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Experimental Therapeutics |
Departments of Anatomy [C. D. R., L. M. M.], Chemistry and Oceanography (EOS) [D. E. W., R. J. A.], and Biochemistry and Molecular Biology [L. M. M., M. R.], University of British Columbia, Vancouver, British Columbia V6T 1Z3; Departments of Pathology and Medical Biophysics, British Columbia Cancer Agency, Vancouver, British Columbia V5Z IL3 [K. G. L., A. K.]; and Jack Bell Research Laboratories, Vancouver, British Columbia V6H 3Z6 [A. T., S. D.], Canada
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Invasion, angiogenesis, and metastasis all require cells to modify their adhesion to other cells and to the extracellular matrix, to break down the matrix, and to migrate through the breaches thus created (3 , 4) . Therefore, agents that inhibit the movement of tumor cells and endothelial cells through the extracellular matrix have the potential to be of considerable therapeutic value.
We have developed a quantitative assay suitable for testing crude natural extracts for inhibitors of this process. In an initial small-scale screen, we found that a family of marine sponge alkaloids, the motuporamines, inhibit the invasion of metastatic MDA-231 breast carcinoma cells. In subsequent testing, we demonstrate that motuporamine C interferes with the migration and leading edge ruffling of human breast cancer cells, prostate carcinoma cells, and glioma cells. In addition, motuporamine C inhibits angiogenesis in both an in vitro sprouting assay and an in vivo chick chorioallantoic membrane assay. These properties, combined with its low cytotoxicity and ease of synthesis, make motuporamine C an attractive drug candidate.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Marine Organism Collection and Extract Preparation.
Approximately 250 g each of marine sponges were collected by hand, using scuba, from tropical Pacific Ocean reefs off Motupore and Madang in Papua, New Guinea. Samples were deep frozen on site and transported to Vancouver, British Columbia, Canada over dry ice. Voucher samples of each are kept in methanol at -20°C at the University of British Columbia for taxonomic identification. Extracts were prepared by homogenizing 200 g of each sponge sample in methanol. The homogenates were filtered and concentrated in vacuo to give a gummy residue. About 1 mg was dissolved in 100 µl of DMSO for use in the invasion inhibition screen.
Three-step Assay for Invasion Inhibitors.
Reconstituted basement membrane (Matrigel; Collaborative Biomedical Products) was diluted 1:1 in ice-cold DMEM:Ham’s F-12, and 50 µl was distributed into each well of ice-cold 96-well cell culture plates. The plates were transferred to a 37°C incubator overnight to allow the Matrigel to polymerize and adhere to the plastic. On top of the Matrigel was added 100 µl of growth medium warmed to 37°C, with or without 1 µl of sponge extract dissolved in DMSO, followed by 100 µl of medium containing 60,000 highly invasive MDA-231 cells. Addition of 1 µl of DMSO served as a negative control and 125 µM LY294002 served as a positive control. The cells were then incubated for 2.5 h to allow invasion to take place. After incubation, cells had either invaded the Matrigel or failed to invade and settled on the surface of the Matrigel. The cell culture medium was then removed without disturbing the cells using the aspiration function of a Bio-Tek ELx405 96-well plate washer with the aspiration needles positioned about 2 mm above the surface of the Matrigel. The attached cells that failed to invade were recovered by detaching them from the surface of the Matrigel by incubation with 200 µl of 0.125% trypsin in HBSS for 30 min at 37°C. The cells were then suspended by pipetting up and down three times using the 100-µl setting of a hand-held pipettor, and 100 µl were withdrawn and transferred to fresh plates without Matrigel containing 100 µl of medium supplemented with 30% FBS. The cells were then incubated overnight to allow attachment of cells to the plastic surface, and live cells were measured using the MTT assay (5)
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Isolation of Motuporamines.
A portion of the sponge (86 g) was extracted repeatedly with methanol. The combined methanol extracts were concentrated in vacuo and partitioned between water and ethyl acetate to give a water layer active in the assay. n-butyl alcohol extracted the bioactive material from the water layer. Concentration of the n-butyl alcohol extracts in vacuo gave an active residue that was suspended in water adjusted to pH >12 by addition of NaOH. Extraction of the basic aqueous solution with CH2Cl2 followed by evaporation of the combined CH2Cl2 extracts in vacuo gave a residue active in the assay. The bioactive residue was first fractionated via Sephadex LH20 chromatography eluting with methanol followed by a second Sephadex LH20 chromatography eluting with the mixed solvent system ethyl acetate/methanol/water (20:5:2). The active materials were further fractionated by reversed-phase high-performance liquid chromatography [eluent, 2% trifluoroacetic acid in methanol/water (11:9)] to give pure samples of the known compounds motuporamines A and C (6)
, a sample of monoacetylmotuporamine C (an artifact resulting from reaction of motuporamine C with ethyl acetate during purification), and mixtures of a number of minor related analogues. Diacetylated derivatives of the motuporamines were prepared by dissolving them in pyridine/acetic anhydride (3:1) and stirring at room temperature for 16 h. Removal of the reagents by evaporation in vacuo gave diacetylated motuporamines that were purified via reversed-phase high-performance liquid chromatography [eluent, acetonitrile/0.6% trifluoroactic acid in H2O (2:3)].
Integrin Activation State.
Integrin expression on the cell surface was analyzed by flow cytometry. PC3 cells were serum-starved overnight and then incubated for 1 h in medium without serum containing the indicated concentrations of motuporamine C. Cells were washed and harvested by scraping. Cells were suspended in 200 µl of PBS (pH 7.4), 20 mM glucose, and 1% BSA and incubated with 4 µg of anti-activated ß1 integrin (MAB2079Z; Chemicon) or total-ß1 integrin (Upstate Biotechnology) monoclonal antibody. After a 45-min incubation at room temperature, cells were incubated with 1 µg of FITC-conjugated antimouse secondary antibody (Jackson Laboratories) for 30 min at room temperature. Cells were analyzed on a Coulter EXPO XL4 flow cytometer. Experiments were performed in duplicate and repeated three times.
Endothelial Sprouting Assay.
Endothelial sprouting was assessed by a modification of the method used by Nehls and Drenckhahn (7)
. Briefly, microcarrier beads coated with denatured collagen (Cytodex 3; Sigma Chemical Co.) were seeded with HUVECs and embedded in fibrin gels in 96-well plates. For preparation of fibrin gels, bovine fibrinogen was dissolved in MCDB medium at a concentration of 2.5 mg/ml. Aprotinin was added at a concentration of 0.05 mg/ml, and the solution was passed through a 0.22-µm filter. The fibrinogen solution was supplemented with 15 ng/ml VEGF. As a control, fibrinogen solution without VEGF was used. Motuporamine C was also added at different concentrations, and the fibrinogen solutions were transferred to 96-well plates together with HUVEC-coated beads at a density of 50 beads/well. Clotting was induced by the addition of thrombin (1.2 units/ml). After clotting was complete, gels were equilibrated with MCDB medium containing 5% FBS at 37°C. After 60 min of incubation, the medium was replaced with medium with or without motuporamine C. After 3 days of incubation with daily changes of the medium, the number of capillary-like tubes formed/microcarrier bead (sprouts/bead) was counted by microscopy, monitoring at least 150 beads for each treatment. Only sprouts greater that 150 µm in length and composed of at least three endothelial cells were counted.
Chick CAM Assay for Angiogenesis.
Fertilized White Leghorn chicken eggs were incubated at 37°C under conditions of constant humidity. On embryonic day 6, the developing CAM was separated from the shell by opening a small circular window at the broad end of the egg above the air sac. The opening was sealed with Parafilm, and the eggs were incubated for 2 more days. Motuporamine C was prepared in PBS supplemented with 30 ng/ml VEGF. On day 8, 20 µl was loaded onto 2-mm3 gelatin sponges (Gelfoam; Pharmacia Upjohn) that were placed on the surface of the developing CAM. Sponges containing vehicle alone (20 µl of PBS) were used as negative controls, whereas sponges containing 20 µl of 30 ng/ml VEGF in PBS were used as positive controls. Eggs were resealed and returned to the incubator. On day 10, images of CAM were captured digitally using an Olympus SZX9 stereomicroscope equipped with a Spot RT digital imaging system (Diagnostic Instruments).
Cell Viability and Proliferation Assays.
HUVEC viability was determined as follows. Cells were plated in 96-well plates at 1.5 x 105 cells/well. When cells reached 95% confluency, motuporamine C was added at different concentrations and for different times, with daily change of medium and drug, and cell viability was measured by incubating cells with 100 µl of 0.005% neutral red in cell culture medium for 4 h. The medium was removed, and 100 µl of 1% acetic acid in 50% ethanol was added/well to solubilize the dye and absorbance was measured at 550 nm. HUVEC proliferation was determined as follows. Cells were plated in 96-well plates at 5 x 103 cells/well and incubated with different concentrations of motuporamine C, with daily change of medium and drug. Cell proliferation was measured at 0, 24, 48, and 72 h using neutral red as described above. MDA 231 proliferation was determined as follows. Cells were plated in 96-well plates at 1 x 103 cells and treated with different concentrations of motuporamine C for 24 h. Proliferation was measured at different times using the MTT assay (5)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nature is a prime source of drug leads (10) . However, an inherent problem with using crude natural extracts in cell-based screens is that 10–20% of the extracts are toxic to cells at the dilutions that generate optimal hit rates because of the high concentrations of salts and other materials they contain. To avoid unacceptably high numbers of false-positive results, a screen needs to distinguish invasion inhibition attributable to a specific inhibitor from that caused by cell death or toxicity. We have combined the principles of the Matrigel outgrowth and the Boyden chamber assays in a three-step screen for invasion inhibitors. The assay is quantitative and screens sequentially for compounds that prevent invasion of Matrigel, do not prevent cell attachment to Matrigel, and are not cytotoxic.
We first tested the suitability of the assay for drug screening using a small selection of crude methanol extracts from marine sponges. Two-hundred and thirty extracts were tested at 50–100 µg/ml. Two-hundred and twenty-eight extracts showed absorbance readings close to or below those of the DMSO carrier negative controls (0.025). Two extracts showed strong inhibition of invasion (Fig. 1A)
higher than LY294002, a phosphatidylinositol 3-kinase inhibitor known to inhibit invasion (11)
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, two macrocyclic alkaloids with a spermidine-like "tail" (6)
. The compounds showed concentration-dependent activity with IC50 of 3 µM for motuporamine A and 1 µM for motuporamine C. Only motuporamine C was obtained in sufficient quantities for further study. As expected from the design of the screening assay, motuporamine C did not inhibit the proliferation of MDA-231 cells at a concentration close to the IC50 for invasion inhibition (Fig. 2)
. Higher concentrations of 4 and 8 µM showed only a mild inhibition of cell proliferation.
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. HCl treatment of diacetylated motuporamine C removed both acetates and restored activity (Fig. 3)
. Therefore, the terminal amino group is dispensable for activity, but the central amine is absolutely required.
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. It is noteworthy that the motuporamine C concentration that strongly inhibited basement membrane invasion (5 µM) did not cause the cells to detach from the matrix. Instead, the cells remained rounded, attached, and, for the most part, single (Fig. 4A
, part d). Motuporamine C (5 µM) had similar morphological effects on prostate carcinoma (PC-3) and glioma (U-251 and U-87) cells (Table 1)
. Therefore, the activity observed in the initial three-step screen using MDA-231 cells correctly identified motuporamine C as a cancer cell invasion inhibitor, and the follow-up morphological experiments with other cell lines indicates widespread in vitro efficacy.
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. The effects of motuporamine C treatment on the actin cytoskeleton of pre-spread cells was subtle. Cytoplasmic actin stress fibers were still visible, but small discrete "buttons" of actin localization were observed at cell edges (Fig. 4B
, arrowheads). In contrast, treatment of pre-spread cells with the filamentous actin disrupting agent cytochalasin D (1 µg/ml) caused cells to round up and cytoplasmic actin filaments to disassemble (Fig. 4B)
. Therefore, motuporamine C did not cause a global disruption of the actin cytoskeleton in MDA-231 cells.
Motuporamine C Decreases Cell Migration.
The subtle effects of motuporamine C on cell shape and the actin cytoskeleton led us to suspect that it might inhibit invasion, at least in part, by decreasing cell migration. Therefore, we examined cell migration across "wounded" monolayers on a tissue culture plastic substratum. A sterile toothpick was drawn across a confluent monolayer of MDA-231 cells leaving a cell-free gap of about 100 µm. In control cultures, the cells migrated into the gap and obliterated it within 24 h (Fig. 5A)
. In contrast, in cultures treated with motuporamine C (5 µM) a significant gap was still present at 24 h. This inhibition of migration was also observed in prostate carcinoma and one of the glioma cell lines tested (Table 1)
. Although no significant differences in wound closure were observed between control and motuporamine C-treated MDA-231 cell cultures after only 8 h of treatment, we did notice a slight morphological difference at this earlier time point. In control cultures, cells at the edge of the wound had broad leading lamellae that were located in the direction of migration. As expected in migrating cells, the leading edge of control lamellae was continuous and phase-dark, which is indicative of ruffling membranes (Fig. 5B
, part a). This was confirmed by phalloidin staining, which demonstrated filamentous actin condensation in the ruffles (Fig. 5B
, part b). These lamellae were along their leading edges with actin condensations throughout. In contrast, cells treated with motuporamine C had only small and discontinuous ruffles along their leading edges (Fig. 5B
, part c). These small ruffles contained discrete patches of actin condensation (Fig. 5B
, part d), similar to the "button-like" condensations observed in pre-spread nonmigrating cells (see Fig. 4B
, part e). We also observed this fragmentation of leading edge ruffles in prostate carcinoma and glioma cell lines (Table 1)
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, exposure of PC-3 cells to motuporamine C resulted in a small dose-dependent reduction of the binding of the "activated" ß1 antibody compared with the total ß1 antibody. The results show that motuporamine C exposure causes conformational changes in ß1-integrins leading to a decrease of "activated" ß1 on the surface of PC-3 cells. This effect of motuporamine C was subtle but highly reproducible, consistent with the effects on cell spreading and migration (see Figs. 4
and 5
above) in the absence of discernable effects on cell adhesion per se.
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. In two separate experiments, endothelial sprouting quantitated at 72 h was inhibited by 88% and 93% with 5 µM motuporamine C and was inhibited completely with 10 µM motuporamine C (see Fig. 7
for examples). In the CAM assay, angiogenesis in response to VEGF (30 ng/ml) was reduced by treatment with 2.5 µM and 5 µM motuporamine C and was completely inhibited at 10 µM motuporamine C (Fig. 8)
. Motuporamine C was not toxic in the CAM assay when tested at concentrations of up to 25 µM (data not shown).
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. Exposure of confluent, nonproliferating HUVEC monolayers to different motuporamine concentrations for up to 3 days also had no effects on cell survival (Fig. 9B)
. Therefore, as was the case for invasion, motuporamine C does not inhibit angiogenesis through toxic or antiproliferative effects.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The motuporamines are a family of relatively simple macrocyclic alkaloids containing a spermidine-like substructure (6) . Comparison of the activities of the natural compounds and simple chemical modification of the tail of motuporamine C provided initial structure-activity information. The positively charged amine in the middle of the tail is a critical determinant of activity because its acetylation completely abrogated anti-invasion activity. However, acetylation of the terminal amino group had no detectable effect. The size of the macrocyclic ring had an influence on activity because motuporamines with smaller rings were slightly less active. Simple and inexpensive motuporamine synthetic schemes have been published recently (17 , 18) that will make possible further structure-activity study and eliminate dependence on natural sources.
The motuporamines show some resemblance to squalamine, an angiogenesis inhibitor (19) currently in Phase II clinical trials for the treatment of advanced non-small cell lung cancer (20) . Squalamine was isolated from dogfish shark liver and is a much more complex molecule composed of spermidine attached to C-3 of a steroid core with a sulfated side chain. The observation that both classes of compounds decrease cell migration, inhibit angiogenesis, and are composed of a macrocyclic ring attached to a polyamine raises the possibility that they act in a similar fashion. However, although the mechanism of action of squalamine is still unclear, it appears to inhibit both cell proliferation and migration (19) . Motuporamine C inhibits migration with little effect on proliferation, suggesting that the two compounds may have distinct cellular targets.
In this study, we carried out preliminary cell biological studies in an effort to broadly define the mechanism of action of motuporamine C. We found that doses of motuporamine C that inhibit both tumor cell invasion and endothelial cell angiogenesis are not cytotoxic to cancer cell lines and HUVECs. This characteristic, which was built into the original screen, is an important consideration with respect to the possible therapeutic usefulness of the drug. Invasive tumor cells in malleable basement membrane gels and endothelial cells in malleable fibrin gels remained rounded in the presence of motuporamine C. This could have been explained by global disruption of the actin cytoskeleton. However, experiments in monolayer culture do not support this possibility because cells were able to spread and form cytoplasmic actin stress fibers.
The ability of motuporamines to decrease tumor cell movement through basement membrane gels and endothelial cell movement through fibrin gels suggests that the drug acts to inhibit cell-mediated degradation of extracellular matrix and/or inhibit cell motility. It has not yet been determined whether motuporamine C alters matrix degradation. However, we have demonstrated that the compound decreased tumor cell motility and subtly altered the organization of filamentous actin at the cell margin. In particular, we observed an impairment of actin-mediated membrane ruffling in the leading lamellae of cells induced to migrate by wounding. This suggests at least two possible classes of molecular targets. The first class is the Rho-family GTPases, most specifically Rac, the activity of which is required for the formation of leading lamellae and ruffles (21 , 22) . The second class consists of molecules that regulate actin polymerization within ruffles. These include the Arp2/3 complex and regulators such as WASP (23) . Cellular adhesion to the extracellular matrix itself leads to the activation of Rho family GTPases and subcellular actin rearrangements, including those associated with leading edge ruffling (24) . Importantly, these events also provide feedback via an as yet poorly defined "inside-out" signaling mechanism to regulate the affinity of cell surface integrin receptors for their extracellular matrix ligands (25) . It has been proposed that such changes in integrin affinity help regulate the maturation of transient focal complexes at the leading edge of migrating cells into more mature, stable focal complexes in the cell body (26) . Motuporamine C may act to dampen this inside-out signaling because it subtly decreased the affinity state of ß1 integrins. Despite this modest decrease, we did not observe any disruption of tumor cell attachment to either basement membrane gels or tissue culture plastic. To our knowledge, compounds with analogous structure have not been documented to have effects on adhesion. We are currently addressing this by examining focal adhesion complex formation and signaling on a number of defined extracellular matrices.
Compounds that have the potential for inhibiting tumor cell invasion and angiogenesis are attractive candidates for cancer therapy. In combination with conventional cytotoxic chemotherapy agents, they may prove to be efficacious in controlling cancer progression. We are now carrying out experiments to evaluate the antitumor, antiangiogenesis, and antimetastasis activity of motuporamine C in animals and structure-activity studies to identify additional compounds for in vivo testing. Motuporamines showing in vivo activity will become attractive novel drug candidates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Canadian Breast Cancer Research Initiative (to C. D. R., M. R.), the Natural Sciences and Engineering Research Council of Canada (to R. J. A.), and the National Cancer Institute of Canada (S. D., A. K.). 
2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Phone: (604) 822-2304; Fax: (604) 822-5227; E-mail: michel{at}otter.biochem.ubc.ca
3 The abbreviations used are: FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CAM, chorioallantoic membrane.
Received 4/ 5/01. Accepted 7/18/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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