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The New Sulindac Derivative IND 12 Reverses Ras-induced Cell Transformation¹

Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany [I-M. K., P. H., S. C., A. K., U. H., M-R. A., O. M.]; Laboratory of Experimental Cancerology, Ghent University Hospital, B-9000 Ghent, Belgium [P. D., M. M.]; Squarix GmbH, 45768 Marl, Germany [K-H. G.]; Klinikum Kreis Herford, Herford, 32045 Germany [G. W.]; and Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, 45122 Essen, Germany [T. M.]

	ABSTRACT

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The nonsteroidal anti-inflammatory drug Sulindac has chemopreventiveand antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound ß-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.

	INTRODUCTION

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Sulindac and other nonsteroidal anti-inflammatory drugs reduce the overall risk to develop cancer and can inhibit the growth of tumors (1, 2, 3) . Nonsteroidal anti-inflammatory drugs inhibit the key enzymes of the eicosanoid metabolism, the COX³ 1 and 2, and thereby decrease the levels of proliferation-activating eicosanoids (4) . The COX inhibitory effect of Sulindac is mediated by its main physiological metabolite Sulindac sulfide, whereas Sulindac itself and its sulfone metabolite do not affect the COX pathway (5) . Nevertheless, Sulindac sulfone, which is ineffective on COX, can induce apoptosis and is able to inhibit tumor growth (6) . Additional findings that Sulindac metabolites cause apoptosis in epithelial tissue and in cultured cancer cells independently from the eicosanoid metabolism indicated that Sulindac might exert its antitumorigenic effects independently from the eicosanoid pathway (7, 8, 9) . Sulindac is used in the therapy and the prevention of tumors in patients with the cancer predisposition familial adenomatous polyposis caused by inherited mutations in the APC gene (7 , 10) . Experiments performed with the Min mouse, the animal model for familial adenomatous polyposis, also demonstrated that the antitumor effect of Sulindac is independent of prostaglandin biosynthesis (11) .

It has been shown that Sulindac metabolites can inhibit Ras-induced signal transduction. The consequences of this inhibition are reduced activation levels of MAP/ERK kinase 1/2, p44-MAPK, and p42-MAPK and decreased levels of Ras-activated gene expression (12, 13, 14) . Along this line, oncogenic Ras interferes with the induction of cell death by Sulindac metabolites (15 , 16) . In vitro, Sulindac sulfide directly blocks the interaction between p21ras and its main effector Raf kinase (12) . These results offer an explanation for the finding that Sulindac inhibits the growth of tumors harboring Ras gene mutations more effectively than the growth of tumors with the wild-type Ras gene (17) .

Recently, we identified a novel Sulindac derivative, named IND 12, which inhibited the proliferation of Ras-transformed cells at significantly lower concentrations than were needed to inhibit the proliferation of untransformed cells (18) . In addition, IND 12 inhibited COX activity and the p21ras/Raf kinase interaction at lower concentrations than Sulindac sulfide. On the basis of these results, IND 12 represented a promising candidate in the search for a new antiproliferative drug with inhibitory activity on the Ras pathway. In this study, we asked whether IND 12 can interfere with cell transformation induced by the oncogenic Ras gene. As a model system, we used MDCK cells, which undergo epithelial-to-mesenchymal transition on transformation with oncogenic Ras. Cells of the Ras-transformed subclone MDCK-f3 can be distinguished from the untransformed MDCK parent cell line by several morphological and biochemical parameters (cell morphology, cell growth, cell aggregation, E-cadherin/ß-catenin interaction; Ref. 19 ).

	MATERIALS AND METHODS

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Overall Experimental Strategy.
On the first level of analysis, we screened for drug-induced alterations of cell morphology, cell growth, cell aggregation, and cell invasion behavior. We visualized these parameters by microscopy or in cell biological assays. On the next level, we asked for the molecular background of the detected drug-induced effects. To address this question, we analyzed the levels and the activities of proteins, which are known to play a pivotal role in the regulation of cell adhesion and cytoskeletal arrangement. At this point, our results indicated that the drug directly inhibits the Ras-signaling pathway. Therefore, we looked for the activity of the Ras pathway under drug influence by measuring the levels of Ras-activated effector proteins and Ras-induced gene expression.

The Drug IND 12 and the Cell Lines.
The synthesis and the chemical properties of IND 12 have been described (18) . The drug was dissolved in DMSO as a 1000 x stock solution. In all experiments, DMSO controls showed no significant effects. The linear correlation between drug concentration and UV absorption at the wavelength of the characteristic absorption maximum revealed no hints of insolubility or micelle formation in buffered aqueous solutions at the applied concentrations.

Epithelial MDCK cells and MDCK-f3 cells were kindly provided by John G. Collard (Amsterdam). The MDCK-f3 cell line is a subclone of the MDCK-ras-f cell line, which is a virally Ras-transformed derivative of the MDCK cell line (20) . MDCK-f3 cells are cytokeratin positive and show a fibroblast-like morphotype (19) . Rabbit kidney epithelial-like RK13 cells were purchased from American Type Culture Collection. Cells were cultured under standard conditions.

Fluorescence Microscopy and SEM.
The cellular morphology was analyzed by light microscopic or confocal microscopic analysis of cells immunolabeled with a monoclonal mouse rhodamine-coupled antibody directed against F-actin or polyclonal rat antibodies directed against E-cadherin (Sigma Chemical Co.). Binding sites of the primary rat antibodies were detected using a secondary antirat dichlorotriazinylaminofluorescein-coupled antibody (Transduction Laboratories). For SEM analysis, cells were grown on thermanox slides and fixed by incubation in 4% paraformaldehyde and 1% glutaraldehyde in 200 mM HEPES (pH 7.4) for 45 min. Fixed cells were dehydrated in an ascending ethanol series and then critical point dried using methanol as intermedium and CO₂ as drying medium. To avoid charging artifacts, the dried cells were covered with gold by sputter coating. Covered cells were studied with a Hitachi S-800 SEM equipped with a field emission gun. Cell surface morphology was imaged by the signal of secondary electrons at an accelerating voltage of 20 kV and a working distance of 10 mm.

Analysis of the E-cadherin/ß-catenin Complex by Immunoprecipitation and Western Blot.
Cells were washed with PBS and lysed in cold PBS containing 1% Triton x 100, 1% NP40, and protease inhibitors. Cell debris was precipitated by centrifugation, and supernatants were adjusted to equal concentrations of total protein. Aliquots of the supernatants were retained for the analysis of the total amount of ß-catenin. The E-cadherin/ß-catenin complex was immunoprecipitated from the supernatants using antibodies against E-cadherin (Transduction Laboratories). The precipitates were analyzed by Western blot probed with a primary monoclonal antibody against ß-catenin, or against E-cadherin as a control (Transduction Laboratories), and a secondary rabbit antimouse antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech). Signals were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Cell Aggregation Assay and Collagen Invasion Assay.
Slow cell aggregation assay was performed as described (21 , 22) . Briefly, 20,000 cells/ml were incubated with or without 100 µM IND 12 after seeding on soft agar in wells of a 96-well plate. After 24 h, aggregation was evaluated under an inverted microscope. Collagen invasion assay was performed as described (23 , 24) . Briefly, 10⁵ cells/ml were seeded with or without 100 µM IND 12 on top of collagen type I gels (from rat tail; Upstate Biotechnology), which were prepared in DMEM (0.22% w/v) in six-well plates. After 24 h, cell invasion was evaluated under an inverted microscope. Cells on or above the surface of the collagen gel were regarded as superficial cells. Single individual cells found 10 µm or deeper inside the collagen gel matrix were interpreted as invasive cells that have migrated into the collagen gel. The number of superficial cells and invasive cells was counted in 15 microscope fields. The invasion index was calculated as a percentage of invasive cells referred to the total number of superficial and invasive cells.

Rac and Rho Activity Assay.
Sustained activation of the Ras pathway leads to the decrease of the relative level of active GTP-bound Rac what in turn increases the level of GTP-bound Rho (25) . The balance of the levels of active Rac and Rho is crucial for cellular morphology (26) . The levels of the active small GTPases Rac and Rho were measured as described (25 , 26) . GTP-bound active Rac or Rho molecules were precipitated by p21-activated kinase or C21, each fused to glutathione-S transferase precoupled to glutathione-Sepharose beads, respectively. The total amount of the GTPases in the cell lysate and the precipitated amount of the GTPases were detected by Western blot analysis using monoclonal antibodies against Rac1 (Transduction Laboratories) or RhoA (Santa Cruz Biotechnology).

MAP Kinase Assay.
The total levels and the levels of the phosphorylated active kinases p44-MAP and p42-MAP (p44-MAPK/p42-MAPK, also called MAPK1/2 or ERK1/2) were analyzed by Western blot of cell lysates as described (25 , 27) . The phosphorylated p44-MAPK and p42-MAPK were detected by antibodies (Cell Signaling), which bind to the proteins only when activated catalytically by phosphorylation. The total levels of p44-MAPK and p42-MAPK were analyzed on a parallel Western blot and detected by antibodies, which bind to p44-MAPK and p42-MAPK independently of phosphorylation (New England Biolabs).

Reporter Gene Assay.
The reporter gene assay was performed as described (12) . RK13 cells were transfected by the calcium phosphate coprecipitation method using the vectors, which have been described (28, 29, 30) . As controls, the reporter gene was stimulated by transfection with the constitutively active forms of Raf and Raf-CAAX and of MAP-KK and MAP-KK(EE), respectively (12) . The MAP-KK(EE) transfection vector was kindly provided by Chris Marshall (London). To test a potential inhibitory effect of the drug on the expression of the reporter gene already from the beginning of the induction, we added 100 µM IND 12 to the medium right after transfection. After transfection (36 h), cells were lysed, and luciferase and ß-galactosidase activities were determined. Relative luciferase activity was calculated by normalizing luminescence to ß-galactosidase activity.

	RESULTS

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IND 12 Restores Epithelial Morphology in Malignantly Transformed MDCK-f3 Cells, Reactivates Cell Aggregation, and Inhibits Cell Invasion.
MDCK cells display the typical cellular and cell surface morphology of polarized transporting epithelial cells as revealed by scanning electron and light microscopy (Figs. 2 and 3 ). MDCK cells are characterized by numerous cell surface microvilli and well-organized cell-cell contacts and grow in an epithelium-like monolayer. In contrast, oncogenically transformed MDCK-f3 cells show the spindle-shaped form of functionally dedifferentiated tumor cells only sparsely covered with microvilli, strong membrane ruffling, and a reduced number of cell-cell contacts. They grow in single cell clones and partly overlap each other indicative for the loss of contact inhibition (Figs. 1 2 3) . To test the effective concentration and the time-dependent effects of IND 12 on MDCK-f3 cells, we monitored morphological changes after IND 12 incubation. We observed that the morphology of MDCK-f3 cells changed in an IND 12 concentration-dependent manner toward the epithelioid morphotype that is characteristic for untransformed MDCK cells (Fig. 1) . The maximum effect was reached at 100 µM IND 12. Further increase of the IND 12 concentration showed no additional effect on cellular morphology. Therefore, we chose 100 µM as incubation concentration in the additional assays. IND 12-treated MDCK-f3 cells also resembled untransformed MDCK cells in their growing manner and number of cell contacts (Fig. 2) . The treatment of untransformed MDCK cells had no effect. The morphological reversion of MDCK-f3 cells from the fibroblastoid to the epithelioid phenotype was confirmed by electron microscopy to be dependent on the drug-incubation time (Fig. 3) . The fibroblastoid MDCK-f3 cells changed to cells with smooth, flattened cell surface with less filopodia compared with untreated MDCK-f3 cells. The extent of this reversion increased time dependently up to an incubation time of 8 h. Longer incubation had no additional effects. Even after 24 h, the IND 12-induced reversion of the transformed into the untransformed phenotype was not complete, e.g., IND 12-treated MDCK-f3 cells did not show as many microvilli as MDCK cells (Fig. 3) .

View larger version (133K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. IND 12 treatment has no effect on MDCK cells, whereas MDCK-f3 cells change their phenotype and their growing and aggregation behavior in the presence of IND 12. MDCK (top two rows) and MDCK-f3 cells (bottom two rows) were visualized by fluorescent immunostaining for F-actin (left column), E-cadherin (middle column), and both F-actin and E-cadherin (right column) and confocal fluorescence light microscopy at a primary magnification of x400. MDCK cells grew in an epithelium-like monolayer with a high level of the membrane-bound cell adhesion protein E-cadherin and regularly arranged cell contacts. Treatment of MDCK cells with 100 µM IND 12 had no effect. Transformed MDCK-f3 cells showed an elongated fibroblast-like phenotype with no regular cell contacts and no significant staining of E-cadherin. Treatment with 100 µM IND 12 led MDCK-f3 cells to change their phenotype toward the untransformed MDCK cells; at the same time, the level of the membrane-bound E-cadherin increased. Arrowheads, two cell contacts with E-cadherin staining of a single IND 12-treated MDCK-f3 cell.

View larger version (152K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. IND 12 reconstitutes the untransformed morphology of Ras-induced MDCK-f3 cells in a time-dependent manner. MDCK-f3 cells were incubated with 100 µM IND 12 for the indicated time and visualized by SEM at a primary magnification of x1500. Top row, left, MDCK cells showed the typical morphology of epithelial cells with flat and large cell bodies and numerous microvilli present on the cell surface. Untreated MDCK-f3 cells displayed a spindle-shaped fibroblastoid morphology with distinct membrane ruffling. MDCK-f3 cells grew in single cell foci overlapping each other. After IND 12 treatment for 4 h, MDCK-f3 cells showed already first morphological changes toward the epithelioid character of MDCK cells. Cell bodies are larger, and cells form more cell contacts. After 8-h incubation, MDCK-f3 cells recovered more morphological aspects of epithelioid MDCK cells. Cell bodies are round and large. After incubation times of 16 and 24 h, MDCK-f3 cells strongly resembled untransformed MDCK cells and were able to form regular cell-to-cell contacts as the parent MDCK cells. However, the restoration of the epithelial character was not complete in IND 12-treated MDCK-f3 cells, as shown by the number of microvilli, which did not reach the number found on untransformed MDCK cells even after 24 h of incubation.

View larger version (87K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Ras-transformed MDCK-f3 cells change their phenotype in the presence of IND 12 in a concentration-dependent manner. MDCK-f3 cells were treated with IND 12 at the indicated micromolar concentrations. To visualize cellular morphology, cells were immunostained for F-actin and analyzed by fluorescence microscopy at a primary magnification of x400. Untreated MDCK-f3 cells are small, fibroblast-like, and spindle-shaped cells. IND 12 treatment led to large, round cells with epithelioid character. The maximum effect was reached at 100 µM IND 12. Further increase of IND 12 concentration had no additional effects on cellular morphology.

View larger version (85K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. IND 12 reactivates the aggregation ability of MDCK-f3 cells in a cell aggregation assay. MDCK (left), MDCK-f3 (middle), or MDCK-f3 cells in the presence of 100 µM IND 12 (right) were grown on soft agar and visualized by light microscopy at x250 magnification. MDCK cells were adhesive and grew attached to each other within large aggregations, in opposite to MDCK-f3 cells, which formed only very small aggregations of cells. In contrast, in the presence of IND 12, MDCK-f3 cells formed large aggregations of cells similar to untransformed MDCK cells.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. IND 12 reduces the invasive potential of MDCK-f3 cells in a cell invasion assay. Given is the invasion index that represents the percentage of invasive cells among the total number of cells. In contrast to untransformed MDCK cells, MDCK-f3 cells are highly invasive. In the presence of IND 12 at 50 or 100 µM, the invasion index of MDCK-f3 cells was drastically reduced.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. IND 12 restores the level of the cell adhesion complex between E-cadherin and ß-catenin, but the levels of immunoprecipitated total E-cadherin and of total ß-catenin remain unchanged by IND 12. Western blot analysis (WB) of ß-catenin in anti-E-cadherin immunoprecipitates (IP; middle row) from MDCK, MDCK-f3, and MDCK-f3 cells after treatment with 100 µM IND 12. In MDCK-f3 cells, the level of the E-cadherin-bound ß-catenin was reduced in comparison with the level in untransformed MDCK cells (middle row). IND 12 treatment restored the level of the E-cadherin-bound ß-catenin to levels found in untreated MDCK cells. This restoration is not attributable to an increase of the level of the complexed E-cadherin (top row) and not to an increase of the level of total ß-catenin in the cell lysates (bottom row), as shown by WB using anti-E-cadherin or anti-ß-catenin antibodies, respectively.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. The reciprocal levels of active Rac and Rho in MDCK-f3 cells are reconstituted partly by IND 12 to levels found in untreated MDCK cells. A, Western blot analysis showing the p21-activated kinase-complexed active Rac (top row) and the level of total Rac (bottom row) from MDCK, MDCK-f3, and MDCK-f3 cells after treatment with 100 µM IND 12 for the indicated time. In MDCK-f3 cells, the level of active Rac relative to the amount of total Rac was lower than in untransformed MDCK cells. IND 12 treatment of MDCK-f3 cells induced the increase of the level of active Rac relative to its total amount after 4 and 8 h. The level of total Rac remained unchanged during IND 12 treatment. Although, the relative level of active Rac to total Rac did not reach the relative level of active Rac in untransformed MDCK cells. B, Western blot analysis showing the C21-complexed active Rho (top row) and the level of total Rho (bottom row) from MDCK, MDCK-f3, and MDCK-f3 cells after treatment with 100 µM IND 12 for the indicated time. The level of active Rho was decreased in IND 12-treated MDCK-f3 cells reaching the level of active Rho in untransformed MDCK cells after 8 h, whereas the level of total Rho remained unchanged.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. IND 12 reduces the levels of the active MAP kinases. The levels of the phosphorylated active p44-MAPK and p42-MAPK (top row) and of the total levels of p44-MAPK and p42-MAPK (bottom row) were analyzed by Western blot of protein extracts from MDCK, MDCK-f3, and MDCK-f3 cells treated with 100 µM IND 12 for the indicated time using specific antibodies. As expected, the levels of active phospho-p44-MAPK and phospho-p42-MAPK were increased in Ras-transformed MDCK-f3 cells. Treatment of MDCK-f3 cells with IND 12 led to the decrease of the levels of the active MAP kinases (top row), whereas the total level of MAP kinases remained unchanged (bottom row).

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. IND 12 inhibits Ras-dependent transactivation. Relative activities of the luciferase reporter gene are shown. The influence of IND 12 was measured on RasG12V-, Raf-CAAX-, or MAP-KK(EE)-induced reporter gene activity, respectively. The activities of the untreated controls were set as 100% of the relative reporter activity. IND 12 inhibits the transcriptional transactivation induced by activated RasG12V in a concentration-dependent manner. IND 12 inhibits also transcriptional transactivation stimulated by Raf-CAAX, whereas it has no effect on the reporter gene activity activated by MAP-KK(EE). Each experiment was performed in triplicates, and average values are given.

	DISCUSSION

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As a first sign for the reversion of transformation, IND 12 changed the fibroblastoid phenotype of MDCK-f3 cells toward the epithelioid morphology of the untransformed, normal MDCK cells. Cell-cell contacts are formed by membrane proteins of the cadherin superfamily, which are the starting point of the molecular cascade that leads to the formation of junctional desmosomes and tight junctions (31) . On the cytoplasmic side, cadherins bind to the catenins, which is a prerequisite for the link between cell-cell contacts and the cytoskeleton and, thus, for the stability of cell-cell adhesion. Ras-transformed MDCK-f3 cells are highly invasive as a result of reduced E-cadherin-mediated cell-cell adhesion (19 , 20) . A reversion of MDCK cell transformation is accompanied by the translocation of cytosolic ß-catenin to the membrane and the increase of E-cadherin/ß-catenin complex (26) . The reversal of the transformed phenotype by IND 12 correlated with a partial restoration of the levels of membrane-bound E-cadherin and of E-cadherin-bound ß-catenin. This suggests that the observed morphological changes induced by IND 12 in these cells are based on a reorganization of both molecules at the membrane or in complexes, respectively. These results also show that IND 12 can initiate restoration of membrane-bound E-cadherin, retranslocation of ß-catenin to the cell membrane, and the reformation of complexes between these molecules. Thus, it is conceivable that IND 12-treated cells regain the ability for contact inhibition and lose their invasive potential. The exact molecular mechanism by which IND 12 can exert this reversal of phenotype and the restoration of the levels of the membrane-bound E-cadherin and of the E-cadherin/ß-catenin complex remains to be determined. However, the morphological differences between MDCK-f3 and MDCK cells have been shown to be a consequence of Ras-induced transformation of MDCK-f3 cells (19 , 20) . In this model, sustained Ras activation leads to the down-regulation of the relative level of active Rac in MDCK-f3 cells (25) . Rac plays a role as a suppressor of invasion of epithelial cells and regulates cytoskeletal rearrangement and epithelial morphology and, thus, plays a role in the control of cell morphology and migration (26 , 32) . As a result of down-regulated Rac activity, the level of the active GTPase Rho increases. We show here that in the presence of IND 12, the ratio of active Rac and Rho levels relative to the total levels of the two proteins is altered in MDCK-f3 cells and resembles the ratios normally found in untransformed MDCK cells. These findings suggest that the observed effects of IND 12 on the transformed MDCK-f3 cells are associated with the expression or activity of these small G-proteins. Because the alteration of the levels of active Rac and Rho in MDCK-f3 cells is because of the sustained activation of the Ras pathway, an inhibition of the Ras pathway by IND 12 would offer an attractive explanation for the restoration of the Rac and Rho levels and the observed effects of IND 12 on MDCK-f3 cells. A number of other findings suggest that IND 12 exerts its effect by directly interfering with Ras-initiated signal transduction: (a) the levels of the phosphorylated active downstream effectors p44-MAPK and p42-MAPK decreased in the presence of IND 12; (b) IND 12 lowered the gene expression activated by oncogenic Ras but not the gene expression activated by the Ras downstream effector MAP-KK(EE); and (c) in a recent study, we showed that IND 12 inhibits the interaction between the proto-oncoprotein p21ras and the Raf kinase in vitro (18) . Altogether, our findings make IND 12 a promising candidate with high potential in the all-important search for new antitumorigenic drugs with effects on the Ras pathway. Future studies have to demonstrate the effects of this drug on tumors in vivo.

	ACKNOWLEDGMENTS

 
We thank Alfred Wittinghofer for continuous support.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by grants from the Deutsche Forschungsgemeinschaft (Grant MU 1091/8-1) and the Deutsche Krebshilfe (Grant 10-1474-Mü2). Philip Debruyne is a research assistant with the "Bijzonder Onderzoeksfonds Universiteit Gent."

² To whom requests for reprints should be addressed, at Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. Phone: +49-231-1332158; Fax: +49-231-1332199; E-mail: oliver.mueller{at}mpi-dortmund.mpg.de

³ The abbreviations used are: COX, cyclooxygenase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MDCK, Madin-Darby Canine Kidney; IND 12, (Z)-5-Fluoro-2-methyl-1-(2-furanyl)-indene-3-acetic acid; SEM, scanning electron microscopy; MAP-KK, mitogen-activated protein kinase kinase; F-actin, filamentous-actin.

Received 6/15/01. Accepted 1/16/02.

	REFERENCES

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1. Rao C. V., Rivenson A., Simi B., Zang E., Kelloff G., Steele V., Reddy B. S. Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent. Cancer Res., 55: 1464-1472, 1995.[Abstract/Free Full Text]
2. Luk G. D. Prevention of gastrointestinal cancer–the potential role of NSAIDs in colorectal cancer. Schweiz. Med. Wochenschr., 126: 801-812, 1996.[Medline]
3. Janne P. A., Mayer R. J. Chemoprevention of colorectal cancer. N. Engl. J. Med., 342: 1960-1968, 2000.[Free Full Text]
4. Vane J. R., Botting R. M. Mechanism of action of anti-inflammatory drugs. Scand. J. Rheumatol. Suppl., 102: 9-21, 1996.[Medline]
5. Duggan D. E., Hooke K. F., Risley E. A., Shen T. Y., Arman C. G. Identification of the biologically active form of sulindac. J. Pharmacol. Exp. Ther., 201: 8-13, 1977.[Abstract/Free Full Text]
6. Piazza G. A., Alberts D. S., Hixson L. J., Paranka N. S., Li H., Finn T., Bogert C., Guillen J. M., Brendel K., Gross P. H., Sperl G., Ritchie J., Burt R. W., Ellsworth L., Ahnen D. J., Pamukcu R. Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels. Cancer Res., 57: 2909-2915, 1997.[Abstract/Free Full Text]
7. Pasricha P. J., Bedi A., O’Connor K., Rashid A., Akhtar A. J., Zahurak M. L., Piantadosi S., Hamilton S. R., Giardiello F. M. The effects of sulindac on colorectal proliferation and apoptosis in familial adenomatous polyposis. Gastroenterology, 109: 994-998, 1995.[Medline]
8. Hanif R., Pittas A., Feng Y., Koutsos M. I., Qiao L., Staiano-Coico L., Shiff S. I., Rigas B. Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem. Pharmacol., 52: 237-245, 1996.[Medline]
9. Piazza G. A., Rahm A. K., Finn T. S., Fryer B. H., Li H., Stoumen A. L., Pamukcu R., Ahnen D. J. Apoptosis primarily accounts for the growth-inhibitory properties of sulindac metabolites and involves a mechanism that is independent of cyclooxygenase inhibition, cell cycle arrest, and p53 induction. Cancer Res., 57: 2452-2459, 1997.[Abstract/Free Full Text]
10. Winde G., Schmid K. W., Schlegel W., Fischer R., Osswald H., Bunte H. Complete reversion and prevention of rectal adenomas in colectomized patients with familial adenomatous polyposis by rectal low-dose sulindac maintenance treatment. Advantages of a low-dose nonsteroidal anti-inflammatory drug regimen in reversing adenomas exceeding 33 months. Dis. Colon Rectum, 38: 813-830, 1995.[Medline]
11. Chiu C. H., McEntee M. F., Whelan J. Sulindac causes rapid regression of preexisting tumors in Min/+ mice independent of prostaglandin biosynthesis. Cancer Res., 57: 4267-4273, 1997.[Abstract/Free Full Text]
12. Herrmann C., Block C., Geisen C., Haas K., Weber C., Winde G., Moroy T., Müller O. Sulindac sulfide inhibits Ras signaling. Oncogene, 17: 1769-1776, 1998.[Medline]
13. Taylor M. T., Lawson K. R., Ignatenko N. A., Marek S. E., Stringer D. E., Skovan B. A., Gerner E. W. Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expression in human colon cancer cells. Cancer Res., 60: 6607-6610, 2000.[Abstract/Free Full Text]
14. Rice P. L., Goldberg R. J., Ray E. C., Driggers L. J., Ahnen D. J. Inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and induction of apoptosis by sulindac metabolites. Cancer Res., 61: 1541-1547, 2001.[Abstract/Free Full Text]
15. Arber N., Han E. K., Sgambato A., Piazza G. A., Delohery T. M., Begemann M., Weghorst C. M., Kim N. H., Pamukcu R., Ahnen D. J., Reed J. C., Weinstein I. B., Holt P. R. A K-ras oncogene increases resistance to sulindac-induced apoptosis in rat enterocytes. Gastroenterology, 113: 1892-1900, 1997.[Medline]
16. Lawson K. R., Ignatenko N. A., Piazza G. A., Cui H., Gerner E. W. Influence of K-ras activation on the survival responses of Caco-2 cells to the chemopreventive agents sulindac and difluoromethylornithine. Cancer Epidemiol. Biomarkers Prev., 9: 1155-1162, 2000.[Abstract/Free Full Text]
17. Thompson H. J., Jiang C., Lu J., Mehta R. G., Piazza G. A., Paranka N. S., Pamukcu R., Ahnen D. J. Sulfone metabolite of sulindac inhibits mammary carcinogenesis. Cancer Res., 57: 267-271, 1997.[Abstract/Free Full Text]
18. Karaguni, I. M., Glüsenkamp, K. H., Langerak, A., Geisen, C., Ullrich, V., Winde, G., Moeroey, T., and Müller, O. New indene-derivatives with anti-proliferative properties. Bioorg. Med. Chem. Lett., in press.
19. Behrens J., Mareel M. M., Van Roy F. M., Birchmeier W. Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol., 108: 2435-2447, 1989.[Abstract/Free Full Text]
20. Vleminckx K., Vakaet L., Jr., Mareel M., Fiers W., van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell, 66: 107-119, 1991.[Medline]
21. Takeda H., Nagafuchi A., Yonemura S., Tsukita S., Behrens J., Birchmeier W., Tsukita S. V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift. J. Cell Biol., 131: 1839-1847, 1995.[Abstract/Free Full Text]
22. Boterberg T., Bracke M. E., Bruyneel E. A., Mareel M. Cell aggregation assays Brooks S. A. Schumacher U. eds. . Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols Vol. 2: Cell behavior in vitro and in vivo, : 33-45, Humana Press New York 2001.
23. Vakaet L., Jr., Vleminckx K., van Roy F., Mareel M. Numerical evaluation of the invasion of closely related cell lines into collagen type I gels. Invasion Metastasis, 11: 249-260, 1991.[Medline]
24. Bracke M. E., Boterberg T., Bruyneel E. A., Mareel M. Collagen invasion assays Brooks S. A. Schumacher U. eds. . Methods in Molecular Medicine Vol. 58: Metastasis Research Protocols Vol. 2: Cell behavior in vitro and in vivo, : 81-89, Humana Press New York 2001.
25. Zondag G. C., Evers E. E., ten Klooster J. P., Janssen L., van der Kammen R. A., Collard J. G. Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition. J. Cell Biol., 149: 775-782, 2000.[Abstract/Free Full Text]
26. Sander E. E., van Delft S., ten Klooster J. P., Reid T., van der Kammen R. A., Michiels F., Collard J. G. Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J. Cell Biol., 143: 1385-1398, 1998.[Abstract/Free Full Text]
27. Cowley S., Paterson H., Kemp P., Marshall C. J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell, 77: 841-852, 1994.[Medline]
28. Janknecht R. Analysis of the ERK-stimulated ETS transcription factor ER81. Mol. Cell. Biol., 16: 1550-1556, 1996.[Abstract]
29. Wixler V., Smola U., Schuler M., Rapp U. Differential regulation of Raf isozymes by growth versus differentiation inducing factors in PC12 pheochromocytoma cells. FEBS Lett., 385: 131-137, 1996.[Medline]
30. Flory E., Weber C. K., Chen P., Hoffmeyer A., Jassoy C., Rapp U. R. Plasma membrane-targeted Raf kinase activates NF-B and human immunodeficiency virus type 1 replication in T lymphocytes. J. Virol., 72: 2788-2794, 1998.[Abstract/Free Full Text]
31. Gumbiner B. M. Regulation of cadherin adhesive activity. J. Cell Biol., 148: 399-404, 2000.[Abstract/Free Full Text]
32. Hordijk P. L., ten Klooster J. P., van der Kammen R. A., Michiels F., Oomen L. C., Collard J. G. Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science (Wash. DC), 278: 1464-1466, 1997.[Abstract/Free Full Text]

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