Sceptrin, a marine natural compound, inhibits cell motility in a variety of cancer cell lines

Abstract

Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility.

Figure 1

Sceptrin inhibits HGF-induced cell motility in HeLa cells. a) HeLa cells were treated with sceptrin at indicated concentrations, and cell motility was induced by hepatocyte growth factor (HGF) (10 ng mL⁻¹). Cells were allowed to migrate for 24 h before fixation. A representative picture (10x objective) of one well for each condition is shown. Three independent experiments done in triplicate for each condition were carried out. b) ImageJ software was used to segment and quantify cell tracks of the cell motility assay shown in panel a. Graph represents surface on arbitrary units of the cell tracks. c) Effect of synthetic and natural sceptrin (40 μM) in cell motility is compared. HeLa cells were induced to migrate as described for panel a. Each condition was tested in triplicate. Cell tracks were analyzed as described for panel b. d) Dose-dependent experiment was carried out in HeLa cells at indicated sceptrin concentrations. Each condition was tested in triplicate. Quantification of cell motility was done as described in panel b. Surface tracks were plotted, and a logarithmic trend line is shown.

Figure 2

Effect of sceptrin on cell motility in different cancer cell lines. a−c) Indicated cells were seeded on top of fluorescence beads and treated with sceptrin (40 μM). Basal random motility was measured for cells in panels a and b. Motility was stimulated by hepatocyte growth factor (HGF) (10 ng mL⁻¹) in panel c. Cells were fixed after 24 h, a picture of each well (all the conditions were assayed in triplicate) was taken, and the surface of the cell tracks was segmented and quantified.

Figure 3

Effect of sceptrin on apoptosis and cell proliferation. a) Effect of sceptrin on cell proliferation was tested in HeLa cells, which were seeded at low density and treated with vehicle, sceptrin (40 μM), or positive control aphidicolin (5 nM). Proliferation was quantified by cell counting at indicated times. b) Putative induction of apoptosis by sceptrin was assayed in HeLa cells, which were treated with sceptrin at indicated concentrations or with the positive control staurosporine (5 nM) for 24 h. Apoptosis was quantified by immunochemical determination of histone-complexed DNA fragments in a microplate well.

Figure 4

Structure of synthesized sceptrin-derived compounds. Five different sceptrin-derived compounds were synthesized to test their ability to block cell motility. In summary, extra bromide groups were added or original bromides were removed from the pyrrole group of sceptrin to generate the bromo, dibromo, or debromo compounds. Nakamuric compounds lack one of the aminoimidazole groups of sceptrin. On the oxysceptrin compound an oxygen is added to one of the aminoimidazole groups.

Figure 5

Effect on cell motility of the various sceptrin-derived compounds. HeLa cells were allowed to migrate in the presence of hepatocyte growth factor (HGF) (10 ng mL⁻¹). As indicated cells were treated with sceptrin or sceptrin-derivatives (40 μM) for 24 h as described above. Control cells were treated with vehicle (H₂O). Cell motility was quantified as described in Methods.

Figure 6

Time-lapse confocal video microscopy of cells treated with sceptrin. a) Effect of sceptrin on cell motility dynamics was tested on MDA-MB-231 cells transiently transfected with GFP-actin construct. Cells that had been seeded on fibronectin-coated LabTek chambers were pictured (20x objective) every 2 min up to 3 h. Panel a shows an RGB composite still image taken at time 0, 1, 2, and 3 h, where red represents 0 h, green 1 h, blue 2 h and white 3 h. Actual movies can be seen in online Supporting Information. b) Effect of sceptrin on HeLa cells was assayed as described for panel a. HeLa cells were induced to migrate by addition of hepatocyte growth factor (HGF) (10 ng mL⁻¹). A 40x objective was used to take the pictures in this case.

Figure 7

Inhibition of cell contractility and actin binding by sceptrin. a) Cell contractility was assayed using the clot retraction assay. CHOα_IIbβ₃ cells treated with vehicle, sceptrin (40 μM), or 10 μM Calpain inhibitor XI (positive control) were mixed with human serum, thrombin (2 U mL⁻¹), and CaCl₂ (5 mM). Clot was allowed to retract for 2 h, and still images were taken. All conditions were tested in triplicate and assayed in three independent experiments. Panel a shows a representative picture of each condition. b) 2D size of clots was segmented and quantified using the NIH ImageJ software. c) Isothermal titration calorimetry (ITC) binding curve after subtraction of the heat associated with sceptrin dilution. K_d = 19.4 ± 0.2 μM for sceptrin binding to G-actin is an average obtained from two independent measurements.