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Sennoside B inhibits PDGF receptor signaling and cell proliferation induced by PDGF-BB in human osteosarcoma cells
Life Sciences 84 (2009) 915–922
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Sennoside B inhibits PDGF receptor signaling and cell proliferation induced by PDGF-BB in human osteosarcoma cells Yen-Chun Chen a,b,⁎, Chia-Ni Chang a, Hui-Chun Hsu a, Shu-Jiau Chiou a, Lain-Tze Lee a, Tzong-Hsiung Hseu b a b
Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
a r t i c l e
i n f o
Article history: Received 19 December 2008 Accepted 4 April 2009 Keywords: Sennoside B Osteosarcoma Signaling pathway Platelet-derived growth factor (PDGF) Platelet-derived growth factor receptor (PDGFR)
a b s t r a c t Aims: To address the possibility that sennoside B inhibition of cell proliferation is mediated via interference with platelet-derived growth factor (PDGF) signaling. Main methods: Human osteosarcoma MG63 cells were treated with PDGF in the presence or absence of sennoside B. Activation of the PDGF signaling pathway was monitored using western immunoblotting with specific antibodies against the PDGF receptor, phosphotyrosine and components of the downstream signaling cascade. Activation of cell metabolism and proliferation was assessed by chromogenic reduction of MTT. Key findings: Sennoside B was found to inhibit PDGF-BB-induced phosphorylation of the PDGF receptor (PDGFR) in human MG63 osteosarcoma cells. Downstream signaling was also affected; pre-incubation of PDGF-BB with sennoside B inhibited the phosphorylation of pathway components including Ak strain transforming protein (AKT), signal transducer and activator of transcription 5 (STAT-5) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further, we found that sennoside B can bind directly to the extracellular domains of both PDGF-BB and the PDGF-β receptor (PDGFR-β). The effect was specific for sennoside B; other similar compounds including aloe-emodin, rhein and the meso isomer (sennoside A) failed to inhibit PDGFR activation or downstream signaling. Sennoside B also inhibited PDGF-BB stimulation of MG63 cell proliferation. Significance: These results indicate that sennoside B can inhibit PDGF-stimulated cell proliferation by binding to PDGF-BB and its receptor and by down-regulating the PDGFR-β signaling pathway. Sennoside B is therefore of potential utility in the treatment of proliferative diseases in which PDGF signaling plays a central role. © 2009 Elsevier Inc. All rights reserved.
Introduction Sennosides have been used as natural, safe laxatives in traditional and modern systems of medicine. In addition, aloe-emodin and rhein, degradation products of sennosides, have been reported to inhibit tumor cell growth (Cardenas et al. 2006; Huang et al. 2007). Doxorubicin, a structurally related anthraquinone glycoside, is an important antineoplastic drug (Huang et al. 2007), while emodin has been reported to induce apoptotic cell death in proliferating cells (Pecere et al. 2000; Huang et al. 2007). In view of the planar structure of these molecules, it has been suggested that anthraquinones may be able to intercalate into DNA and that this could underlie their cytoxicity.
⁎ Corresponding author. Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan. Tel.: +886 3 5732561; fax: +886 3 5732359. E-mail address: [email protected] (Y.-C. Chen). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.04.003
Sennosides and related molecules may also exert their effects by interfering with the activation of cell-surface receptors. Emodin preferentially suppresses the phosphorylation of the human epidermal growth factor receptor 2 (HER-2/neu) in breast cancer cells (Zhang et al. 1995, 1998). Tyrosine kinase phosphorylation induced by epidermal growth factor (EGF) was also suppressed (Huang et al. 2005). Furthermore, it has been suggested that the anti-angiogenic activity of emodin may be due to inhibition of the phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2) and of downstream molecules (Kaneshiro et al. 2006; Srinivas et al. 2007; Lu et al. 2008). Platelet-derived growth factors (PDGFs) and their receptors have been implicated in the pathogenesis of a number of tumor types and play an important role in angiogenesis (Board and Jayson 2005; Pietras et al. 2003; Alvarez et al. 2006). Extensive experimental data highlight the potential therapeutic advantage of targeting the platelet-derived growth factor receptor (PDGFR). In humans, the PDGF signaling network consists of four ligands, PDGFs A through D. These factors interact with two receptors, PDGFR-α and PDGFR-β. Ligand-bound receptors undergo autophosphorylation, resulting in the activation of
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multiple intracellular signaling cascades. Regarding PDGF, the best characterized intracellular signaling cascade comprises mitogenactivated protein kinase kinase/extracellular regulated kinase (MEK/ ERK), phosphatidylinositol 3-kinase/Ak strain transforming protein (PI3K/AKT) and signal transducer and activator of transcription 5 (STAT-5) (Sandy et al. 1998; Graves et al. 1984; Thomas et al. 1997; Paukku et al. 2000; Valgeirsdottir et al. 1998; Zhang et al. 2007). Because this pathway has been widely implicated in the regulation of tumor growth, agents interfering with PDGF-BB signaling have great potential for the development of new anti-neoplastic agents. MG63 osteosarcoma cells present a functional PDGFR but do not secrete PDGF-like mitogens (Graves et al. 1984); these cells have been used for many years in osteoblast research (Sandy et al. 1998). In the present study, we used MG63 cells to investigate the effects of sennoside B on PDGF-BB-induced signaling. Our results indicate that sennoside B specifically binds to PDGF-BB and PDGFR and can inhibit PDGFR activation and downstream signaling induced by PDGF-BB. In a cell-based functional study, we also found that sennoside B can inhibit MG63 proliferation induced by PDGF-BB. Sennoside B may have significant potential for anti-cancer therapy.
were then cultured for 24 h, and then incubated in serum-free medium overnight before preparation of cell lysates.
Materials and methods
Dot-binding assay
Materials
Aliquots (2.5 µl) of vehicle (DMSO) and different concentrations of sennoside, PDGF-BB or PDGFR-β/Fc chimera in vehicle were spotted onto gridded PVDF membranes and air-dried. Membranes were blocked with BSA (5% in PBS) for 0.5 h. After washing in PBS, membranes were incubated with PDGF-BB or the PDGFR-β/Fc chimera (1 µg/ml) for 1 h at room temperature in PBS. Following a brief wash, the membrane was incubated with anti-PDGF-BB or PDGFR-β extracellular domain antibody (2 µg/ml in PBS containing 1% BSA) for 1 h at room temperature. After another brief wash, the membrane was incubated with HRP-conjugated secondary antibody and developed by ECL.
Sennoside A, Sennoside B, aloe-emodin and rhein were obtained from Sigma Chemical Co (St. Louis, MO, USA). These compounds were dissolved in DMSO (Sigma Chemical Co) and further diluted in Dulbecco's Modified Eagle's Medium (DMEM) without fetal bovine serum (FBS; Biological Industries). Penicillin, DMEM, and streptomycin were obtained from GIBCO™/Invitrogen Life Technologies (CA, USA). Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma Chemical Co. Antibodies raised against PDGFR-β and anti-phosphotyrosine antibody (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotech (Lake Placid, NY, USA). Recombinant human PDGF-BB, the PDGFR-β/Fc chimera, EGF and antibodies raised against native PDGF-BB, PDGFR-β and ERK1/2, AKT, STAT5 were from R&D Systems (MN, USA). Antibodies raised against phospho-ERK1/2 (Thr202/Tyr204), phospho-AKT (Ser473, Thr308), phospho-STAT5 (Tyr694), Akt and horseradish peroxidase (HRP)-linked anti-goat and anti-mouse IgG antibodies, as well as the cell lysis buffer, were from Cell Signaling Technology (Danvers, MA, USA). Antibodies raised against EGFR were from LabVision (Fremont CA, USA). Pre-stained protein markers were from Fermentas Life Sciences (Glen Burnie, MD, USA). SuperSignal® West Pico HRP chemiluminescence substrate, the Micro BCA™ Protein Assay Reagent protein assay kit and Western Blot Stripping Buffer were from Pierce (Rockford, IL, USA). Nitrocellulose membranes (Hybond-C Extra) were from Amersham Biosciences (Sunnyvale, CA, USA), and PVDF membranes were from Millipore (Billerica, MA, USA).
Cell lysate preparation and western blot analysis Osteosarcoma cells were cultured with PDGF-BB and sennosides for the times and at the different concentrations indicated in the figure legends. Lysates were prepared by the addition of cell lysis buffer and were clarified by centrifugation (14,000 g, 10 min, 4 °C). Lysate protein contents were quantified using the Pierce protein assay kit according to the manufacturer's instructions, using bovine serum albumin (BSA) as the reference standard. Proteins were separated by electrophoresis on SDS-polyacrylamide gels, electroblotted onto PVDF or NC membranes and probed using primary anti-phosphotyrosine, anti-p-ERK, anti-p-AKT, and anti-p-STAT5 antibodies. Immunoblots were developed using enhanced chemiluminescence (ECL) with HRP-labeled secondary antibodies and the SuperSignal® HRP substrate. The membranes were stripped (Western Blot Stripping Buffer), washed and reprobed with anti-PDGFR-β, anti-ERK1/2, anti-AKT or anti-STAT5 antibodies and developed as described above.
Cell proliferation assays Activation of cell metabolism and proliferation as reflected by NAD-dependent dehydrogenase activity was determined via chromogenic reduction of MTT. Osteosarcoma cells in a 96-well plates were allowed to reach 50% confluence (5 × 103 cells/well), starved for 24 h and incubated with different concentrations of sennoside B or vehicle (final concentration 0.5% DMSO) in the absence or presence of PDGFBB (20 ng/ml) in quintuplicate. After 24 and 48 h, cells were incubated with 0.5 mg/ml MTT for 2 h at 37 °C. Formazan crystals resulting from MTT reduction were dissolved by adding 200 µl DMSO and gently
Osteosarcoma cell culture Human osteoblast-like cells MG63 (BCRC-60279) and U-2OS (BCRC-60187) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Saos-2 cells (HTB-85) were obtained from The American Type Culture Collection (VA, USA) and grown in DMEM supplemented with 5–10% FBS, penicillin (100 units/ ml) and streptomycin (100 µg/ml). Unless otherwise indicated, cells reaching 80–90% confluence were starved and synchronized in DMEM without FBS at 37 °C for 24 h before further analysis. For western blot analysis, osteosarcoma cells were seeded into 6-well plates (Orange Scientific, Belgium) such that each well contained 1 ml of a 5 × 105 cells/ml suspension (as assessed by hemocytometry). Cells
Fig. 1. PDGFR phosphorylation in MG63 cells treated with PDGF-BB and sennosides A and B. MG63 cells were treated with medium containing PDGF-BB (20 ng/ml), with vehicle or the indicated concentrations of sennoside A or B for 5 min. Multiple kinase inhibitor SU11248 was included as a positive control. Cell lysates were collected, and protein tyrosine phosphorylation (p-Tyr) and total PDGFR-β were determined by western blotting. The 180-kDa phosphotyrosine band (detected by PY99) in the upper panel was identified as tyrosine-phosphorylated PDGFR. Relative band densities were quantified by scanning densitometry and normalized to the relative value of total PDGFR-β. The data shown are representative of three or more independent experiments.
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Fig. 2. Dot-binding assay of the interaction between PDGF-BB and the PDGFR's extracellular domain with immobilized sennoside B. Compounds were suspended in DMSO vehicle (Veh) and applied to a PVDF membrane. After incubation with PDGF-BB (panel A) or the PDGF extracellular domain (panel B), membranes were developed with peroxidase-linked secondary antibodies specific for PDGF-BB or the PDGF-β extracellular domain. Binding was detected using a chemiluminescent peroxidase substrate. ⁎Positive controls were immobilized PDGF-BB and the PDGF-β extracellular domain fused to Fc (extraPDGFR-IgG).
agitating for 20 min. The absorbance of the supernatant was then measured spectrophotometrically at 570 nm using an ELISA reader.
Results Sennoside B selectively inhibits PDGF-BB-induced PDGFR phosphorylation in osteosarcoma cells
Statistical analysis Data were expressed as means ± the standard error of the mean (SEM). Comparison of the means and SEMS used the unpaired, twotailed Student's t test. P-values b0.05 were considered significant. All statistical calculations were performed using GraphPad prism v5.0 (GraphPad Software Inc., CA, USA) or Grafit v5.0 (Erithacus Software Ltd., UK).
Several anthraquinones, including emodin, aloe-emodin and rhein, have been reported to exert anti-neoplastic effects by inducing apoptosis. Potential mechanisms of apoptosis induction include inhibition of VEGFR 2 (KDR/Flk-1) phosphorylation and suppression of HER2/neu tyrosine kinase autophosphorylation. The effects on PDGFR phosphorylation, however, have not been previously addressed. To investigate whether sennoside B can modulate PDGFR signaling, human
Fig. 3. Effects of sennoside B on signaling induced by PDGF-BB, EGF and FBS in MG63 cells. Panel A, MG63 cells treated with medium containing PDGF-BB (20 ng/ml); Panel B, cells treated with EGF (20 ng/ml); Panel C, cells treated with 10% FBS. All cells were incubated in the absence or presence of increasing concentrations (1–1000 nM) of sennoside B for 5 min (panel A) or 15 min (panels B, C). The levels of protein and protein phosphorylation for PDGFR, EGFR, STAT-5, AKT and ERK were analyzed by western blotting. Each blot is representative of two to three independent experiments.
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Fig. 4. Time course and concentration-dependence of sennoside B down-regulation of PDGFR-β phosphorylation in MG63 cells. Confluent cells were co-cultured in the absence or presence of sennoside B (100–500 nM) in serum-free medium for the time indicated. Levels of PDGFR-β and tyrosine-phosphorylated receptor (p-Tyr) were determined using 7.5% SDS-PAGE and immunoblotting. Relative band densities were quantified by scanning densitometry and normalized to total protein levels. Each blot is representative of two to three independent experiments.
osteoblast-like MG63 cells were treated with PDGF-BB in the presence or absence of different concentrations of sennoside B and PDGFR-β tyrosine phosphorylation was measured. We also studied the effects of the related compounds sennoside A, aloe-emodin and rhein. Expression and tyrosine phosphorylation of osteosarcoma cell PDGFRs were determined by western blotting using primary antibodies specific for PDGFR-β and phosphotyrosine, respectively. MG63 cells were found to express high levels of PDGFR-β (Fig. 1). Stimulation with PDGF-BB resulted in a significant increase in the level of tyrosine phosphorylation in a molecular weight band identified as PDGFR-β (Fig. 1, lane 2). Incubation of PDGF-BB with sennoside B resulted in a marked inhibition of PDGF-BB-induced PDGFR-β phosphorylation (Fig. 1, lanes 7 to 9). The extent of inhibition was concentration-dependent; the IC50 (19.2 ± 3.6 nM) was comparable to the inhibition associated with the multiple kinase inhibitor SU11248 (lane 3). Sennoside B therefore appears to be a potent inhibitor of PDGFR-β activation. In contrast, sennoside A, the meso form of the sennoside molecule, only minimally inhibited PDGF-BBinduced PDGFR-β phosphorylation (Fig. 1, lanes 4 and 5) and was far less potent than the trans form, sennoside B. Aloe-emodin and rhein at concentrations of 10 µM did not show any inhibitory effect on PDGFRβ phosphorylation induced by PDGF-BB (data not shown).
by 50% (data not shown). This result argues against rapid diffusion, and instead suggests that sennoside B binds to other cellular target sites. We hypothesized that sennoside B might antagonize PDGFR-β activation by binding to PDGF-BB or to the PDGFR extracellular domain. Accordingly, we used a direct dot-binding assay to study possible binding of sennoside B to PDGF-BB or to the PDGFR extracellular domain. Sennoside A, sennoside B, aloe-emodin, rhein and recombinant PDGFBB were immobilized on a PVDF membrane. After incubation with PDGF-BB, the membrane was incubated with an antibody directed against PDGF-BB and then developed. Control immobilized PDGF-BB was efficiently recognized by the antibody (Fig. 2A, row 5). The antibody also detected immobilized PDGF-BB in sennoside B spots, and the antibody binding was dependent on the concentration of sennoside B solution applied (Fig. 2A, row 2). No reactivity was observed with sennoside A, aloe-emodin or rhein at 10 µM (Fig. 2A, rows 1, 4, and 3). These results indicate that PDGF-BB specifically and directly binds to sennoside B.
Sennoside B binds to the PDGF-BB and PDGFR extracellular domains Inhibition of PDGF-BB-induced PDGFR-β phosphorylation in MG63 cells was only observed when PDGF-BB was co-incubated with sennoside B. When cells were first treated with sennoside B followed by PDGF-BB, no inhibition of PDGFR-β phosphorylation was observed until the concentration reached ~ 500 nM (data not shown). This suggested that sennoside B could reversibly bind to PDGF or could rapidly diffuse into the culture medium. We also observed that when MG63 cells were pre-treated with sennoside B (1 µM) followed by the removal of sennoside by changing the culture medium and the addition of PDGF-BB, the extent of inhibition was only reduced
Fig. 5. PDGFR phosphorylation in osteosarcoma cells treated with PDGF-BB and sennoside B. Osteosarcoma cells were treated with medium containing PDGF-BB (20 ng/ml), with vehicle or 10 μM sennoside B for 5 min. Cell lysates were collected and protein tyrosine phosphorylation (p-Tyr) and total PDGFR-β determined by western blotting. The 180-kDa phosphotyrosine band (detected by PY99) in the upper panel was identified as tyrosine-phosphorylated PDGFR. Relative band densities were quantified by scanning densitometry and normalized to the relative value of total PDGFR-β. The data shown are representative of three or more independent experiments.
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We also used a similar dot-binding assay in conjunction with an antibody against PDGFR to address whether sennoside B can bind to the extracellular domain of PDGFR. Unexpectedly, specific binding was also observed between sennoside B and the PDGFR extracellular domain (Fig. 2B). To evaluate whether sennoside binding to PDGFR blocks ligand binding, we examined the retention of 125I-labeled PDGF-BB by mouse 3T3 cells in the presence or absence of sennoside B. Here it was found that sennoside B strongly inhibited PDGF-BB binding, with an estimated IC50 of 2.35 µM and a Ki of 0.883 µM (data
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not shown). We therefore suggest that sennoside B binds to both PDGF-BB and PDGFR and disturbs the ligand/receptor interaction. PDGF-BB-induced phosphorylation of PDGFR-β, STAT-5, AKT and ERK: inhibition by sennoside B We next examined the effects of sennoside B on downstream signaling induced by PDGF-BB in MG63 cells. Western blot analysis using phospho-specific antibodies revealed that the downstream
Fig. 6. Sennoside B inhibition of osteosarcoma cell metabolism and proliferation induced by PDGF-BB. (A) MG63 cells were precultured in serum-free medium for 24 h and stimulated with 20 ng/ml PDGF-BB in the absence or presence of sennoside B (0.3–5.0 µM). Cell metabolism/proliferation was assayed by a chromogenic MTT assay after 24 and 48 h. Absorbance (A570) was proportional to the number of viable cells. Data are expressed as means ± SEM (n = 5); ⁎⁎⁎P b 0.001 compared to PDGF-BB alone. The GI50 = 1.07 ± 0.126 µM was calculated by Grafit 5.0. (B) U-2OS cells were cultured in the absence or presence of sennoside B and assayed by MTT assay after 24 and 48 h. ###P b 0.001 compared to serum free medium (without PDGF-BB). ⁎P b 0.05, ⁎⁎⁎P b 0.001 compared to PDGF-BB alone. (C) Saos-2 cells were cultured in the absence or presence of sennoside B and assayed by MTT assay after 24 and 48 h. No significant differences were observed in Saos-2 cells.
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signaling molecules STAT-5, AKT and ERK were phosphorylated after addition of PDGF-BB (Fig. 3A). Sennoside B blocked PDGF-BBdependent phosphorylation of PDGFR-β, STAT-5, AKT and ERK in a concentration-dependent manner, but without altering the expression levels of these proteins (Fig. 3A). In contrast, although both EGF and FBS could induce STAT-5, AKT and ERK1/2 phosphorylation in MG63 cells, sennoside B pretreatment did not inhibit this phosphorylation (Fig. 3B, C). Sennoside B therefore abrogates ligand-induced activation of PDGFR and downstream signaling molecules (Fig. 3A). No significant inhibition of phosphorylation of c-KIT induced by stem cell factor (SCF) and KDR phosphorylation induced by vascular endothelial growth factor (VEGF) was observed at sennoside B concentrations below 10 µM (data not shown). Because sennoside B did not affect these other signaling pathways, we conclude that the inhibition in MG63 cells is specific for PDGF-BB and its receptor. Sennoside B slows the rate of PDGFR-β activation We studied the concentration- and time-dependence of sennoside B inhibition. MG63 cells were treated with PDGF-BB (20 ng/ml) and PDGFR-β tyrosine phosphorylation was recorded at different time points following ligand addition (Fig. 4A, B). PDGFR-β tyrosine phosphorylation reached a maximum 5–10 min after ligand stimulation, followed by a slow decline. We then explored whether sennoside B inhibition blocked or only delayed PDGF-BB stimulation. Sennoside B alone did not stimulate PDGFR-β phosphorylation under any condition tested (data not shown), and instead reduced receptor phosphorylation in an abolished-dependent manner. When measured 5 min after the addition of PDGF-BB plus sennoside B, 100 nM sennoside inhibited and 200 nM abolished PDGFR-β phosphorylation. When measured 1 h after cotreatment with both ligands, however, no inhibition of receptor phosphorylation was observed at these concentrations. Nevertheless, after 1 h of cotreatment with PDGF-BB and 500 nM sennoside B, receptor phosphorylation remained at a low level. These results indicate that sennoside B does not irreversibly block receptor activation but that, instead, sennoside slows the rate receptor activation in a concentration-dependent manner. Sennoside B inhibits osteosarcoma cell proliferation induced by PDGF-BB To investigate whether sennoside B can inhibit the proliferation of osteosarcoma cells, we used an MTT assay to evaluate the effects of sennoside B on PDGF-BB activation of cell growth and metabolism. Osteosarcoma cells MG63, U2-OS and Saos-2 were characterized by stimulating with PDGF-BB, in which Saos-2 cells were found to have low phosphorylated PDGFR-β and PDGFR expression (Fig. 5). When stimulated with PDGF-BB, the metabolic activity of growth-arrested MG63 cells increased, consistent with the initiation of cellular proliferation. This proliferative effect was observed 24–48 h after stimulation. Nanomolar concentration levels of sennoside B did not significantly inhibit proliferation (Fig. 6A), consistent with other results showing that sennosides do not interfere with MG63 viability at this concentration. Sennoside B also did not induce cytotoxicity (data not shown), indicating that blocking PDGFR signaling in these cells is not sufficient to induce cell death. In the functional studies reported here, therefore, sennoside B failed to inhibit cell proliferation at concentrations (~100 nM) that abolished PDGF-BB-dependent signaling. High concentrations of sennoside B (1 µM), however, did inhibit the proliferation of human MG63 cells induced by PDGF-BB (Fig. 6A). The concentration of sennoside B required to inhibit PDGF-BB-induced MG63 cell proliferation by 50% versus controls (GI50) was 1.07 ± 0.126 µM (calculated by Grafit 5.0). Similar results are observed in U-2OS cells, which were shown to secrete PDGF (Heldin et al. 1986; Ringbom-Anderson et al. 1995). The sennoside B anti-proliferation effect was observed 24 h after stimulation. We found a significant
difference in serum-free medium without PDGF-BB added (p b 0.001, Fig. 6B). Sennoside B alone inhibited the proliferation of U-2OS cells significantly, which was not observed in MG63 cells. As verification of the inhibition phenomenon, in Saos-2 osteosarcoma cell lines, in which PDGF-induced phosphorylation of PDGFRs is low, we did not observe a significant anti-proliferation effect between experimental groups (Fig. 6C). Moreover, PDGF-BB was unable to stimulate cell proliferation after serum deprivation because of the low PDGFR expression. Taken together, we demonstrate the anti-proliferation effect is directed against PDGF-induced proliferation (Fig. 6A, B, C). These results suggest that sennoside B has potential as an antiproliferation agent in PDGF-driven diseases. Discussion Several studies have indicated that emodin may exert anti-cancer effects via the induction of apoptosis in tumor cells. Although the mechanisms of action of diverse phyto-anthraquinones have yet to be elucidated, evidence is emerging that such old remedies can exert anti-tumor effects both in vivo and in vitro. Nevertheless, there have been no reports to date regarding the anti-cancer effects of sennosides A (meso) and B (trans), both glycosides of rhein homodianthrones. Platelet-derived growth factors are potent growth promoters in cells of mesenchymal origin, and PDGF binding to its receptor, a tyrosine kinase, activates a phosphorylation cascade that culminates in changes in nuclear gene expression. The growth factor modulates diverse processes including mitogenesis, differentiation, migration and cell survival (Valgeirsdottir et al. 1998). Constitutive mitogenic signaling due to overexpression of receptor protein tyrosine kinases or autocrine production of mitogenic growth factors has been implicated in several proliferative diseases, including tumors of epithelial and mesenchymal origin (Panek et al. 1997). Platelet-derived growth factor has been specifically implicated in malignant diseases. For instance, overexpression of PDGF and/or PDGF receptors is often observed in human tumors, including glioblastomas and sarcomas (Pietras et al. 2003; Levitzki 2004; Board and Jayson 2005; Alvarez et al. 2006). Moreover, PDGF is a potent inducer of growth and motility in several cell types including fibroblasts, endothelial cells and smooth muscle cells. In melanoma, PDGF-BB has been suggested to modulate the stromal fibroblast microenvironment, favoring melanoma growth, invasion and metastasis. Several types of PDGF antagonists have been described, including antibodies directed against ligands or against the extracellular domain of the receptor. Low molecular weight PDGFR inhibitors have also been developed, exemplified by SU11248, a multiple receptor kinase inhibitor. This compound has been proposed to inhibit PDGFR-β activation by mimicking the conformation of its ATP-binding site. In addition, epigallocatechin-3 gallate (EGCG) (Weber et al. 2004), lycopene (Lo et al. 2007) and luteolin (Kim et al. 2005), natural substances from green tea, tomato and celery, respectively, have been shown to bind to the PDGFR-β at micromolar concentrations and inhibit its phosphorylation in rat aortic vascular smooth muscle cells (VSMCs). A previous study examined the mechanism by which EGCG inhibits the induction of cell signaling and mitogenesis by PDGF-BB. It was proposed that EGCG, incorporated into cell-surface membranes, leads to a reversible binding of PDGF-BB to non-receptor target sites, thereby reducing PDGF binding to its receptor(s) (Liang et al. 1999; Weber et al. 2004). It has also been reported that synthetic PDGFbinding agents can antagonize PDGFR activation. The growth factor binding molecules GFB-111 and GFB-204 bind to PDGF with a high degree of selectivity. These molecules inhibit PDGFR autophosphorylation and downstream signaling, and can abolish tumor growth in nude mice grafted with different tumor cell lines, possibly by interfering with angiogenesis (Blaskovich et al. 2000; Sun et al. 2005). In the present study, we report that sennoside B can inhibit PDGF signaling in human MG63 osteosarcoma cells. Our results indicate that
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this inhibition takes place through direct binding of sennoside B to PDGF-BB and to the PDGFR extracellular domain. First, incubation of PDGF-BB with sennoside B resulted in a marked inhibition (IC50 = 19.2 ± 3.6 nM) of PDGFR phosphorylation, while sequential treatment of cells with sennoside B then PDGF-BB inhibited PDGFR phosphorylation at concentrations in the micromolar range (IC50 around 500 nM, data not shown). In these cells, PDGF-BB induced the phosphorylation of PDGFR-β and of downstream signaling molecules STAT-5, AKT and ERK1/2. Phosphorylation of all these molecules was inhibited by sennoside B. We also examined the selectivity of sennoside B in PDGF-AA. Sennoside B inhibits PDGF-AA-induced phosphorylation of PDGFR-α in MG63 cells at the same level (IC50 = 12.5 ± 22.6 nM, data not shown). These effects, however, were specific to PDGFs, as signaling by other growth factors was unaffected. Second, dot-binding assays demonstrated that sennoside B is retained by immobilized PDGF-BB and by the PDGFR's extracellular domain. Although there may be other interpretations, we suggest that, despite the lack of obvious sequence homology between the two proteins, sennoside B can bind to both PDGF-BB and the PDGFR and disturbs the ligand/receptor interaction. Third, high concentrations of sennoside B inhibited PDGF-BB-induced cell proliferation. This is the first report demonstrating that sennoside B specifically binds to PDGFBB and the PDGFR's extracellular domain to inhibit downstream signaling and proliferative effects. It was previously proposed that the binding of PDGF-BB to its receptor is predominantly mediated by hydrophobic and basic amino acid residues found in the loops connecting the β-sheets of the PDGF peptide (Oefner et al. 1992). In the present study, the meso and trans forms of sennoside molecules showed different binding affinities. We observed that only the trans form (sennoside B) was able to bind to PDGF-BB and the PDGFR-β extracellular domain and inhibit downstream signaling pathways. The stereo-selectivity of this binding may permit molecules to be designed that can selectively target PDGF-BB or the PDGFR's extracellular domain. Although our results indicate that low concentrations (~100 nM) of sennoside B can abolish PDGFR-β signaling by binding to the receptor and its ligand, higher concentrations (~1 µM) were required to inhibit cell proliferation. This could suggest that the inhibition of cell growth and the blockade of PDGF/PDGFR signaling may involve more than one mechanism. Further experiments will be required to address this possibility. Nonetheless, understanding the nature of the PDGF-BB-sennoside B interaction may lead to the development of a new class of anti-cancer drugs that irreversibly bind to PDGF-BB and have a therapeutic potential in a wide spectrum of human cancers. It is notable that, because sennoside B binds to PDGF-BB and the extracellular domain of the PDGFR, there is no requirement for the compound to enter cells, favoring a therapeutic use at low concentrations. Most tyrosine kinase inhibitors developed to date, in contrast, must enter the cell to target the kinase ATP-binding site. Such inhibitors suffer from a further drawback in that a large number of structurally similar ATP-binding sites are likely to exist within the cell. The clinical outcome of treatment with a molecule such as sennoside B may therefore be very different from the outcome of using inhibitors that target PDGF receptor tyrosine kinases. Conclusions We report that sennoside B is a specific inhibitor of PDGFR-β activation by PDGF-BB and down-regulates downstream signaling and cell proliferation. This molecule may be an attractive candidate for therapeutic use in diseases characterized by excessive PDGF-dependent cell proliferation, including cancer, atherosclerosis and restenosis. Although the specific binding of sennoside B to PDGF-BB/ PDGFR-β clearly interferes with signaling and proliferation in MG63 cells, it is still unclear how sennoside B interacts with two different
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proteins that lack evident sequence homology. Indeed, the molecular mechanism by which sennoside B “traps” the PDGF-BB ligand and/or “antagonizes” its receptor may emerge as a basis for the development of other similar ligand-trapping or antagonist receptor molecules, thereby leading to treatment options for neoplastic conditions in which PDGF signaling is known to play a central role. Conflict of interest statement There are no conflicts of interest. Acknowledgement This work was supported by the Ministry of Economic Affairs, Taiwan. References Alvarez RH, Kantarjian HM, Cortes JE. Biology of platelet-derived growth factor and its involvement in disease. Mayo Clinic Proceedings 81 (9), 1241–1257, 2006. Blaskovich MA, Lin Q, Delarue FL, Sun J, Park HS, Coppola D, Hamilton AD, Sebti AM. Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice. Nature Biotechnology 18 (10), 1065–1070, 2000. Board R, Jayson GC. Platelet-derived growth factor receptor (PDGFR): a target for anticancer therapeutics. Drug Resistance Updates: Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy, vol. 8(1–2), pp. 75–83, 2005. Cardenas C, Quesada AR, Medina MA. Evaluation of the anti-angiogenic effect of aloeemodin. Cellular and Molecular Life Sciences 63 (24), 3083–3089, 2006. Graves DT, Antoniades HN, Williams SR, Owen AJ. Evidence for functional plateletderived growth factor receptors on MG-63 human osteosarcoma cells. Cancer Research 44 (7), 2966–2970, 1984. Heldin CH, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature 319 (6), 511–514, 1986. Huang Q, Shen HM, Ong CN. Emodin inhibits tumor cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway. Cellular and Molecular Life Sciences 62 (10), 1167–1175, 2005. Huang Q, Lu G, Shen HM, Chung MC, Ong CN. Anti-cancer properties of anthraquinones from rhubarb. Medicinal Research Reviews 27 (5), 609–630, 2007. Kaneshiro T, Morioka T, Inamine M, Kinjo T, Arakaki J, Chiba I, Sunagawa N, Suzui M, Yoshimi N. Anthraquinone derivative emodin inhibits tumor-associated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation. European Journal of Pharmacology 553 (1–3), 46–53, 2006. Kim JH, Jin YR, Park BS, Kim TJ, Kim SY, Lim Y, Hong JT, Yoo HS, Yun YP. Luteolin prevents PDGF-BB-induced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation. Biochemical Pharmacology 69 (12), 1715–1721, 2005. Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine & Growth Factor Reviews 15 (4), 229–235, 2004. Liang YC, Chen YC, Lin YL, Lin-Shiau SY, Ho CT, Lin JK. Suppression of extracellular signals and cell proliferation by the black tea polyphenol, theaflavin-3,3′-digallate. Carcinogenesis 20 (4), 733–736, 1999. Lo HM, Hung CF, Tseng YL, Chen BH, Jian JS, Wu WB. Lycopene binds PDGF-BB and inhibits PDGF-BB-induced intracellular signaling transduction pathway in rat smooth muscle cells. Biochemical Pharmacology 74 (1), 54–63, 2007. Lu Y, Zhang J, Qian J. The effect of emodin on VEGF receptors in human colon cancer cells. Cancer Biotherapy & Radiopharmaceuticals 23 (2), 222–228, 2008. Oefner C, D'Arcy A, Winkler FK, Eggimann B, Hosang M. Crystal structure of human platelet-derived growth factor BB. The EMBO Journal 11 (11), 3921–3926, 1992. Panek RL, Lu GH, Klutchko SR, Batley BL, Dahring TK, Hamby JM, Hallak H, Doherty AM, Keiser JA. In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor. The Journal of Pharmacology and Experimental Therapeutics 283 (3), 1433–1444, 1997. Paukku K, Valgeirsdottir S, Saharinen P, Bergman M, Heldin CH, Silvennoinen O. Platelet-derived growth factor (PDGF)-induced activation of signal transducer and activator of transcription (Stat) 5 is mediated by PDGF beta-receptor and is not dependent on c-src, fyn, jak1 or jak2 kinases. The Biochemical Journal 345 (3), 759–766, 2000. Pecere T, Gazzola MV, Mucignat C, Parolin C, Vecchia FD, Cavaggioni A, Basso G, Diaspro A, Salvato B, Carli M, Palu G. Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors. Cancer Research 60 (11), 2800–2804, 2000. Pietras K, Sjoblom T, Rubin K, Heldin CH, Ostman A. PDGF receptors as cancer drug targets. Cancer Cells 3 (5), 439–443, 2003. Ringbom-Anderson T, Sandberg M, Andersson G, Akerman KE. Phenotypic modification of human osteosarcoma cells with the phorbol ester 1 2-O-tetradecanoylphorbol-1 3-acetate. Cell Growth & Differentiation 457 (6), 457–464, 1995. Sandy J, Davies M, Prime S, Farndale R. Signal pathways that transduce growth factorstimulated mitogenesis in bone cells. Bone 23 (1), 17–26, 1998.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Sennoside B inhibits PDGF receptor signaling and cell proliferation induced by PDGF-BB in human osteosarcoma cells Yen-Chun Chen a,b,⁎, Chia-Ni Chang a, Hui-Chun Hsu a, Shu-Jiau Chiou a, Lain-Tze Lee a, Tzong-Hsiung Hseu b a b
Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan
a r t i c l e
i n f o
Article history: Received 19 December 2008 Accepted 4 April 2009 Keywords: Sennoside B Osteosarcoma Signaling pathway Platelet-derived growth factor (PDGF) Platelet-derived growth factor receptor (PDGFR)
a b s t r a c t Aims: To address the possibility that sennoside B inhibition of cell proliferation is mediated via interference with platelet-derived growth factor (PDGF) signaling. Main methods: Human osteosarcoma MG63 cells were treated with PDGF in the presence or absence of sennoside B. Activation of the PDGF signaling pathway was monitored using western immunoblotting with specific antibodies against the PDGF receptor, phosphotyrosine and components of the downstream signaling cascade. Activation of cell metabolism and proliferation was assessed by chromogenic reduction of MTT. Key findings: Sennoside B was found to inhibit PDGF-BB-induced phosphorylation of the PDGF receptor (PDGFR) in human MG63 osteosarcoma cells. Downstream signaling was also affected; pre-incubation of PDGF-BB with sennoside B inhibited the phosphorylation of pathway components including Ak strain transforming protein (AKT), signal transducer and activator of transcription 5 (STAT-5) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further, we found that sennoside B can bind directly to the extracellular domains of both PDGF-BB and the PDGF-β receptor (PDGFR-β). The effect was specific for sennoside B; other similar compounds including aloe-emodin, rhein and the meso isomer (sennoside A) failed to inhibit PDGFR activation or downstream signaling. Sennoside B also inhibited PDGF-BB stimulation of MG63 cell proliferation. Significance: These results indicate that sennoside B can inhibit PDGF-stimulated cell proliferation by binding to PDGF-BB and its receptor and by down-regulating the PDGFR-β signaling pathway. Sennoside B is therefore of potential utility in the treatment of proliferative diseases in which PDGF signaling plays a central role. © 2009 Elsevier Inc. All rights reserved.
Introduction Sennosides have been used as natural, safe laxatives in traditional and modern systems of medicine. In addition, aloe-emodin and rhein, degradation products of sennosides, have been reported to inhibit tumor cell growth (Cardenas et al. 2006; Huang et al. 2007). Doxorubicin, a structurally related anthraquinone glycoside, is an important antineoplastic drug (Huang et al. 2007), while emodin has been reported to induce apoptotic cell death in proliferating cells (Pecere et al. 2000; Huang et al. 2007). In view of the planar structure of these molecules, it has been suggested that anthraquinones may be able to intercalate into DNA and that this could underlie their cytoxicity.
⁎ Corresponding author. Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan. Tel.: +886 3 5732561; fax: +886 3 5732359. E-mail address: [email protected] (Y.-C. Chen). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.04.003
Sennosides and related molecules may also exert their effects by interfering with the activation of cell-surface receptors. Emodin preferentially suppresses the phosphorylation of the human epidermal growth factor receptor 2 (HER-2/neu) in breast cancer cells (Zhang et al. 1995, 1998). Tyrosine kinase phosphorylation induced by epidermal growth factor (EGF) was also suppressed (Huang et al. 2005). Furthermore, it has been suggested that the anti-angiogenic activity of emodin may be due to inhibition of the phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2) and of downstream molecules (Kaneshiro et al. 2006; Srinivas et al. 2007; Lu et al. 2008). Platelet-derived growth factors (PDGFs) and their receptors have been implicated in the pathogenesis of a number of tumor types and play an important role in angiogenesis (Board and Jayson 2005; Pietras et al. 2003; Alvarez et al. 2006). Extensive experimental data highlight the potential therapeutic advantage of targeting the platelet-derived growth factor receptor (PDGFR). In humans, the PDGF signaling network consists of four ligands, PDGFs A through D. These factors interact with two receptors, PDGFR-α and PDGFR-β. Ligand-bound receptors undergo autophosphorylation, resulting in the activation of
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multiple intracellular signaling cascades. Regarding PDGF, the best characterized intracellular signaling cascade comprises mitogenactivated protein kinase kinase/extracellular regulated kinase (MEK/ ERK), phosphatidylinositol 3-kinase/Ak strain transforming protein (PI3K/AKT) and signal transducer and activator of transcription 5 (STAT-5) (Sandy et al. 1998; Graves et al. 1984; Thomas et al. 1997; Paukku et al. 2000; Valgeirsdottir et al. 1998; Zhang et al. 2007). Because this pathway has been widely implicated in the regulation of tumor growth, agents interfering with PDGF-BB signaling have great potential for the development of new anti-neoplastic agents. MG63 osteosarcoma cells present a functional PDGFR but do not secrete PDGF-like mitogens (Graves et al. 1984); these cells have been used for many years in osteoblast research (Sandy et al. 1998). In the present study, we used MG63 cells to investigate the effects of sennoside B on PDGF-BB-induced signaling. Our results indicate that sennoside B specifically binds to PDGF-BB and PDGFR and can inhibit PDGFR activation and downstream signaling induced by PDGF-BB. In a cell-based functional study, we also found that sennoside B can inhibit MG63 proliferation induced by PDGF-BB. Sennoside B may have significant potential for anti-cancer therapy.
were then cultured for 24 h, and then incubated in serum-free medium overnight before preparation of cell lysates.
Materials and methods
Dot-binding assay
Materials
Aliquots (2.5 µl) of vehicle (DMSO) and different concentrations of sennoside, PDGF-BB or PDGFR-β/Fc chimera in vehicle were spotted onto gridded PVDF membranes and air-dried. Membranes were blocked with BSA (5% in PBS) for 0.5 h. After washing in PBS, membranes were incubated with PDGF-BB or the PDGFR-β/Fc chimera (1 µg/ml) for 1 h at room temperature in PBS. Following a brief wash, the membrane was incubated with anti-PDGF-BB or PDGFR-β extracellular domain antibody (2 µg/ml in PBS containing 1% BSA) for 1 h at room temperature. After another brief wash, the membrane was incubated with HRP-conjugated secondary antibody and developed by ECL.
Sennoside A, Sennoside B, aloe-emodin and rhein were obtained from Sigma Chemical Co (St. Louis, MO, USA). These compounds were dissolved in DMSO (Sigma Chemical Co) and further diluted in Dulbecco's Modified Eagle's Medium (DMEM) without fetal bovine serum (FBS; Biological Industries). Penicillin, DMEM, and streptomycin were obtained from GIBCO™/Invitrogen Life Technologies (CA, USA). Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma Chemical Co. Antibodies raised against PDGFR-β and anti-phosphotyrosine antibody (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotech (Lake Placid, NY, USA). Recombinant human PDGF-BB, the PDGFR-β/Fc chimera, EGF and antibodies raised against native PDGF-BB, PDGFR-β and ERK1/2, AKT, STAT5 were from R&D Systems (MN, USA). Antibodies raised against phospho-ERK1/2 (Thr202/Tyr204), phospho-AKT (Ser473, Thr308), phospho-STAT5 (Tyr694), Akt and horseradish peroxidase (HRP)-linked anti-goat and anti-mouse IgG antibodies, as well as the cell lysis buffer, were from Cell Signaling Technology (Danvers, MA, USA). Antibodies raised against EGFR were from LabVision (Fremont CA, USA). Pre-stained protein markers were from Fermentas Life Sciences (Glen Burnie, MD, USA). SuperSignal® West Pico HRP chemiluminescence substrate, the Micro BCA™ Protein Assay Reagent protein assay kit and Western Blot Stripping Buffer were from Pierce (Rockford, IL, USA). Nitrocellulose membranes (Hybond-C Extra) were from Amersham Biosciences (Sunnyvale, CA, USA), and PVDF membranes were from Millipore (Billerica, MA, USA).
Cell lysate preparation and western blot analysis Osteosarcoma cells were cultured with PDGF-BB and sennosides for the times and at the different concentrations indicated in the figure legends. Lysates were prepared by the addition of cell lysis buffer and were clarified by centrifugation (14,000 g, 10 min, 4 °C). Lysate protein contents were quantified using the Pierce protein assay kit according to the manufacturer's instructions, using bovine serum albumin (BSA) as the reference standard. Proteins were separated by electrophoresis on SDS-polyacrylamide gels, electroblotted onto PVDF or NC membranes and probed using primary anti-phosphotyrosine, anti-p-ERK, anti-p-AKT, and anti-p-STAT5 antibodies. Immunoblots were developed using enhanced chemiluminescence (ECL) with HRP-labeled secondary antibodies and the SuperSignal® HRP substrate. The membranes were stripped (Western Blot Stripping Buffer), washed and reprobed with anti-PDGFR-β, anti-ERK1/2, anti-AKT or anti-STAT5 antibodies and developed as described above.
Cell proliferation assays Activation of cell metabolism and proliferation as reflected by NAD-dependent dehydrogenase activity was determined via chromogenic reduction of MTT. Osteosarcoma cells in a 96-well plates were allowed to reach 50% confluence (5 × 103 cells/well), starved for 24 h and incubated with different concentrations of sennoside B or vehicle (final concentration 0.5% DMSO) in the absence or presence of PDGFBB (20 ng/ml) in quintuplicate. After 24 and 48 h, cells were incubated with 0.5 mg/ml MTT for 2 h at 37 °C. Formazan crystals resulting from MTT reduction were dissolved by adding 200 µl DMSO and gently
Osteosarcoma cell culture Human osteoblast-like cells MG63 (BCRC-60279) and U-2OS (BCRC-60187) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Saos-2 cells (HTB-85) were obtained from The American Type Culture Collection (VA, USA) and grown in DMEM supplemented with 5–10% FBS, penicillin (100 units/ ml) and streptomycin (100 µg/ml). Unless otherwise indicated, cells reaching 80–90% confluence were starved and synchronized in DMEM without FBS at 37 °C for 24 h before further analysis. For western blot analysis, osteosarcoma cells were seeded into 6-well plates (Orange Scientific, Belgium) such that each well contained 1 ml of a 5 × 105 cells/ml suspension (as assessed by hemocytometry). Cells
Fig. 1. PDGFR phosphorylation in MG63 cells treated with PDGF-BB and sennosides A and B. MG63 cells were treated with medium containing PDGF-BB (20 ng/ml), with vehicle or the indicated concentrations of sennoside A or B for 5 min. Multiple kinase inhibitor SU11248 was included as a positive control. Cell lysates were collected, and protein tyrosine phosphorylation (p-Tyr) and total PDGFR-β were determined by western blotting. The 180-kDa phosphotyrosine band (detected by PY99) in the upper panel was identified as tyrosine-phosphorylated PDGFR. Relative band densities were quantified by scanning densitometry and normalized to the relative value of total PDGFR-β. The data shown are representative of three or more independent experiments.
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Fig. 2. Dot-binding assay of the interaction between PDGF-BB and the PDGFR's extracellular domain with immobilized sennoside B. Compounds were suspended in DMSO vehicle (Veh) and applied to a PVDF membrane. After incubation with PDGF-BB (panel A) or the PDGF extracellular domain (panel B), membranes were developed with peroxidase-linked secondary antibodies specific for PDGF-BB or the PDGF-β extracellular domain. Binding was detected using a chemiluminescent peroxidase substrate. ⁎Positive controls were immobilized PDGF-BB and the PDGF-β extracellular domain fused to Fc (extraPDGFR-IgG).
agitating for 20 min. The absorbance of the supernatant was then measured spectrophotometrically at 570 nm using an ELISA reader.
Results Sennoside B selectively inhibits PDGF-BB-induced PDGFR phosphorylation in osteosarcoma cells
Statistical analysis Data were expressed as means ± the standard error of the mean (SEM). Comparison of the means and SEMS used the unpaired, twotailed Student's t test. P-values b0.05 were considered significant. All statistical calculations were performed using GraphPad prism v5.0 (GraphPad Software Inc., CA, USA) or Grafit v5.0 (Erithacus Software Ltd., UK).
Several anthraquinones, including emodin, aloe-emodin and rhein, have been reported to exert anti-neoplastic effects by inducing apoptosis. Potential mechanisms of apoptosis induction include inhibition of VEGFR 2 (KDR/Flk-1) phosphorylation and suppression of HER2/neu tyrosine kinase autophosphorylation. The effects on PDGFR phosphorylation, however, have not been previously addressed. To investigate whether sennoside B can modulate PDGFR signaling, human
Fig. 3. Effects of sennoside B on signaling induced by PDGF-BB, EGF and FBS in MG63 cells. Panel A, MG63 cells treated with medium containing PDGF-BB (20 ng/ml); Panel B, cells treated with EGF (20 ng/ml); Panel C, cells treated with 10% FBS. All cells were incubated in the absence or presence of increasing concentrations (1–1000 nM) of sennoside B for 5 min (panel A) or 15 min (panels B, C). The levels of protein and protein phosphorylation for PDGFR, EGFR, STAT-5, AKT and ERK were analyzed by western blotting. Each blot is representative of two to three independent experiments.
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Fig. 4. Time course and concentration-dependence of sennoside B down-regulation of PDGFR-β phosphorylation in MG63 cells. Confluent cells were co-cultured in the absence or presence of sennoside B (100–500 nM) in serum-free medium for the time indicated. Levels of PDGFR-β and tyrosine-phosphorylated receptor (p-Tyr) were determined using 7.5% SDS-PAGE and immunoblotting. Relative band densities were quantified by scanning densitometry and normalized to total protein levels. Each blot is representative of two to three independent experiments.
osteoblast-like MG63 cells were treated with PDGF-BB in the presence or absence of different concentrations of sennoside B and PDGFR-β tyrosine phosphorylation was measured. We also studied the effects of the related compounds sennoside A, aloe-emodin and rhein. Expression and tyrosine phosphorylation of osteosarcoma cell PDGFRs were determined by western blotting using primary antibodies specific for PDGFR-β and phosphotyrosine, respectively. MG63 cells were found to express high levels of PDGFR-β (Fig. 1). Stimulation with PDGF-BB resulted in a significant increase in the level of tyrosine phosphorylation in a molecular weight band identified as PDGFR-β (Fig. 1, lane 2). Incubation of PDGF-BB with sennoside B resulted in a marked inhibition of PDGF-BB-induced PDGFR-β phosphorylation (Fig. 1, lanes 7 to 9). The extent of inhibition was concentration-dependent; the IC50 (19.2 ± 3.6 nM) was comparable to the inhibition associated with the multiple kinase inhibitor SU11248 (lane 3). Sennoside B therefore appears to be a potent inhibitor of PDGFR-β activation. In contrast, sennoside A, the meso form of the sennoside molecule, only minimally inhibited PDGF-BBinduced PDGFR-β phosphorylation (Fig. 1, lanes 4 and 5) and was far less potent than the trans form, sennoside B. Aloe-emodin and rhein at concentrations of 10 µM did not show any inhibitory effect on PDGFRβ phosphorylation induced by PDGF-BB (data not shown).
by 50% (data not shown). This result argues against rapid diffusion, and instead suggests that sennoside B binds to other cellular target sites. We hypothesized that sennoside B might antagonize PDGFR-β activation by binding to PDGF-BB or to the PDGFR extracellular domain. Accordingly, we used a direct dot-binding assay to study possible binding of sennoside B to PDGF-BB or to the PDGFR extracellular domain. Sennoside A, sennoside B, aloe-emodin, rhein and recombinant PDGFBB were immobilized on a PVDF membrane. After incubation with PDGF-BB, the membrane was incubated with an antibody directed against PDGF-BB and then developed. Control immobilized PDGF-BB was efficiently recognized by the antibody (Fig. 2A, row 5). The antibody also detected immobilized PDGF-BB in sennoside B spots, and the antibody binding was dependent on the concentration of sennoside B solution applied (Fig. 2A, row 2). No reactivity was observed with sennoside A, aloe-emodin or rhein at 10 µM (Fig. 2A, rows 1, 4, and 3). These results indicate that PDGF-BB specifically and directly binds to sennoside B.
Sennoside B binds to the PDGF-BB and PDGFR extracellular domains Inhibition of PDGF-BB-induced PDGFR-β phosphorylation in MG63 cells was only observed when PDGF-BB was co-incubated with sennoside B. When cells were first treated with sennoside B followed by PDGF-BB, no inhibition of PDGFR-β phosphorylation was observed until the concentration reached ~ 500 nM (data not shown). This suggested that sennoside B could reversibly bind to PDGF or could rapidly diffuse into the culture medium. We also observed that when MG63 cells were pre-treated with sennoside B (1 µM) followed by the removal of sennoside by changing the culture medium and the addition of PDGF-BB, the extent of inhibition was only reduced
Fig. 5. PDGFR phosphorylation in osteosarcoma cells treated with PDGF-BB and sennoside B. Osteosarcoma cells were treated with medium containing PDGF-BB (20 ng/ml), with vehicle or 10 μM sennoside B for 5 min. Cell lysates were collected and protein tyrosine phosphorylation (p-Tyr) and total PDGFR-β determined by western blotting. The 180-kDa phosphotyrosine band (detected by PY99) in the upper panel was identified as tyrosine-phosphorylated PDGFR. Relative band densities were quantified by scanning densitometry and normalized to the relative value of total PDGFR-β. The data shown are representative of three or more independent experiments.
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We also used a similar dot-binding assay in conjunction with an antibody against PDGFR to address whether sennoside B can bind to the extracellular domain of PDGFR. Unexpectedly, specific binding was also observed between sennoside B and the PDGFR extracellular domain (Fig. 2B). To evaluate whether sennoside binding to PDGFR blocks ligand binding, we examined the retention of 125I-labeled PDGF-BB by mouse 3T3 cells in the presence or absence of sennoside B. Here it was found that sennoside B strongly inhibited PDGF-BB binding, with an estimated IC50 of 2.35 µM and a Ki of 0.883 µM (data
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not shown). We therefore suggest that sennoside B binds to both PDGF-BB and PDGFR and disturbs the ligand/receptor interaction. PDGF-BB-induced phosphorylation of PDGFR-β, STAT-5, AKT and ERK: inhibition by sennoside B We next examined the effects of sennoside B on downstream signaling induced by PDGF-BB in MG63 cells. Western blot analysis using phospho-specific antibodies revealed that the downstream
Fig. 6. Sennoside B inhibition of osteosarcoma cell metabolism and proliferation induced by PDGF-BB. (A) MG63 cells were precultured in serum-free medium for 24 h and stimulated with 20 ng/ml PDGF-BB in the absence or presence of sennoside B (0.3–5.0 µM). Cell metabolism/proliferation was assayed by a chromogenic MTT assay after 24 and 48 h. Absorbance (A570) was proportional to the number of viable cells. Data are expressed as means ± SEM (n = 5); ⁎⁎⁎P b 0.001 compared to PDGF-BB alone. The GI50 = 1.07 ± 0.126 µM was calculated by Grafit 5.0. (B) U-2OS cells were cultured in the absence or presence of sennoside B and assayed by MTT assay after 24 and 48 h. ###P b 0.001 compared to serum free medium (without PDGF-BB). ⁎P b 0.05, ⁎⁎⁎P b 0.001 compared to PDGF-BB alone. (C) Saos-2 cells were cultured in the absence or presence of sennoside B and assayed by MTT assay after 24 and 48 h. No significant differences were observed in Saos-2 cells.
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signaling molecules STAT-5, AKT and ERK were phosphorylated after addition of PDGF-BB (Fig. 3A). Sennoside B blocked PDGF-BBdependent phosphorylation of PDGFR-β, STAT-5, AKT and ERK in a concentration-dependent manner, but without altering the expression levels of these proteins (Fig. 3A). In contrast, although both EGF and FBS could induce STAT-5, AKT and ERK1/2 phosphorylation in MG63 cells, sennoside B pretreatment did not inhibit this phosphorylation (Fig. 3B, C). Sennoside B therefore abrogates ligand-induced activation of PDGFR and downstream signaling molecules (Fig. 3A). No significant inhibition of phosphorylation of c-KIT induced by stem cell factor (SCF) and KDR phosphorylation induced by vascular endothelial growth factor (VEGF) was observed at sennoside B concentrations below 10 µM (data not shown). Because sennoside B did not affect these other signaling pathways, we conclude that the inhibition in MG63 cells is specific for PDGF-BB and its receptor. Sennoside B slows the rate of PDGFR-β activation We studied the concentration- and time-dependence of sennoside B inhibition. MG63 cells were treated with PDGF-BB (20 ng/ml) and PDGFR-β tyrosine phosphorylation was recorded at different time points following ligand addition (Fig. 4A, B). PDGFR-β tyrosine phosphorylation reached a maximum 5–10 min after ligand stimulation, followed by a slow decline. We then explored whether sennoside B inhibition blocked or only delayed PDGF-BB stimulation. Sennoside B alone did not stimulate PDGFR-β phosphorylation under any condition tested (data not shown), and instead reduced receptor phosphorylation in an abolished-dependent manner. When measured 5 min after the addition of PDGF-BB plus sennoside B, 100 nM sennoside inhibited and 200 nM abolished PDGFR-β phosphorylation. When measured 1 h after cotreatment with both ligands, however, no inhibition of receptor phosphorylation was observed at these concentrations. Nevertheless, after 1 h of cotreatment with PDGF-BB and 500 nM sennoside B, receptor phosphorylation remained at a low level. These results indicate that sennoside B does not irreversibly block receptor activation but that, instead, sennoside slows the rate receptor activation in a concentration-dependent manner. Sennoside B inhibits osteosarcoma cell proliferation induced by PDGF-BB To investigate whether sennoside B can inhibit the proliferation of osteosarcoma cells, we used an MTT assay to evaluate the effects of sennoside B on PDGF-BB activation of cell growth and metabolism. Osteosarcoma cells MG63, U2-OS and Saos-2 were characterized by stimulating with PDGF-BB, in which Saos-2 cells were found to have low phosphorylated PDGFR-β and PDGFR expression (Fig. 5). When stimulated with PDGF-BB, the metabolic activity of growth-arrested MG63 cells increased, consistent with the initiation of cellular proliferation. This proliferative effect was observed 24–48 h after stimulation. Nanomolar concentration levels of sennoside B did not significantly inhibit proliferation (Fig. 6A), consistent with other results showing that sennosides do not interfere with MG63 viability at this concentration. Sennoside B also did not induce cytotoxicity (data not shown), indicating that blocking PDGFR signaling in these cells is not sufficient to induce cell death. In the functional studies reported here, therefore, sennoside B failed to inhibit cell proliferation at concentrations (~100 nM) that abolished PDGF-BB-dependent signaling. High concentrations of sennoside B (1 µM), however, did inhibit the proliferation of human MG63 cells induced by PDGF-BB (Fig. 6A). The concentration of sennoside B required to inhibit PDGF-BB-induced MG63 cell proliferation by 50% versus controls (GI50) was 1.07 ± 0.126 µM (calculated by Grafit 5.0). Similar results are observed in U-2OS cells, which were shown to secrete PDGF (Heldin et al. 1986; Ringbom-Anderson et al. 1995). The sennoside B anti-proliferation effect was observed 24 h after stimulation. We found a significant
difference in serum-free medium without PDGF-BB added (p b 0.001, Fig. 6B). Sennoside B alone inhibited the proliferation of U-2OS cells significantly, which was not observed in MG63 cells. As verification of the inhibition phenomenon, in Saos-2 osteosarcoma cell lines, in which PDGF-induced phosphorylation of PDGFRs is low, we did not observe a significant anti-proliferation effect between experimental groups (Fig. 6C). Moreover, PDGF-BB was unable to stimulate cell proliferation after serum deprivation because of the low PDGFR expression. Taken together, we demonstrate the anti-proliferation effect is directed against PDGF-induced proliferation (Fig. 6A, B, C). These results suggest that sennoside B has potential as an antiproliferation agent in PDGF-driven diseases. Discussion Several studies have indicated that emodin may exert anti-cancer effects via the induction of apoptosis in tumor cells. Although the mechanisms of action of diverse phyto-anthraquinones have yet to be elucidated, evidence is emerging that such old remedies can exert anti-tumor effects both in vivo and in vitro. Nevertheless, there have been no reports to date regarding the anti-cancer effects of sennosides A (meso) and B (trans), both glycosides of rhein homodianthrones. Platelet-derived growth factors are potent growth promoters in cells of mesenchymal origin, and PDGF binding to its receptor, a tyrosine kinase, activates a phosphorylation cascade that culminates in changes in nuclear gene expression. The growth factor modulates diverse processes including mitogenesis, differentiation, migration and cell survival (Valgeirsdottir et al. 1998). Constitutive mitogenic signaling due to overexpression of receptor protein tyrosine kinases or autocrine production of mitogenic growth factors has been implicated in several proliferative diseases, including tumors of epithelial and mesenchymal origin (Panek et al. 1997). Platelet-derived growth factor has been specifically implicated in malignant diseases. For instance, overexpression of PDGF and/or PDGF receptors is often observed in human tumors, including glioblastomas and sarcomas (Pietras et al. 2003; Levitzki 2004; Board and Jayson 2005; Alvarez et al. 2006). Moreover, PDGF is a potent inducer of growth and motility in several cell types including fibroblasts, endothelial cells and smooth muscle cells. In melanoma, PDGF-BB has been suggested to modulate the stromal fibroblast microenvironment, favoring melanoma growth, invasion and metastasis. Several types of PDGF antagonists have been described, including antibodies directed against ligands or against the extracellular domain of the receptor. Low molecular weight PDGFR inhibitors have also been developed, exemplified by SU11248, a multiple receptor kinase inhibitor. This compound has been proposed to inhibit PDGFR-β activation by mimicking the conformation of its ATP-binding site. In addition, epigallocatechin-3 gallate (EGCG) (Weber et al. 2004), lycopene (Lo et al. 2007) and luteolin (Kim et al. 2005), natural substances from green tea, tomato and celery, respectively, have been shown to bind to the PDGFR-β at micromolar concentrations and inhibit its phosphorylation in rat aortic vascular smooth muscle cells (VSMCs). A previous study examined the mechanism by which EGCG inhibits the induction of cell signaling and mitogenesis by PDGF-BB. It was proposed that EGCG, incorporated into cell-surface membranes, leads to a reversible binding of PDGF-BB to non-receptor target sites, thereby reducing PDGF binding to its receptor(s) (Liang et al. 1999; Weber et al. 2004). It has also been reported that synthetic PDGFbinding agents can antagonize PDGFR activation. The growth factor binding molecules GFB-111 and GFB-204 bind to PDGF with a high degree of selectivity. These molecules inhibit PDGFR autophosphorylation and downstream signaling, and can abolish tumor growth in nude mice grafted with different tumor cell lines, possibly by interfering with angiogenesis (Blaskovich et al. 2000; Sun et al. 2005). In the present study, we report that sennoside B can inhibit PDGF signaling in human MG63 osteosarcoma cells. Our results indicate that
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this inhibition takes place through direct binding of sennoside B to PDGF-BB and to the PDGFR extracellular domain. First, incubation of PDGF-BB with sennoside B resulted in a marked inhibition (IC50 = 19.2 ± 3.6 nM) of PDGFR phosphorylation, while sequential treatment of cells with sennoside B then PDGF-BB inhibited PDGFR phosphorylation at concentrations in the micromolar range (IC50 around 500 nM, data not shown). In these cells, PDGF-BB induced the phosphorylation of PDGFR-β and of downstream signaling molecules STAT-5, AKT and ERK1/2. Phosphorylation of all these molecules was inhibited by sennoside B. We also examined the selectivity of sennoside B in PDGF-AA. Sennoside B inhibits PDGF-AA-induced phosphorylation of PDGFR-α in MG63 cells at the same level (IC50 = 12.5 ± 22.6 nM, data not shown). These effects, however, were specific to PDGFs, as signaling by other growth factors was unaffected. Second, dot-binding assays demonstrated that sennoside B is retained by immobilized PDGF-BB and by the PDGFR's extracellular domain. Although there may be other interpretations, we suggest that, despite the lack of obvious sequence homology between the two proteins, sennoside B can bind to both PDGF-BB and the PDGFR and disturbs the ligand/receptor interaction. Third, high concentrations of sennoside B inhibited PDGF-BB-induced cell proliferation. This is the first report demonstrating that sennoside B specifically binds to PDGFBB and the PDGFR's extracellular domain to inhibit downstream signaling and proliferative effects. It was previously proposed that the binding of PDGF-BB to its receptor is predominantly mediated by hydrophobic and basic amino acid residues found in the loops connecting the β-sheets of the PDGF peptide (Oefner et al. 1992). In the present study, the meso and trans forms of sennoside molecules showed different binding affinities. We observed that only the trans form (sennoside B) was able to bind to PDGF-BB and the PDGFR-β extracellular domain and inhibit downstream signaling pathways. The stereo-selectivity of this binding may permit molecules to be designed that can selectively target PDGF-BB or the PDGFR's extracellular domain. Although our results indicate that low concentrations (~100 nM) of sennoside B can abolish PDGFR-β signaling by binding to the receptor and its ligand, higher concentrations (~1 µM) were required to inhibit cell proliferation. This could suggest that the inhibition of cell growth and the blockade of PDGF/PDGFR signaling may involve more than one mechanism. Further experiments will be required to address this possibility. Nonetheless, understanding the nature of the PDGF-BB-sennoside B interaction may lead to the development of a new class of anti-cancer drugs that irreversibly bind to PDGF-BB and have a therapeutic potential in a wide spectrum of human cancers. It is notable that, because sennoside B binds to PDGF-BB and the extracellular domain of the PDGFR, there is no requirement for the compound to enter cells, favoring a therapeutic use at low concentrations. Most tyrosine kinase inhibitors developed to date, in contrast, must enter the cell to target the kinase ATP-binding site. Such inhibitors suffer from a further drawback in that a large number of structurally similar ATP-binding sites are likely to exist within the cell. The clinical outcome of treatment with a molecule such as sennoside B may therefore be very different from the outcome of using inhibitors that target PDGF receptor tyrosine kinases. Conclusions We report that sennoside B is a specific inhibitor of PDGFR-β activation by PDGF-BB and down-regulates downstream signaling and cell proliferation. This molecule may be an attractive candidate for therapeutic use in diseases characterized by excessive PDGF-dependent cell proliferation, including cancer, atherosclerosis and restenosis. Although the specific binding of sennoside B to PDGF-BB/ PDGFR-β clearly interferes with signaling and proliferation in MG63 cells, it is still unclear how sennoside B interacts with two different
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proteins that lack evident sequence homology. Indeed, the molecular mechanism by which sennoside B “traps” the PDGF-BB ligand and/or “antagonizes” its receptor may emerge as a basis for the development of other similar ligand-trapping or antagonist receptor molecules, thereby leading to treatment options for neoplastic conditions in which PDGF signaling is known to play a central role. Conflict of interest statement There are no conflicts of interest. Acknowledgement This work was supported by the Ministry of Economic Affairs, Taiwan. References Alvarez RH, Kantarjian HM, Cortes JE. Biology of platelet-derived growth factor and its involvement in disease. Mayo Clinic Proceedings 81 (9), 1241–1257, 2006. Blaskovich MA, Lin Q, Delarue FL, Sun J, Park HS, Coppola D, Hamilton AD, Sebti AM. Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice. Nature Biotechnology 18 (10), 1065–1070, 2000. Board R, Jayson GC. Platelet-derived growth factor receptor (PDGFR): a target for anticancer therapeutics. Drug Resistance Updates: Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy, vol. 8(1–2), pp. 75–83, 2005. Cardenas C, Quesada AR, Medina MA. Evaluation of the anti-angiogenic effect of aloeemodin. Cellular and Molecular Life Sciences 63 (24), 3083–3089, 2006. Graves DT, Antoniades HN, Williams SR, Owen AJ. Evidence for functional plateletderived growth factor receptors on MG-63 human osteosarcoma cells. Cancer Research 44 (7), 2966–2970, 1984. Heldin CH, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature 319 (6), 511–514, 1986. Huang Q, Shen HM, Ong CN. Emodin inhibits tumor cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway. Cellular and Molecular Life Sciences 62 (10), 1167–1175, 2005. Huang Q, Lu G, Shen HM, Chung MC, Ong CN. Anti-cancer properties of anthraquinones from rhubarb. Medicinal Research Reviews 27 (5), 609–630, 2007. Kaneshiro T, Morioka T, Inamine M, Kinjo T, Arakaki J, Chiba I, Sunagawa N, Suzui M, Yoshimi N. Anthraquinone derivative emodin inhibits tumor-associated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation. European Journal of Pharmacology 553 (1–3), 46–53, 2006. Kim JH, Jin YR, Park BS, Kim TJ, Kim SY, Lim Y, Hong JT, Yoo HS, Yun YP. Luteolin prevents PDGF-BB-induced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation. Biochemical Pharmacology 69 (12), 1715–1721, 2005. Levitzki A. PDGF receptor kinase inhibitors for the treatment of PDGF driven diseases. Cytokine & Growth Factor Reviews 15 (4), 229–235, 2004. Liang YC, Chen YC, Lin YL, Lin-Shiau SY, Ho CT, Lin JK. Suppression of extracellular signals and cell proliferation by the black tea polyphenol, theaflavin-3,3′-digallate. Carcinogenesis 20 (4), 733–736, 1999. Lo HM, Hung CF, Tseng YL, Chen BH, Jian JS, Wu WB. Lycopene binds PDGF-BB and inhibits PDGF-BB-induced intracellular signaling transduction pathway in rat smooth muscle cells. Biochemical Pharmacology 74 (1), 54–63, 2007. Lu Y, Zhang J, Qian J. The effect of emodin on VEGF receptors in human colon cancer cells. Cancer Biotherapy & Radiopharmaceuticals 23 (2), 222–228, 2008. Oefner C, D'Arcy A, Winkler FK, Eggimann B, Hosang M. Crystal structure of human platelet-derived growth factor BB. The EMBO Journal 11 (11), 3921–3926, 1992. Panek RL, Lu GH, Klutchko SR, Batley BL, Dahring TK, Hamby JM, Hallak H, Doherty AM, Keiser JA. In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor. The Journal of Pharmacology and Experimental Therapeutics 283 (3), 1433–1444, 1997. Paukku K, Valgeirsdottir S, Saharinen P, Bergman M, Heldin CH, Silvennoinen O. Platelet-derived growth factor (PDGF)-induced activation of signal transducer and activator of transcription (Stat) 5 is mediated by PDGF beta-receptor and is not dependent on c-src, fyn, jak1 or jak2 kinases. The Biochemical Journal 345 (3), 759–766, 2000. Pecere T, Gazzola MV, Mucignat C, Parolin C, Vecchia FD, Cavaggioni A, Basso G, Diaspro A, Salvato B, Carli M, Palu G. Aloe-emodin is a new type of anticancer agent with selective activity against neuroectodermal tumors. Cancer Research 60 (11), 2800–2804, 2000. Pietras K, Sjoblom T, Rubin K, Heldin CH, Ostman A. PDGF receptors as cancer drug targets. Cancer Cells 3 (5), 439–443, 2003. Ringbom-Anderson T, Sandberg M, Andersson G, Akerman KE. Phenotypic modification of human osteosarcoma cells with the phorbol ester 1 2-O-tetradecanoylphorbol-1 3-acetate. Cell Growth & Differentiation 457 (6), 457–464, 1995. Sandy J, Davies M, Prime S, Farndale R. Signal pathways that transduce growth factorstimulated mitogenesis in bone cells. Bone 23 (1), 17–26, 1998.
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