approximately 82.4%
Fig. 2. P2 receptor antagonist suramin blocks ATP-induced ERK1/2 (A) and p38 (B) activation in 1E8 and 2B4 cells. Serum-starved cells were pretreated with 300 mM suramin for 30 min prior to stimulation with 100 mM ATP for 15 min. (C) Different levels of ERK1/2 and p38 phosphorylation by ATP in 1E8 and 2B4 cells. (D) KA-MEK1 transfection inhibits ERK1/2 activation. (E) MKP-5 transfection inhibits p38 phosphorylation.
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(P!0.001) and 80.7% (P!0.001) in 1E8 and 2B4 cells, respectively. ATP-induced p38 activation was also inhibited by suramin (1E8 cell: 71.6%, P! 0.01; 2B4 cell: 69.2%, P!0.01) (Fig. 2B). These results confirmed our finding that P2Y receptors were coupled to ERK1/2 and p38 pathways in prostate cancer cells. 3.2. Activation of P2Y receptors stimulated invasion of prostate cancer cells in vitro Using the in vitro invasion assay, incubation with 100 mM ATP for 12 h enhanced the invasion of metastatic 1E8 cells (63.4% increases) through Matrigel when compared with control (Fig. 3A). The invasion of non-metastatic 2B4 cells was also stimulated by ATP (46.4% increase). In a parallel experiment, AMP-PNP showed similar effect to that of ATP (data not shown). Since P2Y receptor stimulates activation of ERK1/2 and p38, we examined the effects of blocking ERK1/2 and p38 pathways on ATP-induced invasion. Pretreatment of the cells with p38 inhibitor SB203580 significantly attenuated the stimulatory effect of ATP on cell invasion (69.9% decreases in 1E8, 54% decreases in 2B4). Blocking the ERK1/2 pathway with PD98059 showed the same effect (79.8% decrease in 1E8, 61% decrease in 2B4) (Fig. 3A). These data suggest that both the ERK1/2 and p38 cascades were involved in mediating the invasive phenotype seen in human prostate carcinoma cells. 3.3. ERK1/2 and p38 pathways were involved in P2Y receptor-mediated prostate cancer invasion To further confirm the involvement of the ERK1/2 and p38 pathways in mediating the P2Y receptorinduced cancer invasion, we further tested individual contribution of each pathway to this effect by modulating gene expression. Transfection with the dominant-negative mutant of MEK1 cDNA (KA-MEK1) inhibited ERK1/2 phosphorylation significantly (Fig. 2D). KA-MEK1 transfectants showed a 40.7% decrease in the number of cells passing through Matrigel-coated membranes as compared with vector control (P!0.01), and a 45.2% decrease as compared with ATP-treated control (P!0.01) (Fig. 3B). Together with the result
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obtained in PD98059 pretreatment as shown above (Fig. 3A), the data suggest that ERK1/2 pathway promotes prostate cancer cell invasion. Treatment of the KA-MEK1-transfected cells with SB203580 further inhibited ATP-stimulated cell invasion by 76.2% as compared with ATP-treated control (a further 21.4% decrease as compared with KA-MEK1-transfected control) (Fig. 3B). In order to confirm that p38 is also involved in P2Y receptormediated cancer cell invasion, we overexpressed MKP-5, a dual-specificity phosphatase specific for p38, by transfection of MKP-5 cDNA into 1E8 cells. Phosphorylation of p38 was inhibited significantly in MKP-5 transfectants (Fig. 2E). A 66.6% decrease in invasive cells was seen in MKP-5 transfected cells as compared with vector control, and a 65.9% decrease as compared with ATP-treated control (Fig. 3C). Pretreatment of the MKP-5-transfected cells with PD98059 could further inhibit cell invasion as compared with ATP-treated control, though not so significantly. These results further confirm that both the ERK1/2 and p38 pathways are required for the P2Y receptor-mediated 1E8 cell invasion. 3.4. Activation levels of ERK1/2 and p38 pathways were correlated with invasive potential of prostate cancer cells 1E8 and 2B4 cells derived from PC-3 differ in their metastatic potentials in nude mice as well as invasive ability in vitro. In order to correlate the difference in their invasive abilities to the levels of ERK1/2 and p38 activation, we compared the levels of ERK1/2 and p38 activation by ATP between the two subclones. As shown in Fig. 2C, the phosphorylation levels of ERK1/2 and p38 stimulated by ATP were significantly higher in 1E8 cells than those in 2B4 cells (2.85 and 2.57 folds, respectively). Since, there was no difference in ERK1/2 and p38 constitutive expression between the two subclones, and 1E8 cells have greater metastatic potentials, the findings suggest that ERK1/2 and p38 signaling pathways are involved in prostate cancer progression and other mechanisms such as coupling of P2Y receptor to the MAPK cascades are required in prostate cancer invasion.
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Fig. 3. Effects of different treatments on ATP-induced in vitro invasion of 1E8 or 2B4 cells in Boyden chamber invasion assay. (A) Effects of PD98059 and SB203580 on in vitro invasion of 1E8 and 2B4 cells. (B) Effect of KA-MEK1 transfection on in vitro invasion of 1E8 cells. (C) Effect of MKP-5 transfection on in vitro invasion of 1E8 cells. (D) 1E8 transfectants migrate through Matrigel coated filters (!200). Cells were plated on filters pre-coated with Matrigel, incubated with or without PD98059 (25 mM for 1 h) or SB203580 (10 mM for 20 min), and then stimulated with 100 mM ATP for 12 h and assayed in vitro invasion. The data are expressed as percent of the number of cells passing through filters in control. Fifteen fields were counted on each filter. Values are the meanGs.d.m. (nZ3).
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4. Discussion Intracellular signaling pathways mediating malignant progression of human prostate cancers are yet to be fully determined. In the present study, we investigated roles of ERK1/2 and p38 in cancer cell invasion using two human prostate carcinoma cell lines with different metastatic potentials. We demonstrate for the first time that P2Y purinoceptor agonists stimulate in vitro invasion of prostate cancer cells, and the stimulated invasion is mediated by ERK1/2 and p38 kinases. Inhibition of ERK1/2 and p38 kinases, either singly or in combination, inhibits the invasive ability of prostate cancer cells, providing a novel baseline of research for applied study in human prostate cancer invasion and metastasis. Although the mechanisms underlying tumor invasion and metastasis are very complex, considerable evidence has indicated that Ras oncogenes and Rasassociated signaling pathways contribute significantly to tumor invasion and metastasis [10,21]. Our findings of the participation of ERK1/2 and p38 in mediating prostate cancer cell invasion are consistent with the current notion that Ras-MAPK signaling pathway plays an important role in tumor invasion and metastasis. Consistently, previous studies have also shown that ERK1/2 is necessary for acquisition of the metastatic phenotype of NIH3T3 cells [22] and required for cells with increased RasGEF to express matrix-degrading activity in vitro and tissue invasiveness in vivo [23]. Furthermore, our data show that there is significant difference in the levels of ERK1/2 and p38 activity between the two human prostate cancer cell lines with or without metastatic capacity, although both kinases can be activated by P2Y receptor agonists. This difference in ERK1/2 and p38 MAP kinase signaling pathways may provide a useful index for prediction of not only the molecular mechanism underlying prostate cancer but also the potential of prostate cancer metastasis. The signaling pathways in mediating the tumorigenic and metastatic activities of Ras have been suggested to be segregated and activation of the p38MAPK cascade plays a part in the process. The p38 pathway is activated in many cell types in response to cell stresses including hyperosmotic shock, UV
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irradiation and exposure to pro-inflammatory cytokines, and plays an important role in cell proliferation, apoptosis, motility and invasive phenotype [10,11,24]. To date, however, limited data are available in the potential roles of p38 kinase in prostate cancer progression. We show here that the p38-signaling cascade is activated by ATP treatment and responsible for P2Y receptor-stimulated in vitro invasion in androgen-independent prostate cancer cells. In addition, previous studies have shown that IL-6 induced an androgen response in LNCaP cells through ERK1/2 and p38 pathways [5]. An androgen responsive product, the STE20/SPS-related kinase, modulated p38 MAP kinase activity in androgen-dependent LNCaP cells [9]. Based on these findings, we propose that the p38 pathway may play an important role in mediating the progression of prostate carcinoma to a more advanced stage. Extracellular purines and pyrimidines are important signaling molecules that mediate diverse biological effects via cell surface receptors. The G protein-coupled P2Y receptor subfamilies have extensive pathophysiological functions in cell growth and differentiation, but their roles in tumor progression have not been adequately investigated. Our results that P2Y receptor stimulated prostate cancer invasion via ERK1/2 and p38 pathways provide insight into the potential role of P2Y receptor in prostate cancer progression. Since there are eight P2Y receptors [25] and such multiple P2Y receptors as P2Y2, P2Y6 and P2Y11 have also been shown to coexist on prostate cancer PC-3 cells [14], it can not be ruled out that multiple P2Y receptor subtypes contribute to the observed invasion of prostate cancer cells. Further studies are required to dissect the functions of different P2Y receptors. Furthermore, ERK1/2 and p38 are only part of the effector pathways down-stream of P2Y receptors, contributions of other pathway(s) such as PI-3K/Akt/PTEN system may also need to be explored. Convincing evidence exists that PI-3K/Akt/PTEN pathway is involved in tumor invasion and metastasis as well as prostate cancer progression [26,27]. In summary, ERK1/2 and p38 kinases are important regulators in P2Y receptorinduced prostate cancer invasion, and may provide diagnostic and therapeutic targets for advanced stage prostate carcinoma.
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Acknowledgements The authors wish to thank Dr Jun-ping Liu for his careful reading of the manuscript. This work was supported by grants to WGF from National Natural Science Foundation of China (30070293 and 30270518) and Key Research Project of Beijing (H020920030390).
[12]
[13]
[14]
References [1] S. Hirohashi, Inactivation of the E-cadherin-mediated cell adhesion system in human cancers, Am. J. Pathol. 153 (1998) 333–339. [2] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [3] T. Genda, M. Sakamoto, T. Ichida, H. Asakura, M. Kojiro, S. Narumiya, S. Hirohashi, Cell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma, Hepatology 30 (1999) 1027–1036. [4] C. Magi-Galluzzi, R. Montironi, M.G. Cangi, K. Wishnow, M. Loda, Mitogen-activated protein kinases and apoptosis in PIN, Virchows Arch. 432 (1998) 407–413. [5] H.L. Kim, D.J. Griend, X. Yang, D.A. Benson, Z. Dubauskas, B.A. Yoshida, et al., Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers, Cancer Res. 61 (2001) 2833–2837. [6] T. Suthiphongchai, P. Promyart, S. Virochrut, R. Tohtong, P. Wilairat, Involvement of ERK1/2 in invasiveness and metastatic development of rat prostatic adenocarcinoma, Oncol. Res. 13 (2003) 253–259. [7] X. Ge, Y. Fu, G.G. Meadows, U0126, a mitogen-activated protein kinase kinase inhibitor, inhibits the invasion of human A375 melanoma cells, Cancer Lett. 179 (2002) 133–140. [8] K. Graf, X.P. Xi, D. Yang, E. Fleck, W.A. Hsueh, R.E. Law, Mitogen-activated protein kinase activation is involved in platelet-derived growth factor directed migration by vascular smooth muscle cells, Hypertension 29 (1997) 334–339. [9] H. Qi, Y. Labrie, J. Grenier, A. Fournier, C. Fillion, C. Labrie, Androgens induce expression of SPAK, a STE20/SPS1related kinase, in LNCaP human prostate cancer cells, Mol. Cell Endocrinol. 182 (2001) 181–192. [10] M.S. Kim, E.J. Lee, H.R. Choi Kim, A. Moon, p38 kinase is a key signaling molecule for H-ras-induced cell motility and invasive phenotype in human breast epithelial cells, Cancer Res. 63 (2003) 5454–5461. [11] T. Matsumoto, K. Yokote, K. Tamura, M. Takemoto, H. Ueno, Y. Saito, S. Mori, Platelet-derived growth factor activates p38
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration, J. Biol. Chem. 274 (1999) 13954–13960. G. Burnstock, M. Williams, P2 purinergic receptors: modulation of cell function and therapeutic potential, J. Pharmacol. Exp. Ther. 295 (2000) 862–869. W.G. Fang, F. Pirnia, Y.J. Bang, C.E. Myers, J.B. Trepel, P2purinergic receptor agonists inhibit the growth of androgenindependent prostate carcinoma cells, J. Clin. Invest. 89 (1992) 191–196. R. Janssens, J.M. Boeynaems, Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells, Br. J. Pharmacol. 132 (2001) 536–546. H. Chaulet, C. Desgranges, M.A. Renault, F. Dupuch, G. Ezan, F. Peiretti, et al., Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin, Circ. Res. 89 (2001) 772–778. M.T. Tu, S.F. Luo, C.C. Wang, C.S. Chien, C.T. Chiu, C.C. Lin, C.M. Yang, P2Y2 receptor-mediated proliferation of C6 glioma cell via activation of Ras/Raf/MEK/MAPK pathway, Br. J. Pharmacol. 129 (2000) 1481–1489. H.Y. He, W.G. Fang, J. Zheng, J.F. You, W.J. Heng, Y. Li, Mechanism of the mitogen-activated protein kinase phosphatase-5 regulating the growth and invasion of a human prostate cancer cell line, Natl Med. J. China 83 (2003) 1812–1817. Y.X. Liu, J. Zheng, W.G. Fang, J.F. You, J.L. Wang, X.L. Cui, B.Q. Wu, Identification of metastasis associated gene G3BP by differential display in human cancer cell sublines with different metastatic potentials—G3BP as highly expressed in non-metastatic cells, Chin. Med. J. 114 (2001) 35–38. R. Seger, D. Seger, A.A. Reszka, E.S. Munar, H. EldarFinkelman, G. Dobrowolska, et al., Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells, J. Biol. Chem. 269 (1994) 25699– 25709. A. Albini, Y. Iwamoto, H.K. Kleinman, G.R. Martin, S.A. Aaronson, J.M. Kozlowski, R.N. McEwan, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (1987) 3239–3245. P.M. Campbell, C.J. Der, Oncogenic Ras and its role in tumor cell invasion and metastasis, Semin. Cancer Biol. 14 (2004) 105–114. D.R. Welch, T. Sakamaki, R. Pioquinto, T.O. Leonard, S.F. Goldberg, Q. Hon, Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells, Cancer Res. 60 (2000) 1552–1556. Y. Ward, W. Wang, E. Woodhouse, I. Linnoila, L. Liotta, K. Kelly, Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and ral- specific guanine exchange factor pathways, Mol. Cell Biol. 21 (2001) 5958–5969. J.M. Kyriakis, J. Avruch, Sounding the alarm: protein kinase cascades activated by stress and inflammation, J. Biol. Chem. 271 (1996) 24313–24316.
L. Chen et al. / Cancer Letters 215 (2004) 239–247 [25] M.P. Abbracchio, J.M. Boeynaems, E.A. Barnard, J.L. Boyer, C. Kennedy, M.T. Miras-Portugal, et al., Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family, Trends Pharmacol. Sci. 124 (2003) 52–55. [26] H. Kobayashi, M. Suzuki, N. Kanayama, T. Terao, Genetic down-regulation of phosphoinositide 3-kinase by bikunin
247
correlates with suppression of invasion and metastasis in human ova-rian cancer HRA cells, J. Biol. Chem. 279 (2004) 6371–6379. [27] M.A. Davies, S.J. Kim, N.U. Parikh, Z. Dong, C.D. Bucana, G.E. Gallick, Adenoviral-mediated expression of MMAC/PTEN inhibits proliferation and metastasis of human prostate cancer cell, Clin. Cancer Res. 8 (2002) 1904–1914.