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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Suramin Promotes Proliferation and Scattering of Renal Epithelial Cells

Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina

Received November 11, 2004; accepted April 12, 2005.

	Abstract

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Primary cultures of renal proximal tubules are known to recapitulate several early events in the process of renal regeneration following injury. In this study, we show that suramin, a polysulfonated naphthylurea, stimulates outgrowth, scattering, and proliferation of primary cultures of renal proximal tubule cells (RPTC). These responses were comparable to those produced by epidermal growth factor (EGF). However, AG-1478 [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline], a specific inhibitor of the EGF receptor, blocked EGF but not suramin-induced RPTC outgrowth, scattering, and proliferation. Suramin stimulated phosphorylation of Akt, a downstream kinase of phosphoinositide 3-kinase (PI3K), extracellular signaling-regulated kinase 1/2 (ERK1/2), and Src, but not the EGF receptor. Blockade of Src, but not the EGF receptor, inhibited Akt and ERK1/2 phosphorylation. Furthermore, inactivation of PI3K with LY294002 [2-(4morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] blocked suramin-induced RPTC outgrowth, scattering, and proliferation, whereas blockade of ERK1/2 had no effect. These data identify novel effects of suramin in RPTC outgrowth, scattering, and proliferation. Furthermore, suramin-induced outgrowth, scattering, and proliferation of RPTC are through Src-mediated activation of the PI3K pathway but not ERK1/2 or the EGF receptor.

Although suramin is effective when it is used as a chemotherapeutic agent for treatment of some tumors (e.g., prostate cancer) (Konety and Getzenberg, 1997), its antitumor activity has not proven successful in all the tumors tested (Eisenberger and Reyno, 1994; Kaur et al., 2002). One explanation for this finding is that suramin may induce other biological actions that are in conflict with its antitumor effect. Indeed, suramin can induce proliferation in certain cancer cell lines, in particular, epithelial tumor cell lines with high expression of the EGF receptor (e.g., A431 cells) (Cardinali et al., 1992) and nonsmall cell lung cancer cells (Lokshin et al., 1999). Furthermore, suramin stimulates proliferation of several nontumor cell lines, such as Chinese hamster ovary (CHO) cells (Nakata, 2004). Study of the mechanisms of such proliferation has suggested that suramin may activate the EGF receptor by stimulating the release of TGF-, an EGF receptor ligand (Cardinali et al., 1992), or directly induce EGF receptor dimerization (Lokshin et al., 1999).

Activation of the EGF receptor leads to multiple intracellular signaling events (Herbst, 2004). Among the signaling enzymes that mediate proliferation, phosphoinositide 3-kinase (PI3K) and extracellular signaling-regulated kinase (ERK) 1/2 pathways have been reported to be activated by suramin and required for the mitogenic response in CHO cells (Nakata, 2004).

Src is a nonreceptor tyrosine kinase that plays an important role in mediating a variety of cellular functions by delivering a signal to downstream effectors, including the PI3K and ERK1/2 pathways. Src activity is tightly controlled by phosphorylation of two tyrosine sites: activation by phosphorylation of Tyr416 and inactivation by phosphorylation of Tyr527 (Roskoski, 2004). A recent study showed that suramin also could stimulate Src phosphorylation in keratinocytes (Brown et al., 2004). The role of Src in suramin-mediated biological responses remains to be determined.

Our recent studies showed that renal proximal tubular cells (RPTC) proliferate and migrate in an EGF receptor-dependent manner in the absence of exogenous growth factors (Zhuang et al., 2004), suggesting the involvement of autocrine mechanisms in this process. Because suramin has been reported to block the interaction of growth factors with their receptors and thereby inhibit cell proliferation (Wang and Williams, 1984; Hosang, 1985; Betsholtz et al., 1986; Coffey et al., 1987; Anderson and Ray, 1998), we initially attempted to assess the autocrine mechanism of RPTC proliferation and migration using suramin. Unexpectedly, suramin stimulated RPTC outgrowth, scattering, and proliferation, and the signaling pathways for these actions were investigated.

	Materials and Methods

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Chemicals and Reagents. Human recombinant EGF was obtained from R&D Systems (Minneapolis, MN). LY294002 and U0126 were purchased from Cell Signaling Technology Inc. (Beverly, MA). AG-1478 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to phospho-EGF Tyr1068 receptor, phospho-Akt (Tyr473), Akt, phospho-ERK1/2 (Thr202/Tyr204), phospho-Src (Tyr416 or Tyr527), and Src were obtained from Cell Signaling Technology Inc. Antibodies to ERK1/2 and EGF receptor were purchased from BD Biosciences (San Jose, CA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Fluorescein-labeled phalloidin was obtained from Molecular Probes (Eugene, OR).

Isolation and Culture of Renal Proximal Tubules. Isolation and culture of renal proximal tubules were performed as described previously (Rodeheaver et al., 1990; Nowak and Schnellmann, 1995, 1996). Female New Zealand White rabbits (1.5-2.5 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). RPTC were isolated using the iron oxide perfusion method and grown in six-well tissue culture dishes under improved conditions. The culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 medium (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 µM), selenium (5 ng/ml), human transferrin (5 µg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 µM) were added daily to fresh culture medium.

Isolated tubules were plated at 1 mg of protein/well in a six-well plate. On day 2, RPTC were incubated in the presence and absence of various pharmacological inhibitors for different time periods as indicated in the figure legends. For some experiments, suramin was added to RPTC on day 3 and then incubated for 24 h in the presence or absence of various inhibitors before samples were taken.

MTT Assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to assess cell proliferation (Kooistra et al., 1997; Maeshima et al., 2002). Following a 48-h exposure to various inhibitors or diluents, MTT was added (final concentration, 0.5 mg/ml) to individual cultures for an additional 30 min. Tetrazolium was released by dimethyl sulfoxide, and the optical density was determined with a spectrophotometer (570 nm). Data were normalized to diluent-treated cultures.

Cell Cycle Analysis. Cell cycle phase was determined using flow cytometry as previously described (Zhuang et al., 2004). Cells were harvested and stained with propidium iodide, and the number of cells in S-phase of the cell cycle was determined.

Preparation of Cell Lysates and Immunoblot Analysis. After different treatments, RPTC were washed twice with phosphate-buffered saline and harvested in lysis buffer (0.25 M Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 1 mg/ml bromophenol blue, and 0.5% 2-mercaptoethanol). Cells were disrupted by sonication for 15 s, and lysates were stored at -20°C. Equal amounts of cellular protein lysate were separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes. After treatment with 5% skim milk at 4°C overnight, membranes were incubated with various antibodies for 1 h and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences Inc., Piscataway, NJ). Bound antibodies were visualized following chemiluminescence detection on autoradiographic film.

Immunocytochemistry. After various treatments, RPTC were fixed with 10% buffered formalin, washed, permeabilized, and blocked with bovine serum albumin. Fluorescein-conjugated phalloidin was added, and RPTC were visualized by fluorescent microscopy.

Statistical Analysis. Renal proximal tubules isolated from an individual rabbit represent a single experiment (n = 1) consisting of data obtained from three wells. Data are presented as means ± S.E.M. and were subjected to one-way analysis of variance. Multiple means were compared using Tukey's test. P < 0.05 was considered a statistically significant difference between mean values.

	Results

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Suramin Induces RPTC Outgrowth, Scattering, and Proliferation. We previously developed primary cultures of RPTC in which isolated tubules are plated and RPTC grow out from the tubules and proliferate to form a monolayer in 6 days in the absence of exogenous growth factor stimulation (Nowak and Schnellmann, 1995). Outgrowth, scattering, and proliferation of RPTC from the tubule fragments were observed within 3 days of plating and reached confluence at 6 days (Fig. 1A and data not shown). Using this system, we recently showed that the EGF receptor is activated and required for RPTC proliferation and migration following plating (Zhuang et al., 2004), suggesting that autocrine production of EGF receptor ligands is involved in the activation of the EGF receptor and RPTC proliferation.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Promotion of RPTC outgrowth and proliferation. A, renal proximal tubules plated (a) and after 24 h exposed to fresh medium alone (b), 50 µM suramin (c), 10 ng/ml EGF (d), or suramin + EGF (e) for 24 h and then photographed. RPTC were cultured for 3 days and then incubated with the above agents (B) or varying concentrations of suramin (C) for 24 h. After staining with propidium iodide, the cell cycle was analyzed by flow cytometry, and the number of cells in S-phase was determined. Data are expressed as means ± S.E.M., n = 3. Bars with different superscripts are significantly different from each other (P < 0.05).

The two major factors that contribute to the movement of cells away from the edge of the explants are cell proliferation and migration (Boland et al., 1996). To investigate the effect of suramin on cell proliferation, we measured the number of cells in S-phase of the cell cycle. Approximately 20% of RPTC were in S-phase 4 days following plating and increased to 29% following a 24-h suramin (50 µM) treatment (Fig. 1B). Incubation with exogenous EGF resulted in 32% of RPTC in S-phase. The combination of suramin and EGF did not significantly increase the number of RPTC in S-phase. Suramin concentrations lower than 50 µM did not statistically increase the number of RPTC in S-phase, whereas 100 µM suramin did not further increase RPTC proliferation (Fig. 1C). Using the MTT assay, a 48-h exposure of suramin also increased the number of RPTC by approximately 40% (Fig. 5A). Thus, suramin has the ability to induce RPTC proliferation.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Effects of PI3K and ERK1/2 pathway inhibitors on RPTC proliferation and scattering following plating and suramin exposure. A, RPTC were cultured for 2 days and then incubated with 50 µM suramin for 48 h in the presence and absence of 20 µM LY294002 (LY) and 10 µM U0126. Cell proliferation was determined using the MTT assay. Data are expressed as means ± S.E.M. of the percentage of MTT activity compared with controls grown with diluent (n = 3). B and C, RPTC were cultured for 3 days and then incubated with 50 µM suramin for 24 h in the presence and absence of 20 µM LY294002 (LY) and 10 µM U0126. Cell proliferation was determined by measuring the number of cells in S-phase of the cell cycle. Data are expressed as means ± S.E.M., n = 3. Bars with different superscripts are significantly different from each other (P < 0.05). Bright field photographs were taken at the edges of the cell islands. Original magnification, 40x.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Suramin promotes RPTC scattering and formation of lamellipodia. RPTC were cultured for 3 days and then treated with diluent, 50 µM suramin, 10 ng/ml EGF, or suramin + EGF. After 24 h, bright field photographs were taken (original magnification, 40x) (A), or RPTC were fixed with methanol and then stained with fluorescein-conjugated phalloidin (B). Original magnification, 80x. Arrows show lamellipodia.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Blockade of the EGF receptor by AG-1478 inhibits EGF but not suramin-induced RPTC proliferation and scattering. A, RPTC cultured for 2 days and then incubated with 10 µM AG-1478 in the absence or presence of 50 µM suramin for 48 h. Cell proliferation was determined using the MTT assay. Data are expressed as means ± S.E.M. of the percentage of MTT activity compared with controls grown with diluent (n = 3). B, RPTC cultured for 3 days and then incubated with diluent, 50 µM suramin, or 10 ng/ml EGF in the presence and absence of 10 µM AG-1478 for 24 h. Bright field photographs were taken at the edge of the cell islands. Original magnification, 40x.

A prominent feature of migrating cells is the formation of lamellipodia and the concomitant reorganization of the actin cytoskeleton (Fukata et al., 2003). Therefore, we examined the effect of suramin on RPTC scattering and motility using immunofluorescence staining of the actin cytoskeleton. Lamellipodia were seen at the edge RPTC in control cultures (Fig. 2B). Incubation of RPTC with suramin or EGF increased the formation of lamellipodia and decreased intercellular contacts, indicating cell scattering. Thus, RPTC scattering and motility increase following treatment with suramin and EGF.

Suramin-Induced RPTC Proliferation and Scattering Are Not Mediated by the EGF Receptor. Our previous studies showed that the EGF receptor is critical for RPTC proliferation (Zhuang et al., 2004). Because it was reported that suramin can induce the release of TGF-, an EGF receptor ligand, and stimulate proliferation in tumor cells (Cardinali et al., 1992), we examined the role of the EGF receptor in suramin-induced RPTC proliferation and scattering using AG-1478, a selective EGF receptor inhibitor. AG-1478 treatment did not affect suramin-induced RPTC proliferation, as measured by MTT assay (Fig. 3A). Similarly, AG-1478 did not affect suramin-induced formation of the fibroblast-like phenotype and scattering in RPTC (Fig. 3B). In contrast, AG-1478 blocked EGF-induced formation of the fibroblast-like phenotype and scattering.

To determine whether suramin caused EGF receptor activation, we performed immunoblot analysis using an antibody against phosphorylated Tyr1068 of the EGF receptor. Total EGF receptor content was measured using immunoblot analysis and an antibody that recognizes the EGF receptor independent of its phosphorylation state to control for gel loading. We did not detect phosphorylated EGF receptor after treatment with suramin but did observe phosphorylated EGF receptor in response to exogenous EGF (Fig. 4A). The combination of suramin and EGF had no additional effect on EGF receptor phosphorylation. Furthermore, AG-1478 inhibited EGF-induced EGF receptor phosphorylation (Fig. 4B). Thus, suramin-induced RPTC proliferation and scattering do not occur through the EGF receptor.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Suramin does not induce EGF receptor phosphorylation. RPTC were cultured for 3 days and then treated with 50 µM suramin or 10 ng/ml EGF for 10 min (A) or pretreated with 10 µM AG-1478 for 1 h and then exposed to EGF for 10 min (B). Cell lysates were prepared and subjected to immunoblot analysis using anti-phospho-EGF receptor antibody (Tyr1068) or anti-EGF receptor antibody. Protein loading was monitored using total EGF receptor levels. Representative immunoblot from three or more experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Suramin induces phosphorylation of Akt and ERK1/2. RPTC were cultured for 3 days and then treated with 50 µM suramin for 0-120 min (A) or pretreated with 20 µM LY294002 (LY) (B) or 10 µM U0126 (C) for 1 h and exposed to suramin for 30 min. Cell lysates were prepared and subjected to immunoblot analysis using antibodies to phospho-Akt, phospho-ERK1/2, total Akt, and total ERK1/2. Representative immunoblot from three or more experiments.

We evaluated whether the PI3K/Akt and/or ERK1/2 pathways are activated in RPTC in response to suramin. The activation of the PI3K and ERK1/2 pathways was measured using immunoblot analysis and antibodies that recognize phosphorylated Akt at Ser473 (a target of PI3K) and ERK1/2, respectively. Total Akt and ERK1/2 content was measured using immunoblot analysis and antibodies that recognize Akt and ERK1/2 independent of their phosphorylation state to control for gel loading. Suramin induced Akt phosphorylation within 10 min, reached a maximum at 30 min, and was sustained through 120 min of treatment (Fig. 6A). ERK1/2 phosphorylation was also induced within 10 min and maximal at 30 min but decreased to the control levels at 120 min.

To confirm that LY294002 and U0126 selectively inhibited their respective kinases, we monitored Akt and ERK activation using immunoblot analysis and antibodies that recognized phosphorylated Akt and ERK1/2 as described above. LY294002 blocked Akt phosphorylation and U0126 blocked ERK1/2 phosphorylation of RPTC following plating in the presence and absence of suramin (Fig. 6, B and C). Thus, whereas suramin activates PI3K/Akt and ERK1/2, PI3K/Akt, but not ERK1/2, is critical for suramin-induced RPTC proliferation and scattering. However, suramin-induced RPTC scattering is not completely dependent on PI3K.

Src Is Required for RPTC Proliferation and Scattering Induced by Suramin. It has been reported that Src activation mediates proliferation of renal epithelial cells in response to arachidonic acid metabolites and that suramin can activate Src phosphorylation in keratinocytes (Chen et al., 1998; Brown et al., 2004). Thus, we examined whether Src is required for RPTC proliferation and scattering using PP1, a specific inhibitor of Src (Liu et al., 1999). Treatment of RPTCs with PP1 inhibited RPTC proliferation in control and suramin-treated cells (Fig. 7, A and B). Suramin-stimulated formation of fibroblast-like phenotype and scattering in RPTC also was blocked by PP1 (Fig. 7C).

View larger version (28K): [in this window] [in a new window]   Fig. 7. Effects of Src inhibition on RPTC proliferation and scattering following plating and suramin exposure. A, RPTC were cultured for 2 days and then incubated with 50 µM suramin for 48 h in the presence and absence of 10 µM PP1. Cell proliferation was determined using the MTT assay. Data are expressed as means ± S.E.M. of the percentage of MTT activity compared with controls grown with diluent (n = 3). B and C, RPTC cultured for 3 days and then incubated with 50 µM suramin for 24 h in the presence and absence of 10 µM PP1. Cell proliferation was determined by measuring the number of cells in the S-phase of the cell cycle. Data are expressed as means ± S.E.M., n = 3. Bars with different superscripts are significantly different from each other (P < 0.05). Bright field photographs were taken at the edge of the cell islands. Original magnification, 40x.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Src, but not the EGF receptor, is required for suramin-induced Akt phosphorylation. RPTC were cultured for 3 days and then treated with 50 µM suramin for 0-60 min (A and B) or pretreated with 10 µM PP1 (C) and then treated with 50 µM suramin for 30 min. Cell lysates were prepared and subjected to immunoblot analysis using antibodies to phospho-Src at Tyr416 (A) and Tyr527 (B), Src, phospho-Akt, and total Akt. Representative immunoblot from three or more experiments.

	Discussion

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The mechanisms underlying suramin stimulation of cell proliferation and scattering are not clear. Because an early study showed that suramin induces the release of TGF- (a EGF receptor ligand), activates the EGF receptor, and induces proliferation of the carcinoma cell line KEsC-II (Cardinali et al., 1992), we initially thought that an EGF receptor-mediated mechanism might account for RPTC proliferation and scattering. However, our data do not support this hypothesis. First, suramin did not induce phosphorylation of the EGF receptor. Second, suramin did not affect exogenous EGF-induced EGF receptor phosphorylation. Finally, treatment of cells with AG-1478, a specific EGF receptor inhibitor, did not affect suramin-induced phosphorylation of Akt, proliferation, or scattering. Consistent with our observation, inhibition of the EGF receptor with AG-1478 did not attenuate suramin-induced ERK activation and proliferation in CHO cells (Nakata, 2004). Although it is possible that suramin may stimulate activation of other growth factor receptors through ligand-dependent mechanisms, and platelet-derived growth factor and fibroblast growth factor receptors are found on RPTC, the ligands for these receptors are not expressed in RPTC (Toback, 1992). Thus, we conclude that suramin-stimulated RPTC proliferation and scattering are not through the production of autocrine growth factors.

Another potential mechanism for suramin-induced RPTC proliferation and scattering may involve interference with growth inhibitory factors. In this regard, it has been reported that suramin can block the interaction of TGF- with the TGF receptor and block the inhibitory effect of TGF- in renal cancer cells (Wade et al., 1992). Previous results from our laboratory showed that RPTC can produce TGF- and that oxidant-induced autocrine production of TGF- has an inhibitory effect on RPTC proliferation (Nowak and Schnellmann, 1997). Furthermore, the addition of exogenous TGF- to RPTC inhibited proliferation following plating and oxidant injury (Counts et al., 1995; Kays et al., 1996; Nowak and Schnellmann, 1997) and inhibited wound healing of renal epithelial cells following mechanical injury (Sponsel et al., 1994). Thus, it is possible that inhibition of TGF- binding to its receptor may result in the enhancement of RPTC proliferation and scattering after suramin treatment. This possibility is currently under investigation in our laboratory.

Our results revealed that Src mediates suramin-induced proliferation and scattering of RPTC. Evidence for this statement is the activation of Src and Akt by suramin, the inhibition of suramin-induced Src and Akt phosphorylation by the Src inhibitor PP1, and the inhibition of RPTC proliferation and scattering by PP1. The mechanism by which suramin induces Src activation is currently unclear. One possibility is that suramin induces activation of protein tyrosine phosphatases (PTPs), and as a result Src is activated. Previous studies have shown that Src activation results from dephosphorylation of Tyr527 and subsequent Src autophosphorylation of Tyr416 (Roskoski, 2004), and PTP- has been reported to positively regulate Src activity via dephosphorylating Src at Tyr527 (Harder et al., 1998; Zheng et al., 2000). Furthermore, suramin induces activation of PTP- in vitro (McCain et al., 2004). Alternatively, suramin may induce Src activation by blocking TGF- binding to its receptor. In this context, it has been reported that TGF- treatment decreases Src activity and cell growth in the prostate carcinoma cell line PC3 and hepatoma cell line HepG2 (Atfi et al., 1994; Fukuda et al., 1998) and prevents hepatocyte growth factor-induced tyrosine phosphorylation of Src and migration in endothelial cells (Manganini and Maier, 2000).

To identify the intracellular signaling mechanism responsible for suramin-induced proliferation and scattering of RPTC, we found that Akt, a downstream target of PI3K, is phosphorylated after suramin treatment, and LY294002, a specific inhibitor of the PI3K signaling pathway, attenuated proliferation and scattering. These data illustrate that PI3K plays an essential role in transducing the signal for RPTC proliferation and scattering in response to suramin stimulation. However, it should be noted that the PI3K/Akt signaling pathway might not be the sole pathway responsible for RPTC scattering because blockade of PI3K does not result in complete inhibition of RPTC scattering in response to suramin stimulation. Although suramin also induced phosphorylation of ERK1/2, inhibition of this signaling pathway did not affect either proliferation or scattering, suggesting that the ERK pathway does not mediate this suramin-induced biological process.

In summary, our studies have shown that suramin stimulates proliferation and scattering of RPTC, which were mediated by Src-dependent activation of the PI3K/Akt pathway. Furthermore, suramin-stimulated biological responses do not involve EGF receptor or ERK1/2 activation. Because proliferation and migration of renal epithelial cells are two crucial processes needed for structural regeneration of nephron following injury, it will be of interest to determine whether suramin has a therapeutic potential in promoting renal recovery after injury.

	Acknowledgements

 
We thank Vanessa Haley for technical assistance.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.080648.

ABBREVIATIONS: EGF, epidermal growth factor; TGF, transforming growth factor; CHO, Chinese hamster ovary; PI3K, phosphoinositide 3-kinase; ERK, extracellular signaling-regulated kinase; RPTC, renal proximal tubular cell(s); LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; AG-1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; PTP, protein tyrosine phosphatase.

Address correspondence to: Rick G. Schnellmann, Department of Pharmaceutical Sciences, Medical University of South Carolina, 280 Calhoun Street, P.O. Box 250140, Charleston, SC 29425. E-mail: schnell{at}musc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Anderson RJ and Ray CJ (1998) Potential autocrine and paracrine mechanisms of recovery from mechanical injury of renal tubular epithelial cells. Am J Physiol 274: F463-F472.[Medline]

Atfi A, Drobetsky E, Boissonneault M, Chapdelaine A, and Chevalier S (1994) Transforming growth factor beta down-regulates Src family protein tyrosine kinase signaling pathways. J Biol Chem 269: 30688-30693.[Abstract/Free Full Text]

Barrett MP, Burchmore RJ, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ, and Krishna S (2003) The trypanosomiases. Lancet 362: 1469-1480.[CrossRef][Medline]

Betsholtz C, Johnsson A, Heldin CH, and Westermark B (1986) Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc Natl Acad Sci USA 83: 6440-6444.[Abstract/Free Full Text]

Boland S, Boisvieux-Ulrich E, Houcine O, Baeza-Squiban A, Pouchelet M, Schoevaert D, and Marano F (1996) TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture. J Cell Sci 109: 2207-2219.[Abstract]

Brown TA, Yang TM, Zaitsevskaia T, Xia Y, Dunn CA, Sigle RO, Knudsen B, and Carter WG (2004) Adhesion or plasmin regulates tyrosine phosphorylation of a novel membrane glycoprotein p80/gp140/CUB domain-containing protein 1 in epithelia. J Biol Chem 279: 14772-14783.[Abstract/Free Full Text]

Cardinali M, Sartor O, and Robbins KC (1992) Suramin, an experimental chemotherapeutic drug, activates the receptor for epidermal growth factor and promotes growth of certain malignant cells. J Clin Investig 89: 1242-1247.

Casanova JE (2002) Epithelial cell cytoskeleton and intracellular trafficking V. Confluence of membrane trafficking and motility in epithelial cell models. Am J Physiol Gastrointest Liver Physiol 283: G1015-G1019.[Abstract/Free Full Text]

Chen JK, Falck JR, Reddy KM, Capdevila J, and Harris RC (1998) Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells. J Biol Chem 273: 29254-29261.[Abstract/Free Full Text]

Coffey RJ Jr, Leof EB, Shipley GD, and Moses HL (1987) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J Cell Physiol 132: 143-148.[CrossRef][Medline]

Counts RS, Nowak G, Wyatt RD, and Schnellmann RG (1995) Nephrotoxicant inhibition of renal proximal tubule cell regeneration. Am J Physiol 269: F274-F281.

Eisenberger MA and Reyno LM (1994) Suramin. Cancer Treat Rev 20: 259-273.[CrossRef][Medline]

Foekens JA, Sieuwerts AM, Stuurman-Smeets EM, Dorssers LC, Berns EM, and Klijn JG (1992) Pleiotropic actions of suramin on the proliferation of human breast-cancer cells in vitro. Int J Cancer 51: 439-444.[Medline]

Fukata M, Nakagawa M, and Kaibuchi K (2003) Roles of Rho-family GTPases in cell polarisation and directional migration. Curr Opin Cell Biol 15: 590-597.[CrossRef][Medline]

Fukuda K, Kawata S, Tamura S, Matsuda Y, Inui Y, Igura T, Inoue S, Kudara T, and Matsuzawa Y (1998) Altered regulation of Src tyrosine kinase by transforming growth factor beta1 in a human hepatoma cell line. Hepatology 28: 796-804.[CrossRef][Medline]

Gill JS, Connolly DC, McManus MJ, Maihle NJ, and Windebank AJ (1996) Suramin induces phosphorylation of the high-affinity nerve growth factor receptor in PC12 cells and dorsal root ganglion neurons. J Neurochem 66: 963-972.[Medline]

Harder KW, Moller NP, Peacock JW, and Jirik FR (1998) Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion. J Biol Chem 273: 31890-31900.[Abstract/Free Full Text]

Herbst RS (2004) Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 59: 21-26.

Hosang M (1985) Suramin binds to platelet-derived growth factor and inhibits its biological activity. J Cell Biochem 29: 265-273.[CrossRef][Medline]

Kaur M, Reed E, Sartor O, Dahut W, and Figg WD (2002) Suramin's development: what did we learn? Investig New Drugs 20: 209-219.[CrossRef][Medline]

Kays SE, Nowak G, and Schnellmann RG (1996) Transforming growth factor-beta 1 inhibits regeneration of renal proximal tubular cells after oxidant exposure. J Biochem Toxicol 11: 79-84.[CrossRef][Medline]

Konety BR and Getzenberg RH (1997) Novel therapies for advanced prostate cancer. Semin Urol Oncol 15: 33-42.[Medline]

Kooistra A, Romijn JC, and Schroder FH (1997) Stromal inhibition of epithelial cell growth in the prostate; overview of an experimental study. Urol Res 25: S97-S105.

Kyosseva SV (2004) Mitogen-activated protein kinase signaling. Int Rev Neurobiol 59: 201-220.[Medline]

Leu TH and Maa MC (2003) Functional implication of the interaction between EGF receptor and c-Src. Front Biosci 1: s28-s38.

Liu Y, Bishop A, Witucki L, Kraybill B, Shimizu E, Tsien J, Ubersax J, Blethrow J, Morgan DO, and Shokat KM (1999) Structural basis for selective inhibition of Src family kinases by PP1. Chem Biol 6: 671-678.[CrossRef][Medline]

Lokshin A, Peng X, Campbell PG, Barsouk A, and Levitt ML (1999) Mechanisms of growth stimulation by suramin in non-small-cell lung cancer cell lines. Cancer Chemother Pharmacol 43: 341-347.[CrossRef][Medline]

Maeshima A, Nojima Y, and Kojima I (2002) Activin A: an autocrine regulator of cell growth and differentiation in renal proximal tubular cells. Kidney Int 62: 446-454.[CrossRef][Medline]

Manganini M and Maier JA (2000) Transforming growth factor beta2 inhibition of hepatocyte growth factor-induced endothelial proliferation and migration. Oncogene 19: 124-133.[CrossRef][Medline]

McCain DF, Wu L, Nickel P, Kassack MU, Kreimeyer A, Gagliardi A, Collins DC, and Zhang ZY (2004) Suramin derivatives as inhibitors and activators of protein-tyrosine phosphatases. J Biol Chem 279: 14713-14725.[Abstract/Free Full Text]

Nakata H (2004) Stimulation of extracellular signal-regulated kinase pathway by suramin with concomitant activation of DNA synthesis in cultured cells. J Pharmacol Exp Ther 308: 744-753.[Abstract/Free Full Text]

Nowak G and Schnellmann RG (1995) Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells. Am J Physiol 268: C1053-C1061.

Nowak G and Schnellmann RG (1996) L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture. Am J Physiol 271: C2072-C2080.

Nowak G and Schnellmann RG (1997) Renal cell regeneration following oxidant exposure: inhibition by TGF-beta1 and stimulation by ascorbic acid. Toxicol Appl Pharmacol 145: 175-183.[CrossRef][Medline]

Richardson CJ, Schalm SS, and Blenis J (2004) PI3-kinase and TOR: PIKTORing cell growth. Semin Cell Dev Biol 15: 147-159.[CrossRef][Medline]

Rodeheaver DP, Aleo MD, and Schnellmann RG (1990) Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: comparison in long-term incubation. In Vitro Cell Dev Biol 26: 898-904.[Medline]

Roskoski R Jr (2004) Src protein-tyrosine kinase structure and regulation. Biochem Biophys Res Commun 324: 1155-1164.[CrossRef][Medline]

Sponsel HT, Breckon R, Hammond W, and Anderson RJ (1994) Mechanisms of recovery from mechanical injury of renal tubular epithelial cells. Am J Physiol 267: F257-F264.

Stein CA (1993) Suramin: a novel antineoplastic agent with multiple potential mechanisms of action. Cancer Res 53: 2239-2248.[Free Full Text]

Toback FG (1992) Regeneration after acute tubular necrosis. Kidney Int 41: 226-246.[Medline]

Wade TP, Kasid A, Stein CA, LaRocca RV, Sargent ER, Gomella LG, Myers CE, and Linehan WM (1992) Suramin interference with transforming growth factor-beta inhibition of human renal cell carcinoma in culture. J Surg Res 53: 195-198.[Medline]

Wang JY and Williams LT (1984) A v-sis oncogene protein produced in bacteria competes for platelet-derived growth factor binding to its receptor. J Biol Chem 259: 10645-10648.[Abstract/Free Full Text]

Zheng XM, Resnick RJ, and Shalloway D (2000) A phosphotyrosine displacement mechanism for activation of Src by PTPalpha. EMBO (Eur Mol Biol Organ) J 19: 964-978.[CrossRef][Medline]

Zhuang S, Dang Y, and Schnellmann RG (2004) Requirement of the epidermal growth factor receptor in renal epithelial cell proliferation and migration. Am J Physiol Renal Physiol 287: F365-F372.[Abstract/Free Full Text]

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