Zampanolide, a Microtubule-Stabilizing Agent, Is Active in Resistant Cancer Cells and Inhibits Cell Migration

International Journal of Molecular Sciences Article Zampanolide, a Microtubule-Stabilizing Agent, Is Active in Resistant Cancer Cells and Inhibits Cell Migration Jessica J. Field 1,2,†, Peter T. Northcote 1,3, Ian Paterson 4, Karl-Heinz Altmann 5, J. Fernando Díaz 6and John H. Miller 1,2,* 1Centre for Biodiscovery and Schools, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand; [email protected] (J.J.F.); [email protected] (P.T.N.) 2Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand 3Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand 4Department of Chemistry, Cambridge University, Cambridge CB2 1EW, UK; [email protected] 5Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH), Zürich 8093, Switzerland; [email protected] 6Centro de Investigaciones Biológicas (CIB), CSIC, Madrid 28040, Spain; [email protected] *Correspondence: [email protected]; Tel.: +64-4-463-6082 † Present address: Amgen Inc., 1120 Veterans Blvd, South San Francisco, CA 94080, USA. Academic Editor: Bing Yan Received: 1 April 2017; Accepted: 28 April 2017; Published: 3 May 2017 Abstract: Zampanolide, first discovered in a sponge extract in 1996 and later identified as a microtubule-stabilizing agent in 2009, is a covalent binding secondary metabolite with potent, low nanomolar activity in mammalian cells. Zampanolide was not susceptible to single amino acid mutations at the taxoid site of β -tubulin in human ovarian cancer 1A9 cells, despite evidence that it selectively binds to the taxoid site. As expected, it did not synergize with other taxoid site microtubule-stabilizing agents (paclitaxel, ixabepilone, discodermolide), but surprisingly also did not synergize in 1A9 cells with laulimalide/peloruside binding site agents either. Efforts to generate a zampanolide-resistant cell line were unsuccessful. Using a standard wound scratch assay in cell culture, it was an effective inhibitor of migration of human umbilical vein endothelial cells (HUVEC) and fibroblast cells (D551). These properties of covalent binding, the ability to inhibit cell growth in paclitaxel and epothilone resistant cells, and the ability to inhibit cell migration suggest that it would be of interest to investigate zampanolide in preclinical animal models to determine if it is effective in vivo at preventing tumor growth and metastasis. Keywords: anticancer; cell migration; discodermolide; ixabepilone; microtubule; paclitaxel; zampanolide 1. Introduction Zampanolide (ZMP) is a marine sponge secondary metabolite that stabilizes microtubules (MTs), arrests cells in mitosis, and inhibits cell proliferation in the low nanomolar range [ 1 ]. ZMP binds covalently to its primary target β -tubulin [ 2 ], similar to two other microtubule-stabilizing agents (MSAs) cyclostreptin [ 3 ] and taccalonolide AJ [ 4 ]. Because of its covalent binding [ 5 ], ZMP may evade multi-drug resistance that results from overexpression of the p-glycoprotein (P-gp) drug efflux pump, since any ZMP that covalently binds is no longer available to interact with the drug efflux pumps [ 5 ]. Previous work in the A2780AD human ovarian carcinoma cell line showed that the resistance ratio was 1.4 for ZMP and 208 for paclitaxel (PTX) [ 1 ]. Resistance ratio = (IC 50 in resistant cells)/(IC 50 in parental cells). A2780AD cells overexpress the P-gp drug efflux pump. Cell resistance to drugs can Int. J. Mol. Sci. 2017,18, 971; doi:10.3390/ijms18050971 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017,18, 971 2 of 18 also arise, however, as a result of changes in β -tubulin isotype expression and mutations in the tubulin gene, particularly those mutations that affect the binding pocket of an MSA. Cabral et al. [ 6 ] were the first to show resistance to PTX resulting from a tubulin mutation induced by irradiation, in this case a temperature-sensitive mutation in α -tubulin that conferred 2–3-fold resistance to PTX in Chinese hamster ovary (CHO) cells. Schibler and Cabral [ 7 ] later showed in CHO cells that 59 out of 139 PTX-resistant mutants displayed absolute requirements for PTX, presumably due to unstable MTs, and 13 of these mutants had point mutations in α - and β -tubulin. Yin et al. [ 8 ] later generated β 1-tubulin mutants by site-directed mutagenesis and found that three mutations, Ala187Thr, Ala250Val, and Arg308Cys, caused PTX and epothilone resistance, as well as increased sensitivity to MT-destabilizing agents. Giannakakou et al. [ 9 , 10 ], using ovarian carcinoma 1A9 cells, investigated the effect of different point mutations induced by the selection in high concentrations of the MSAs PTX and epothilone. Single point mutations were found in β 1-tubulin that led to resistance to one or the other MSA, or to both. Later studies by Kanakkanthara et al. [ 11 ] and Begaye et al. [ 12 ] described the effect of similar mutations at the laulimalide/peloruside binding site. Arguments for and against point mutations significantly contributing to cancer cell resistance in the clinic exist; however, it is now generally accepted that mutations can lead to the resistance of tumor cells in patients [8,13,14]. One area of interest in MSA anticancer chemotherapy is the ability of two drugs, when given in combination, to synergize together and have a greater than additive effect on growth inhibition than when given alone. The delivery of two drugs also reduces the likelihood that a cancer cell can acquire resistance to one of the two drugs when the other is still present to prevent survival of the cell. Two binding sites for MSAs have been identified, the taxoid site on β -tubulin that faces the inside of the assembled MT [ 15 ] and the laulimalide/peloruside site on the external face of the assembled MT [ 16 ]. The methods of stabilization differ between these two sites [ 15 , 16 ]; hence, PTX synergizes with peloruside or laulimalide, but not with epothilone or docetaxel [ 17 – 19 ]. Discodermolide, another taxoid site MSA, synergizes with PTX, presumably because the two MSAs occupy distinct positions in the taxoid binding pocket and have different mechanisms of stabilization [ 20 – 23 ]. Discodermolide has recently been shown to stabilize the M-loop of tubulin in a different way to PTX, and this at least partially explains the synergy between the two taxoid site ligands [ 24 ]. Clinical interest is strong on combination therapy between MSAs; however, most combination trials in the literature that involve MSAs are in combination with other classes of anti-cancer drugs that do not target the MT. Metastasis of cancer cells from a primary tumor to other sites in the body is a major, and lethal, problem in cancer. Metastasis involves a complex cascade of events, beginning with the formation of a tumor blood supply, followed by escape of tumor cells from the primary tumor mass, invasion and migration through the extracellular matrix/basal lamina utilizing heparanase and matrix metalloproteinase enzymes, entry into the tumor blood supply, and later extravasation at a distant site to form secondary tumor foci [ 25 ]. Cell migratory ability is an important component of the process and is also necessary for angiogenesis and tumor vascularization [ 26 ]. Metastatic ability can be monitored in culture by measuring cell migration using methods such as the wound scratch assay [ 27 ], Boyden chambers to measure transmembrane migration and invasion [ 28 ], as well as binding to and migration into a collagenous basement membrane matrix such as Matrigel ™ that models the extracellular matrix of cells [ 29 ]. MSAs have been shown to inhibit cell migration in culture, a property that may enhance their overall inhibitory effect on cancer cell proliferation and improve patient survival [ 30 – 33 ]. The inhibition of MT dynamics and cytoskeletal regulatory molecules such as Rho-GTPases have been shown to play a role in cell movement over a substratum [ 34 , 35 ]. Ganguly et al. [ 36 , 37 ] directly investigated the effects of MT-targeting drugs on cell migration and showed that their effects on cell movement occur at concentrations lower than those required to block cell proliferation or to alter tubulin polymer formation. The MTs at the leading edge of the cell are more static than the dynamic MTs at the trailing edge of the cell. The latter allow reorganisation and remodeling of the MT skeleton. Blocking MT dynamicity does not stop the movement of cells but makes it more random, since MTs restrain cell movement by controlling the trailing portion of the migrating cell. Treatment with a Int. J. Mol. Sci. 2017,18, 971 3 of 18 MT-targeting drug prevents tail retraction, but lamillipodia extension still occurs, and the cell can still elongate. The aim of the present study was to further characterize the action of ZMP in cultured cells, comparing its activity to other MSAs, both taxoid site and laulimalide/peloruside site binding agents. The MSAs investigated included the taxoid site ligands, ZMP, PTX, (Taxol ® Bristol-Myers Squibb), docetaxel (Taxotere ® , Rhone Poulenc Rorer), ixabepilone (Ixempra ® , Bristol-Myers Squibb), and discodermolide, and the laulimalide/peloruside site ligands, laulimalide and peloruside A (see Figure 1for structures of the compounds). The ability of ZMP to remain active in tubulin mutant cell lines was investigated. In addition, the ability of ZMP to synergize with other MSAs was tested, and its effects on cell migration in a wound scratch assay were determined. Int. J. Mol. Sci. 2017, 18, 971 3 of 18 migrating cell. Treatment with a MT-targeting drug prevents tail retraction, but lamillipodia extension still occurs, and the cell can still elongate. The aim of the present study was to further characterize the action of ZMP in cultured cells, comparing its activity to other MSAs, both taxoid site and laulimalide/peloruside site binding agents. The MSAs investigated included the taxoid site ligands, ZMP, PTX, (Taxol ® Bristol-Myers Squibb), docetaxel (Taxotere ® , Rhone Poulenc Rorer), ixabepilone (Ixempra ® , Bristol-Myers Squibb), and discodermolide, and the laulimalide/peloruside site ligands, laulimalide and peloruside A (see Figure 1 for structures of the compounds). The ability of ZMP to remain active in tubulin mutant cell lines was investigated. In addition, the ability of ZMP to synergize with other MSAs was tested, and its effects on cell migration in a wound scratch assay were determined. Figure 1. Structure of the compounds. 2. Results 2.1. Growth Inhibition by Zampanolide and Other Microtubule-Stabilizing Agents in Different Cell Lines Human 1A9 ovarian carcinoma cells were treated with MSAs, including ZMP, three other taxoid site MSAs, and two laulimalide/peloruside site MSAs, and the IC 50 values for inhibition of proliferation were calculated (Table 1). The IC 50 values ranged from 3.6 to 23.3 nM. ZMP was then tested in other cell lines to determine the consistency of its action and to compare natural ZMP isolated and purified from a marine sponge [1] with chemically synthesized ZMP [38] (Table 2). Figure 1. Structure of the compounds. 2. Results 2.1. Growth Inhibition by Zampanolide and Other Microtubule-Stabilizing Agents in Different Cell Lines Human 1A9 ovarian carcinoma cells were treated with MSAs, including ZMP, three other taxoid site MSAs, and two laulimalide/peloruside site MSAs, and the IC 50 values for inhibition of proliferation were calculated (Table 1). The IC 50 values ranged from 3.6 to 23.3 nM. ZMP was then tested in other cell lines to determine the consistency of its action and to compare natural ZMP isolated and purified from a marine sponge [ 1 ] with chemically synthesized ZMP [ 38 ] (Table 2). Although the IC 50 values Int. J. Mol. Sci. 2017,18, 971 4 of 18 varied to some extent between different cell lines, there was no major difference between the natural ZMP and the synthetic ZMP. Table 1. IC50 values for microtubule-stabilizing agents in 1A9 cells. Compound IC50 ±SEM (nM) Taxoid site ligands Zampanolide 9.54 ±0.85 Paclitaxel 3.71 ±0.30 Docetaxel 3.55 ±0.43 Ixabepilone 6.65 ±0.33 Discodermolide 138 ±12 Laulimalide/Peloruside site ligands Peloruside A 23.3 ±1.1 Laulimalide 9.71 ±0.28 IC 50 values in 1A9 cells (mean ± SEM) after 48 h of drug treatment are presented (n= the number of independent biological replicates). Table 2. Cytotoxicity of zampanolide (ZMP) in different cell lines. Cell Line Source of ZMP IC50 ±SEM (nM) Duration (h) 1A9 natural 8.2 ±0.1 72 1A9 synthetic 4.6 ±1.3 72 1A9 [1] natural 14.3 ±2.4 72 HL-60 [1] natural 4.3 ±1.1 48 D551 synthetic 7.3 ±1.2 72 HUVEC synthetic 0.6 ±0.1 72 HUVEC synthetic 1.0 ±0.4 120 IC 50 values of zampanolide in different cell lines determined using the MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay. Duration is the time in which each cell line was treated with zampanolide before MTT was added; nis the number of independent biological replicates. 2.2. Action of Zampanolide on Cells with β-Tubulin Mutations The effect of mutant tubulins on the activity of ZMP was investigated using a collection of 1A9 cell lines that were generated by treatment for extended periods of time to step-wise increases in an MSA, resulting in single amino acid mutations in β 1-tubulin [ 9 – 11 ]. The spontaneous, stable mutations were either located at the taxoid site or at the laulimalide/peloruside site on tubulin (Table 3). The resistance ratios (IC 50 mutant/IC 50 parent) are graphed in Figure 2, and the IC 50 values are presented in Table 3. The actual values for the resistance ratios are presented in Supplementary Data Table S1. There was some crossover in the specificity of the mutations generated by high concentrations of PTX or epothilone A, with the PTX10 and A8 cell lines being resistant to both PTX and ixabepilone. B10, the mutant cell line generated by high concentrations of epothilone B, also showed significant crossover with both PTX and ixabepilone showing reduced potency in that cell line. A similar crossover was seen for the 1A9-L4 cell line generated in the presence of high concentrations of laulimalide which was resistant to both laulimalide and peloruside. None of the mutant taxoid site cell lines showed any major resistance to zampanolide, although the resistance ratio for PTX22 was 2.4 ± 0.2 (p-value just greater than the cut-off for significance of p< 0.05) and the resistance ratio for B10 was 3.2 ± 0.6 (p< 0.02). Int. J. Mol. Sci. 2017,18, 971 5 of 18 Table 3. IC50 values for MSAs in 1A9 parental cells and β-tubulin mutant cell lines. Cell Line Resistance to Paclitaxel Ixabepilone Zampanolide Peloruside A Laulimalide 1A9 4.2 ±0.3 7.3 ±0.6 8.2 ±1.0 20.1 ±0.9 8.3 ±0.5 PTX10 PTX and EPO 91.7 ±8.2 54.9 ±9.6 2.3 ±0.9 17.5 ±1.2 11.0 ±1.0 PTX22 PTX 100 ±14.1 11.4 ±1.7 9.2 ±3.9 19.4 ±5.4 10.6 ±2.7 A8 EPO and PTX 94.4 ±5.6 99.8 ±0.6 14.9 ±4.6 14.0 ±2.4 7.2 ±1.1 B10 EPO 17.2 ±4.3 106 ±6.5 8.6 ±3.2 24.9 ±1.9 10.8 ±1.0 1A9-R1 PLA 8.8 ±2.5 14.7 ±3.3 5.9 ±1.6 90.9 ±8.5 9.8 ±1.5 1A9-L4 LAU and PLA 4.2 ±0.1 4.4 ±0.4 4.7 ±1.0 351 ±126 344 ±150 The average 72 h IC 50 values of different MSAs in the parental 1A9 cell line and cloned mutant 1A9 cell lines are presented as the mean IC 50 value ± SEM (n= 3 or more biological replicates). The specific mutations for each cell line are: PTX10 Phe272Val; PTX22 Ala374Thr; A8 Thr276Ile; B10 Arg284Gln; 1A9-R1 Ala298Thr; 1A9-L4 Arg308His(70%)/Cys(30%). Resistance ratios are presented in Figure 2and Supplementary Data Table S1. PTX = paclitaxel, EPO = epothilone, PLA = peloruside A, and LAU = laulimalide. Int. J. Mol. Sci. 2017, 18, 971 5 of 18 Figure 2. Resistance ratios of MSAs in β-tubulin mutant cell lines. β-Tubulin mutant cell lines and the parental 1A9 cell line were treated with serial dilutions of MSAs for 3 days, and the IC50 values were calculated. Resistance ratios (mutant cell IC50/parental cell IC50) for (A) Paclitaxel; (B) Ixabepilone; (C) Laulimalide; (D) Peloruside A, and (E) zampanolide are presented as the mean ± SEM, n ≥ 3 independent experiments. The specific IC50 values are included in Table 3. A one-sample Student’s ttest was carried out to determine if the resistance ratios were significantly different from 1.0 (* p < 0.05; ** p < 0.01; *** p < 0.001). Table 3. IC50 values for MSAs in 1A9 parental cells and β-tubulin mutant cell lines. Cell Line Resistance to Paclitaxel Ixabepilone Zampanolide Peloruside A Laulimalide 1A9 4.2 ± 0.3 7.3 ± 0.6 8.2 ± 1.0 20.1 ± 0.9 8.3 ± 0.5 PTX10 PTX and EPO 91.7 ±8.2 54.9 ± 9.6 2.3 ± 0.9 17.5 ± 1.2 11.0 ± 1.0 PTX22 PTX 100 ± 14.1 11.4 ± 1.7 9.2 ± 3.9 19.4 ± 5.4 10.6 ± 2.7 A8 EPO and PTX 94.4 ± 5.6 99.8 ± 0.6 14.9 ± 4.6 14.0 ± 2.4 7.2 ± 1.1 B10 EPO 17.2 ± 4.3 106 ± 6.5 8.6 ± 3.2 24.9 ± 1.9 10.8 ± 1.0 1A9-R1 PLA 8.8 ± 2.5 14.7 ± 3.3 5.9 ± 1.6 90.9 ± 8.5 9.8 ± 1.5 1A9-L4 LAU and PLA 4.2 ± 0.1 4.4 ± 0.4 4.7 ± 1.0 351 ± 126 344 ± 150 The average 72 h IC50 values of different MSAs in the parental 1A9 cell line and cloned mutant 1A9 cell lines are presented as the mean IC50 value ± SEM (n = 3 or more biological replicates). The specific mutations for each cell line are: PTX10 Phe272Val; PTX22 Ala374Thr; A8 Thr276Ile; B10 Arg284Gln; 1A9-R1 Ala298Thr; 1A9-L4 Arg308His(70%)/Cys(30%). Resistance ratios are presented in Figure 2 and Supplementary Data Table S1. PTX = paclitaxel, EPO = epothilone, PLA = peloruside A, and LAU = laulimalide. Figure 2. Resistance ratios of MSAs in β -tubulin mutant cell lines. β -Tubulin mutant cell lines and the parental 1A9 cell line were treated with serial dilutions of MSAs for 3 days, and the IC 50 values were calculated. Resistance ratios (mutant cell IC 50 /parental cell IC 50 ) for ( A ) Paclitaxel; ( B ) Ixabepilone; ( C ) Laulimalide; ( D ) Peloruside A, and ( E ) zampanolide are presented as the mean ± SEM, n ≥ 3 independent experiments. The specific IC 50 values are included in Table 3. A one-sample Student’s t-test was carried out to determine if the resistance ratios were significantly different from 1.0 ( *p< 0.05 ; ** p< 0.01; *** p< 0.001). Int. J. Mol. Sci. 2017,18, 971 6 of 18 An attempt was made to generate a ZMP-resistant cell line by culturing 1A9 cells for approximately one year in gradually increasing concentrations of ZMP, similar to the procedure used to generate the PTX-, epothilone-, peloruside-, and laulimalide-resistant 1A9 cell lines. The pretreatment with ZMP, however, failed to generate a ZMP-resistant cell line and actually led to a cell line that was slightly more sensitive to ZMP (resistance ratio of 0.59). Despite not being resistant to ZMP, the cells acquired significant resistance to PTX (resistance ratio of 11.2), suggesting a mutation in β -tubulin at or near the taxoid site. However, there was no resistance to ixabepilone (resistance ratio 0.49), nor to peloruside A and laulimalide (resistance ratios of 0.66 and 0.40, respectively). ZMP has been shown by both Flutax competition experiments [ 2 , 39 ] and X-ray crystallography [ 15 ] to bind at the taxoid site, yet taxoid site amino acid mutations had little effect on its interactions with tubulin. We previously showed that a high concentration of PTX could compete for bound Flutax-2 but not at a low concentration, whereas because ZMP binds covalently to the taxoid site [ 2 ], both high and low concentrations of ZMP could displace the Flutax-2 [ 2 , 39 ] (Figure 3). Peloruside A, as expected, was unable to displace Flutax-2 because it binds at a distant, non-taxoid site on β -tubulin [ 16 , 40 ]. In the present study, we therefore tested other MSAs to see if they were effective in displacing Flutax and found that other taxoid site agents, including docetaxel, ixabepilone, and discodermolide, could displace Flutax similar to PTX, but laulimalide, similar to peloruside A, could not (Figure 3). Int. J. Mol. Sci. 2017, 18, 971 6 of 18 An attempt was made to generate a ZMP-resistant cell line by culturing 1A9 cells for approximately one year in gradually increasing concentrations of ZMP, similar to the procedure used to generate the PTX-, epothilone-, peloruside-, and laulimalide-resistant 1A9 cell lines. The pretreatment with ZMP, however, failed to generate a ZMP-resistant cell line and actually led to a cell line that was slightly more sensitive to ZMP (resistance ratio of 0.59). Despite not being resistant to ZMP, the cells acquired significant resistance to PTX (resistance ratio of 11.2), suggesting a mutation in β-tubulin at or near the taxoid site. However, there was no resistance to ixabepilone (resistance ratio 0.49), nor to peloruside A and laulimalide (resistance ratios of 0.66 and 0.40, respectively). ZMP has been shown by both Flutax competition experiments [2,39] and X-ray crystallography [15] to bind at the taxoid site, yet taxoid site amino acid mutations had little effect on its interactions with tubulin. We previously showed that a high concentration of PTX could compete for bound Flutax-2 but not at a low concentration, whereas because ZMP binds covalently to the taxoid site [2], both high and low concentrations of ZMP could displace the Flutax-2 [2,39] (Figure 3). Peloruside A, as expected, was unable to displace Flutax-2 because it binds at a distant, non-taxoid site on β-tubulin [16,40]. In the present study, we therefore tested other MSAs to see if they were effective in displacing Flutax and found that other taxoid site agents, including docetaxel, ixabepilone, and discodermolide, could displace Flutax similar to PTX, but laulimalide, similar to peloruside A, could not (Figure 3). Figure 3. Competition for Flutax-2 binding of cellular microtubules by different microtubulestabilizing agents. Figure 3. Competition for Flutax-2 binding of cellular microtubules by different microtubulestabilizing agents. Int. J. Mol. Sci. 2017,18, 971 7 of 18 HL-60 human promyelocytic leukemic cells were co-treated with a combination of a microtubule-stabilizing agent (MSA) and Flutax-2 (FTX) for 16 h, stained with (4 0 ,6-diamidino-2phenylindole) (DAPI), and the fluorescence of the cells was examined in a confocal microscope. Taxoid site ligands, when in excess at 200 versus 50 nM FTX, inhibit FTX binding since most of the taxoid sites are occupied by the non-fluorescent taxoid site ligand, thus no green fluorescent microtubles (MTs) are seen. When FTX is in excess to the taxoid site ligands, the MTs fluoresce green since FTX occupies most of the binding sites on the MTs. Regardless of the concentration of laulimalide (LAU) or peloruside A (PEL), the MTs always fluoresce green since simultaneous binding of these two MSAs at the LAU/PEL site and Flutax-2 (FTX) at the taxoid site can occur. When zampanolide (ZMP) is in excess over FTX, no green fluorescence of the MT is apparent, as was seen with the other taxoid site ligands. In contrast, when FTX is in excess, no green fluorescence is present, even at a low concentration of ZMP (25 nM). This lack of green fluorescence when FTX is in excess indicates that ZMP can out-compete FTX because it covalently occupies the taxoid binding site, preventing FTX from displacing it. The PTX, PEL, and ZMP images have been previously published [ 39 ] and are shown with permission from the publisher Elsevier. Abbreviations are: PTX = paclitaxel; DTX = docetaxel; IXA = ixabepilone; DSC = discodermolide, and FTX = Flutax-2. 2.3. Lack of Synergistic Interactions between ZMP and Other MSAs MSAs that bind to different sites on tubulin have been shown to synergize together if the appropriate concentrations of the two compounds are combined, as shown in Figure 4. CI values for Figure 4are presented in Supplementary Data Table S2. For example, in 1A9 cells, peloruside A in combination with ixabepilone had a greater effect on cell growth than the sum of the two compounds. PTX, however, did not synergize with ixabepilone, because PTX and ixabepilone bind at the same site, the taxoid site. The exception to the rule is discodermolide, a taxoid site MSA that has previously been shown to synergize with PTX [ 20 – 22 ], despite being able to compete for Flutax binding (Figure 3). The 1A9 ovarian carcinoma cells were therefore treated with ZMP in combination with three other taxoid site MSAs and two laulimalide/peloruside site MSAs to determine if ZMP could synergize with either group of compounds (Figure 5). CI values for Figure 5are presented in Supplementary Data Table S3. No synergy was seen between ZMP and either a taxoid site MSA or a laulimalide/peloruside site MSA. Some antagonism was observed between PTX and ZMP, and laulimalide and ZMP. 2.4. Zampanolide Effect on Cell Migration in Culture To ascertain whether ZMP has the potential to prevent metastasis of cancer cells, its ability to inhibit cell migration in a wound scratch assay was carried out. The ability of human umbilical vein endothelial cells (HUVEC) or D551 human fibroblasts to repair a scratch made in the monolayer with a pipette tip was tested in the absence and presence of ZMP and docetaxel (Figure 6). Both compounds significantly inhibited wound recovery in both cell lines in a concentration-dependent manner. Docetaxel was slightly more potent than ZMP at inhibiting cell migration. Int. J. Mol. Sci. 2017,18, 971 8 of 18 Int. J. Mol. Sci. 2017, 18, 971 8 of 18 Figure 4. Synergistic interactions between MSAs. The Combination Index (CI) is graphed for combinations of MSAs in 1A9 cells given two at a time: (A) Paclitaxel (PTX) and discodermolide (DSC); (B) Paclitaxel and docetaxel (DXT); (C) Paclitaxel and ixabepilone (IXA), and (D) peloruside A (PELA) and ixabepilone are presented as the mean CI value ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; one-sample Student’s t-test compared to 1.0. Values less than 1.0 indicate synergy between the two compounds; values greater than 1.0 indicate antagonism; values equal to 1.0 indicate additivity. B A D C Figure 4. Synergistic interactions between MSAs. The Combination Index (CI) is graphed for combinations of MSAs in 1A9 cells given two at a time: ( A ) Paclitaxel (PTX) and discodermolide (DSC); ( B ) Paclitaxel and docetaxel (DXT); ( C ) Paclitaxel and ixabepilone (IXA), and ( D ) peloruside A (PELA) and ixabepilone are presented as the mean CI value ± SEM. * p< 0.05; ** p< 0.01; *** p< 0.001; one-sample Student’s t-test compared to 1.0. Values less than 1.0 indicate synergy between the two compounds; values greater than 1.0 indicate antagonism; values equal to 1.0 indicate additivity. Int. J. Mol. Sci. 2017,18, 971 9 of 18 1 A B D C E Figure 5. Synergistic interactions of ZMP with selected MSAs. The Combination Index (CI) is graphed for ZMP given in combination with other MSAs: ( A ) Paclitaxel (PTX); ( B ) Ixabepilone (IXE); ( C ) Peloruside A (PEL); ( D ) Laulimalide (LAU), and ( E ) discodermolide (DSC). Data are presented as the mean CI value ± SEM. * p< 0.05; ** p< 0.01; *** p< 0.001; one-sample Student’s t-test compared to 1.0. Values less than 1.0 indicate synergy between the two compounds; values greater than 1.0 indicate antagonism; values equal to 1.0 indicate additivity. Int. J. Mol. Sci. 2017,18, 971 16 of 18 3. Buey, R.M.; Calvo, E.; Barasoain, I.; Pineda, O.; Edler, M.C.; Matesanz, R.; Cerezo, G.; Vanderwal, C.D.; Day, B.W.; Sorensen, E.J.; et al. Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites. Nat. Chem. Biol. 2007,3, 117–125. [CrossRef] [PubMed] 4. Risinger, A.L.; Li, J.; Bennett, M.J.; Rohena, C.C.; Schriemer, D.C.; Mooberry, S.L. Taccalonolide binding to tubulin imparts microtubule stability and potent in vivo activity. Cancer Res. 2013 ,73, 6780–6792. [CrossRef] [PubMed] 5. Singh, J.; Petter, R.C.; Baillie, T.A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011 , 10, 307–317. [CrossRef] [PubMed] 6. Cabral, F.; Abraham, I.; Gottesman, M.M. Isolation of a taxol-resistant Chinese hamster ovary cell mutant that has an alteration in α-tubulin. Proc. Natl. Acad. Sci. USA 1981,78, 4388–4391. [CrossRef] [PubMed] 7. Schibler, M.J.; Cabral, F. Taxol-dependent mutants of Chinese hamster ovary cells with alterations in α - and β-tubulin. J. Cell Biol. 1986,102, 1522–1531. [CrossRef] [PubMed] 8. Yin, S.; Bhattacharya, R.; Cabral, F. Human mutations that confer paclitaxel resistance. Mol. Cancer Ther. 2010,9, 327–335. [CrossRef] [PubMed] 9. Giannakakou, P.; Sackett, D.L.; Kang, Y.K.; Zhan, Z.; Buters, J.T.; Fojo, T.; Poruchynsky, M.S. Paclitaxelresistant human ovarian cancer cells have mutant β -tubulins that exhibit impaired paclitaxel-driven polymerization. J. Biol. Chem. 1997,272, 17118–17125. [CrossRef] [PubMed] 10. Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K.H.; Zaharevitz, D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K.C.; Fojo, T. A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA 2000 ,97, 2904–2909. [CrossRef] [PubMed] 11. Kanakkanthara, A.; Wilmes, A.; O ' Brate, A.; Escuin, D.; Chan, A.; Gjyrezi, A.; Crawford, J.; Rawson, P.; Kivell, B.; Northcote, P.T.; et al. Pelorusideand laulimalide-resistant human ovarian carcinoma cells have β I-tubulin mutations and altered expression of β IIand β III-tubulin isotypes. Mol. Cancer Ther. 2011 ,10, 1419–1429. [CrossRef] [PubMed] 12. Begaye, A.; Trostel, S.; Zhao, Z.; Taylor, R.E.; Schriemer, D.C.; Sackett, D.L. Mutations in the β -tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding. Cell Cycle 2011,10, 3387–3396. [CrossRef] [PubMed] 13. Yin, S.H.; Zeng, C.Q.; Hari, M.; Cabral, F. Random mutagenesis of β -tubulin defines a set of dispersed mutations that confer paclitaxel resistance. Pharm. Res. 2012,29, 2994–3006. [CrossRef] [PubMed] 14. Basciano, P.A.; Matakas, J.; Pecci, A.; Civaschi, E.; Cagioni, C.; Bompiani, N.; Burger, P.; Christos, P.; Snyder, J.P.; Bussel, J.; et al. β -1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia. J. Thromb. Haemost. 2015,13, 651–659. [CrossRef] [PubMed] 15. Prota, A.E.; Bargsten, K.; Zurwerra, D.; Field, J.J.; Díaz, J.F.; Altmann, K.-H.; Steinmetz, M.O. Molecular mechanism of action of microtubule-stabilizing anticancer agents. Science 2013 ,339, 587–590. [CrossRef] [PubMed] 16. Prota, A.E.; Bargsten, K.; Northcote, P.T.; Marsh, M.; Altmann, K.-H.; Miller, J.H.; Díaz, J.F.; Steinmetz, M.O. Structural basis of microtubule stabilization by laulimalide and peloruside A. Angew. Chem. Int. Ed. Engl. 2014,53, 1621–1625. [CrossRef] [PubMed] 17. Clark, E.A.; Hills, P.M.; Davidson, B.S.; Wender, P.A.; Mooberry, S.L. Laulimalide and synthetic laulimalide analogues are synergistic with paclitaxel and 2-methoxyestradiol. Mol. Pharm. 2006 ,3, 457–467. [CrossRef] [PubMed] 18. Wilmes, A.; Bargh, K.; Kelly, C.; Northcote, P.T.; Miller, J.H. Peloruside A synergizes with other microtubule stabilizing agents in cultured cancer cell lines. Mol. Pharm. 2007,4, 269–280. [CrossRef] [PubMed] 19. Wilmes, A.; O’Sullivan, D.; Chan, A.; Chandrahasen, C.; Paterson, I.; Northcote, P.T.; La Flamme, A.C.; Miller, J.H. Synergistic interactions between peloruside A and other microtubule-stabilizing and destabilizing agents in cultured human ovarian carcinoma cells and murine T cells. Cancer Chemother. Pharmacol. 2011 ,68, 117–126. [CrossRef] [PubMed] 20. Giannakakou, P.; Fojo, T. Discodermolide: Just another microtubule-stabilizing agent? No! A lesson in synergy. Clin. Cancer Res. 2000,6, 1613–1615. [PubMed] 21. Martello, L.A.; McDaid, H.M.; Regl, D.L.; Yang, C.P.; Meng, D.; Pettus, T.R.; Kaufman, M.D.; Arimoto, H.; Danishefsky, S.J.; Smith, A.B., 3rd; et al. Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines. Clin. Cancer Res. 2000,6, 1978–1987. [PubMed] Int. J. Mol. Sci. 2017,18, 971 17 of 18 22. Honore, S.; Kamath, K.; Braguer, D.; Horwitz, S.B.; Wilson, L.; Briand, C.; Jordan, M.A. Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells. Cancer Res. 2004,64, 4957–4964. [CrossRef] [PubMed] 23. Khrapunovich-Baine, M.; Menon, V.; Verdier-Pinard, P.; Smith, A.B., 3rd; Angeletti, R.H.; Fiser, A.; Horwitz, S.B.; Xiao, H. Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization. Biochemistry 2009,48, 11664–11677. [CrossRef] [PubMed] 24. Prota, A.E.; Bargsten, K.; Redondo, M.; Smith, A.B., 3rd; Yang, C.H.; McDaid, H.M.; Paterson, I.; Horwitz, S.B.; Díaz, J.F.; Steinmetz, M.O. Structural basis of microtubule stabilization by discodermolide. ChemBioChem 2017. [CrossRef] 25. Brooks, S.A.; Lomax-Browne, H.J.; Carter, T.M.; Kinch, C.E.; Hall, D.M.S. Molecular interactions in cancer cell metastasis. Acta Histochem. 2010,112, 3–25. [CrossRef] [PubMed] 26. Tozer, G.M.; Kanthou, C.; Baguley, B.C. Disrupting tumour blood vessels. Nat. Rev. Cancer 2005 ,5, 423–435. [CrossRef] [PubMed] 27. Liang, C.C.; Park, A.Y.; Guan, J.L. In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007,2, 329–333. [CrossRef] [PubMed] 28. Falasca, M.; Raimondi, C.; Maffucci, T. Boyden chamber. Methods Mol. Biol. 2011,769, 87–95. [PubMed] 29. Hall, D.M.; Brooks, S.A. In vitro invasion assay using Matrigel ™ : A reconstituted basement membrane preparation. Methods Mol. Biol. 2014,1070, 1–11. [PubMed] 30. Belotti, D.; Vergani, V.; Drudis, T.; Borsotti, P.; Pitelli, M.R.; Viale, G.; Giavazzi, R.; Taraboletti, G. The microtubule-affecting drug paclitaxel has antiangiogenic activity. Clin. Cancer Res. 1996 ,2, 1843–1849. [PubMed] 31. Lu, H.; Murtagh, J.; Schwartz, E.L. The microtubule binding drug laulimalide inhibits vascular endothelial growth factor-induced human endothelial cell migration and is synergistic when combined with docetaxel (Taxotere). Mol. Pharmacol. 2006,69, 1207–1215. [CrossRef] 32. Yang, H.; Ganguly, A.; Cabral, F. Inhibition of cell migration and cell division correlates with distinct effects of microtubule inhibiting drugs. J. Biol. Chem. 2010,285, 32242–32250. [CrossRef] [PubMed] 33. Chan, A.; Singh, A.J.; Northcote, P.T.; Miller, J.H. Inhibition of human vascular endothelial cell migration and capillary-like tube formation by the microtubule-stabilizing agent peloruside A. Investig. New Drugs 2015,33, 564–574. [CrossRef] [PubMed] 34. Kamath, K.; Smiyun, G.; Wilson, L.; Jordan, M.A. Mechanisms of inhibition of endothelial cell migration by taxanes. Cytoskeleton 2014,71, 46–60. [CrossRef] [PubMed] 35. Kaverina, I.; Straube, A. Regulation of cell migration by dynamic microtubules. Semin. Cell. Dev. Biol. 2011 , 22, 968–974. [CrossRef] [PubMed] 36. Ganguly, A.; Yang, H.; Sharma, R.; Patel, K.D.; Cabral, F. The role of microtubules and their dynamics in cell migration. J. Biol. Chem. 2012,287, 43359–43369. [CrossRef] [PubMed] 37. Ganguly, A.; Yang, H.; Zhang, H.; Cabral, F.; Patel, K.D. Microtubule dynamics control tail retraction in migrating vascular endothelial cells. Mol. Cancer Ther. 2013,12, 2837–2846. [CrossRef] [PubMed] 38. Zurwerra, D.; Glaus, F.; Betschart, L.; Schuster, J.; Gertsch, J.; Ganci, W.; Altmann, K.-H. Total synthesis of ( − )-zampanolide and structure-activity relationship studies on ( − )-dactylolide derivatives. Chemistry 2012 , 18, 16868–16883. [CrossRef] [PubMed] 39. Field, J.J.; Calvo, E.; Northcote, P.T.; Miller, J.H.; Altmann, K.-H.; Díaz, J.F. Methods for studying microtubule binding site interactions: Zampanolide as a covalent binding agent. In Methods in Cell Biology, Microtubules in Vitro, 2nd ed.; Wilson, L., Correia, J.J., Eds.; Academic Press: Burlington, NJ, USA, 2013; Volume 115, pp. 303–325. 40. Gaitanos, T.N.; Buey, R.M.; Díaz, J.F.; Northcote, P.T.; Teesdale-Spittle, P.; Andreu, J.M.; Miller, J.H. Peloruside A does not bind to the taxoid site on β -tubulin and retains its activity in multidrug-resistant cell lines. Cancer Res. 2004,64, 5063–5067. [CrossRef] [PubMed] 41. Dumontet, C.; Jordan, M.A. Microtubule-binding agents: A dynamic field of cancer therapeutics. Nat. Rev. Drug Discov. 2010,9, 790–803. [CrossRef] [PubMed] 42. Risinger, A.L.; Jackson, E.M.; Polin, L.A.; Helms, G.L.; LeBoeuf, D.A.; Joe, P.A.; Hopper-Borge, E.; Ludueña, R.F.; Kruh, G.D.; Mooberry, S.L. The taccalonolides: Microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms. Cancer Res. 2008,68, 8881–8888. [CrossRef] [PubMed] Int. J. Mol. Sci. 2017,18, 971 18 of 18 43. Kanakkanthara, A.; Teesdale-Spittle, P.H.; Miller, J.H. Cytoskeletal alterations that confer resistance to anti-tubulin chemotherapeutics. Anticancer Agents Med. Chem. 2013,13, 147–158. [CrossRef] [PubMed] 44. Field, J.J.; Díaz, J.F.; Miller, J.H. The binding sites of microtubule-stabilizing agents. Chem. Biol. 2013 ,20, 301–315. [CrossRef] [PubMed] 45. Kanakkanthara, A.; Eras, J.; Northcote, P.T.; Cabral, F.; Miller, J.H. Resistance to peloruside A and laulimalide: Functional significance of acquired β I-tubulin mutations at sites important for drug-tubulin binding. Curr. Cancer Drug Targets. 2014,14, 79–90. [CrossRef] [PubMed] 46. Gapud, E.J.; Bai, R.; Ghosh, A.K.; Hamel, E. Laulimalide and paclitaxel: A comparison of their effects on tubulin assembly and their synergistic action when present simultaneously. Mol. Pharmacol. 2004 ,66, 113–121. [CrossRef] [PubMed] 47. Hamel, E.; Day, B.W.; Miller, J.H.; Jung, M.K.; Northcote, P.T.; Ghosh, A.K.; Curran, D.P.; Cushman, M.; Nicolaou, K.C.; Paterson, I.; et al. Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly. Mol. Pharmacol. 2006,70, 1555–1564. [CrossRef] [PubMed] 48. Photiou, A.; Shah, P.; Leong, L.K.; Moss, J.; Retsas, S. In vitro synergy of paclitaxel (Taxol) and vinorelbine (navelbine) against human melanoma cell lines. Eur. J. Cancer 1997,33, 463–470. [CrossRef] 49. Giannakakou, P.; Villalba, L.; Li, H.; Poruchynsky, M.; Fojo, T. Combinations of paclitaxel and vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: Characterization of a synergistic schedule. Int. J. Cancer 1998,75, 57–63. [CrossRef] 50. Ricker, J.L.; Chen, Z.; Yang, X.P.; Pribluda, V.S.; Swartz, G.M.; van Waes, C. 2-methoxyestradiol inhibits hypoxia-inducible factor 1 α , tumor growth, and angiogenesis and augments paclitaxel efficacy in head and neck squamous cell carcinoma. Clin. Cancer Res. 2004,10, 8665–8673. [CrossRef] [PubMed] 51. Han, G.Z.; Liu, Z.J.; Shimoi, K.; Zhu, B.T. Synergism between the anticancer actions of 2-methoxyestradiol and microtubule-disrupting agents in human breast cancer. Cancer Res. 2005,65, 387–393. [PubMed] 52. Huang, G.S.; Lopez-Barcons, L.; Freeze, B.S.; Smith, A.B., 3rd; Goldberg, G.L.; Horwitz, S.B.; McDaid, H.M. Potentiation of taxol efficacy by discodermolide in ovarian carcinoma xenograft-bearing mice. Clin. Cancer Res. 2006,12, 298–304. [CrossRef] [PubMed] 53. Khrapunovich-Baine, M.; Menon, V.; Yang, C.P.; Northcote, P.T.; Miller, J.H.; Angeletti, R.H.; Fiser, A.; Horwitz, S.B.; Xiao, H. Hallmarks of molecular action of microtubule stabilizing agents: Effects of epothilone B, ixabepilone, peloruside A, and laulimalide on microtubule conformation. J. Biol. Chem. 2011 ,286, 11765–11778. [CrossRef] [PubMed] 54. Ganguly, A.; Cabral, F.; Yang, H.; Patel, K. Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells. Oncoscience 2015,2, 585–595. [CrossRef] [PubMed] 55. Paterson, I.; Florence, G.J.; Gerlach, K.; Scott, J.P. Total synthesis of the antimicrotubule agent (+)-discodermolide using boron-mediated aldol reactions of chiral ketones. Angew. Chem. Int. Ed. Engl. 2000 , 39, 377–380. [CrossRef] 56. West, L.M.; Northcote, P.T.; Battershill, C.N. Peloruside A: A potent cytotoxic macrolide isolated from the New Zealand marine sponge Mycale sp. J. Org. Chem. 2000,65, 445–449. [CrossRef] [PubMed] 57. Hood, K.A.; West, L.M.; Northcote, P.T.; Berridge, M.V.; Miller, J.H. Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine. Apoptosis 2001 ,6, 207–219. [CrossRef] [PubMed] 58. Chou, T.C.; Talalay, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984,22, 27–55. [CrossRef] 59. Berenbaum, M.C. The expected effect of a combination of agents: The general solution. J. Theor. Biol. 1985 , 114, 413–431. [CrossRef] 60. Tanaka, J.; Higa, T. Zampanolide, a new cytotoxic macrolide from a marine sponge. Tetrahedron Lett. 1996 ,37, 5535–5538. [CrossRef] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).