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

In e na ional Jou nal o Molecula Sciences A icle Zampanolide, a Mic o ubule-S abilizing Agen , Is Ac i e in Resis an Cance Cells and Inhibi s Cell Mig a ion Jessica J. Field 1,2,†, Pe e T. No hco e 1,3, Ian Pa e son 4, Ka l-Heinz Al mann 5, J. Fe nando Díaz 6and John H. Mille 1,2,* 1Cen e o Biodisco e y and Schools, Vic o ia Uni e si y o Welling on, PO Box 600, Welling on 6140, New Zealand; [email p o ec ed] (J.J.F.); pe e .no hco e@ uw.ac.nz (P.T.N.) 2Biological Sciences, Vic o ia Uni e si y o Welling on, PO Box 600, Welling on 6140, New Zealand 3Chemical and Physical Sciences, Vic o ia Uni e si y o Welling on, PO Box 600, Welling on 6140, New Zealand 4Depa men o Chemis y, Camb idge Uni e si y, Camb idge CB2 1EW, UK; [email p o ec ed] 5Depa men o Chemis y and Applied Biosciences, Swiss Fede al Ins i u e o Technology (ETH), Zü ich 8093, Swi ze land; [email p o ec ed] 6Cen o de In es igaciones Biológicas (CIB), CSIC, Mad id 28040, Spain; [email p o ec ed] *Co espondence: john.h.mille @ uw.ac.nz; Tel.: +64-4-463-6082 † P esen add ess: Amgen Inc., 1120 Ve e ans Bl d, Sou h San F ancisco, CA 94080, USA. Academic Edi o : Bing Yan Recei ed: 1 Ap il 2017; Accep ed: 28 Ap il 2017; Published: 3 May 2017 Abs ac : Zampanolide, i s disco e ed in a sponge ex ac in 1996 and la e iden i ied as a mic o ubule-s abilizing agen in 2009, is a co alen binding seconda y me aboli e wi h po en , low nanomola ac i i y in mammalian cells. Zampanolide was no suscep ible o single amino acid mu a ions a he axoid si e o β - ubulin in human o a ian cance 1A9 cells, despi e e idence ha i selec i ely binds o he axoid si e. As expec ed, i did no syne gize wi h o he axoid si e mic o ubule-s abilizing agen s (pacli axel, ixabepilone, discode molide), bu su p isingly also did no syne gize in 1A9 cells wi h laulimalide/pelo uside binding si e agen s ei he . E o s o gene a e a zampanolide- esis an cell line we e unsuccess ul. Using a s anda d wound sc a ch assay in cell cul u e, i was an e ec i e inhibi o o mig a ion o human umbilical ein endo helial cells (HUVEC) and ib oblas cells (D551). These p ope ies o co alen binding, he abili y o inhibi cell g ow h in pacli axel and epo hilone esis an cells, and he abili y o inhibi cell mig a ion sugges ha i would be o in e es o in es iga e zampanolide in p eclinical animal models o de e mine i i is e ec i e in i o a p e en ing umo g ow h and me as asis. Keywo ds: an icance ; cell mig a ion; discode molide; ixabepilone; mic o ubule; pacli axel; zampanolide 1. In oduc ion Zampanolide (ZMP) is a ma ine sponge seconda y me aboli e ha s abilizes mic o ubules (MTs), a es s cells in mi osis, and inhibi s cell p oli e a ion in he low nanomola ange [ 1 ]. ZMP binds co alen ly o i s p ima y a ge β - ubulin [ 2 ], simila o wo o he mic o ubule-s abilizing agen s (MSAs) cyclos ep in [ 3 ] and accalonolide AJ [ 4 ]. Because o i s co alen binding [ 5 ], ZMP may e ade mul i-d ug esis ance ha esul s om o e exp ession o he p-glycop o ein (P-gp) d ug e lux pump, since any ZMP ha co alen ly binds is no longe a ailable o in e ac wi h he d ug e lux pumps [ 5 ]. P e ious wo k in he A2780AD human o a ian ca cinoma cell line showed ha he esis ance a io was 1.4 o ZMP and 208 o pacli axel (PTX) [ 1 ]. Resis ance a io = (IC 50 in esis an cells)/(IC 50 in pa en al cells). A2780AD cells o e exp ess he P-gp d ug e lux pump. Cell esis ance o d ugs can In . J. Mol. Sci. 2017,18, 971; doi:10.3390/ijms18050971 www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2017,18, 971 2 o 18 also a ise, howe e , as a esul o changes in β - ubulin iso ype exp ession and mu a ions in he ubulin gene, pa icula ly hose mu a ions ha a ec he binding pocke o an MSA. Cab al e al. [ 6 ] we e he i s o show esis ance o PTX esul ing om a ubulin mu a ion induced by i adia ion, in his case a empe a u e-sensi i e mu a ion in α - ubulin ha con e ed 2–3- old esis ance o PTX in Chinese hams e o a y (CHO) cells. Schible and Cab al [ 7 ] la e showed in CHO cells ha 59 ou o 139 PTX- esis an mu an s displayed absolu e equi emen s o PTX, p esumably due o uns able MTs, and 13 o hese mu an s had poin mu a ions in α - and β - ubulin. Yin e al. [ 8 ] la e gene a ed β 1- ubulin mu an s by si e-di ec ed mu agenesis and ound ha h ee mu a ions, Ala187Th , Ala250Val, and A g308Cys, caused PTX and epo hilone esis ance, as well as inc eased sensi i i y o MT-des abilizing agen s. Giannakakou e al. [ 9 , 10 ], using o a ian ca cinoma 1A9 cells, in es iga ed he e ec o di e en poin mu a ions induced by he selec ion in high concen a ions o he MSAs PTX and epo hilone. Single poin mu a ions we e ound in β 1- ubulin ha led o esis ance o one o he o he MSA, o o bo h. La e s udies by Kanakkan ha a e al. [ 11 ] and Begaye e al. [ 12 ] desc ibed he e ec o simila mu a ions a he laulimalide/pelo uside binding si e. A gumen s o and agains poin mu a ions signi ican ly con ibu ing o cance cell esis ance in he clinic exis ; howe e , i is now gene ally accep ed ha mu a ions can lead o he esis ance o umo cells in pa ien s [8,13,14]. One a ea o in e es in MSA an icance chemo he apy is he abili y o wo d ugs, when gi en in combina ion, o syne gize oge he and ha e a g ea e han addi i e e ec on g ow h inhibi ion han when gi en alone. The deli e y o wo d ugs also educes he likelihood ha a cance cell can acqui e esis ance o one o he wo d ugs when he o he is s ill p esen o p e en su i al o he cell. Two binding si es o MSAs ha e been iden i ied, he axoid si e on β - ubulin ha aces he inside o he assembled MT [ 15 ] and he laulimalide/pelo uside si e on he ex e nal ace o he assembled MT [ 16 ]. The me hods o s abiliza ion di e be ween hese wo si es [ 15 , 16 ]; hence, PTX syne gizes wi h pelo uside o laulimalide, bu no wi h epo hilone o doce axel [ 17 – 19 ]. Discode molide, ano he axoid si e MSA, syne gizes wi h PTX, p esumably because he wo MSAs occupy dis inc posi ions in he axoid binding pocke and ha e di e en mechanisms o s abiliza ion [ 20 – 23 ]. Discode molide has ecen ly been shown o s abilize he M-loop o ubulin in a di e en way o PTX, and his a leas pa ially explains he syne gy be ween he wo axoid si e ligands [ 24 ]. Clinical in e es is s ong on combina ion he apy be ween MSAs; howe e , mos combina ion ials in he li e a u e ha in ol e MSAs a e in combina ion wi h o he classes o an i-cance d ugs ha do no a ge he MT. Me as asis o cance cells om a p ima y umo o o he si es in he body is a majo , and le hal, p oblem in cance . Me as asis in ol es a complex cascade o e en s, beginning wi h he o ma ion o a umo blood supply, ollowed by escape o umo cells om he p ima y umo mass, in asion and mig a ion h ough he ex acellula ma ix/basal lamina u ilizing hepa anase and ma ix me allop o einase enzymes, en y in o he umo blood supply, and la e ex a asa ion a a dis an si e o o m seconda y umo oci [ 25 ]. Cell mig a o y abili y is an impo an componen o he p ocess and is also necessa y o angiogenesis and umo ascula iza ion [ 26 ]. Me as a ic abili y can be moni o ed in cul u e by measu ing cell mig a ion using me hods such as he wound sc a ch assay [ 27 ], Boyden chambe s o measu e ansmemb ane mig a ion and in asion [ 28 ], as well as binding o and mig a ion in o a collagenous basemen memb ane ma ix such as Ma igel ™ ha models he ex acellula ma ix o cells [ 29 ]. MSAs ha e been shown o inhibi cell mig a ion in cul u e, a p ope y ha may enhance hei o e all inhibi o y e ec on cance cell p oli e a ion and imp o e pa ien su i al [ 30 – 33 ]. The inhibi ion o MT dynamics and cy oskele al egula o y molecules such as Rho-GTPases ha e been shown o play a ole in cell mo emen o e a subs a um [ 34 , 35 ]. Ganguly e al. [ 36 , 37 ] di ec ly in es iga ed he e ec s o MT- a ge ing d ugs on cell mig a ion and showed ha hei e ec s on cell mo emen occu a concen a ions lowe han hose equi ed o block cell p oli e a ion o o al e ubulin polyme o ma ion. The MTs a he leading edge o he cell a e mo e s a ic han he dynamic MTs a he ailing edge o he cell. The la e allow eo ganisa ion and emodeling o he MT skele on. Blocking MT dynamici y does no s op he mo emen o cells bu makes i mo e andom, since MTs es ain cell mo emen by con olling he ailing po ion o he mig a ing cell. T ea men wi h a In . J. Mol. Sci. 2017,18, 971 3 o 18 MT- a ge ing d ug p e en s ail e ac ion, bu lamillipodia ex ension s ill occu s, and he cell can s ill elonga e. The aim o he p esen s udy was o u he cha ac e ize he ac ion o ZMP in cul u ed cells, compa ing i s ac i i y o o he MSAs, bo h axoid si e and laulimalide/pelo uside si e binding agen s. The MSAs in es iga ed included he axoid si e ligands, ZMP, PTX, (Taxol ® B is ol-Mye s Squibb), doce axel (Taxo e e ® , Rhone Poulenc Ro e ), ixabepilone (Ixemp a ® , B is ol-Mye s Squibb), and discode molide, and he laulimalide/pelo uside si e ligands, laulimalide and pelo uside A (see Figu e 1 o s uc u es o he compounds). The abili y o ZMP o emain ac i e in ubulin mu an cell lines was in es iga ed. In addi ion, he abili y o ZMP o syne gize wi h o he MSAs was es ed, and i s e ec s on cell mig a ion in a wound sc a ch assay we e de e mined. In . J. Mol. Sci. 2017, 18, 971 3 o 18 mig a ing cell. T ea men wi h a MT- a ge ing d ug p e en s ail e ac ion, bu lamillipodia ex ension s ill occu s, and he cell can s ill elonga e. The aim o he p esen s udy was o u he cha ac e ize he ac ion o ZMP in cul u ed cells, compa ing i s ac i i y o o he MSAs, bo h axoid si e and laulimalide/pelo uside si e binding agen s. The MSAs in es iga ed included he axoid si e ligands, ZMP, PTX, (Taxol ® B is ol-Mye s Squibb), doce axel (Taxo e e ® , Rhone Poulenc Ro e ), ixabepilone (Ixemp a ® , B is ol-Mye s Squibb), and discode molide, and he laulimalide/pelo uside si e ligands, laulimalide and pelo uside A (see Figu e 1 o s uc u es o he compounds). The abili y o ZMP o emain ac i e in ubulin mu an cell lines was in es iga ed. In addi ion, he abili y o ZMP o syne gize wi h o he MSAs was es ed, and i s e ec s on cell mig a ion in a wound sc a ch assay we e de e mined. Figu e 1. S uc u e o he compounds. 2. Resul s 2.1. G ow h Inhibi ion by Zampanolide and O he Mic o ubule-S abilizing Agen s in Di e en Cell Lines Human 1A9 o a ian ca cinoma cells we e ea ed wi h MSAs, including ZMP, h ee o he axoid si e MSAs, and wo laulimalide/pelo uside si e MSAs, and he IC 50 alues o inhibi ion o p oli e a ion we e calcula ed (Table 1). The IC 50 alues anged om 3.6 o 23.3 nM. ZMP was hen es ed in o he cell lines o de e mine he consis ency o i s ac ion and o compa e na u al ZMP isola ed and pu i ied om a ma ine sponge [1] wi h chemically syn hesized ZMP [38] (Table 2). Figu e 1. S uc u e o he compounds. 2. Resul s 2.1. G ow h Inhibi ion by Zampanolide and O he Mic o ubule-S abilizing Agen s in Di e en Cell Lines Human 1A9 o a ian ca cinoma cells we e ea ed wi h MSAs, including ZMP, h ee o he axoid si e MSAs, and wo laulimalide/pelo uside si e MSAs, and he IC 50 alues o inhibi ion o p oli e a ion we e calcula ed (Table 1). The IC 50 alues anged om 3.6 o 23.3 nM. ZMP was hen es ed in o he cell lines o de e mine he consis ency o i s ac ion and o compa e na u al ZMP isola ed and pu i ied om a ma ine sponge [ 1 ] wi h chemically syn hesized ZMP [ 38 ] (Table 2). Al hough he IC 50 alues In . J. Mol. Sci. 2017,18, 971 4 o 18 a ied o some ex en be ween di e en cell lines, he e was no majo di e ence be ween he na u al ZMP and he syn he ic ZMP. Table 1. IC50 alues o mic o ubule-s abilizing agen s in 1A9 cells. Compound IC50 ±SEM (nM) Taxoid si e ligands Zampanolide 9.54 ±0.85 Pacli axel 3.71 ±0.30 Doce axel 3.55 ±0.43 Ixabepilone 6.65 ±0.33 Discode molide 138 ±12 Laulimalide/Pelo uside si e ligands Pelo uside A 23.3 ±1.1 Laulimalide 9.71 ±0.28 IC 50 alues in 1A9 cells (mean ± SEM) a e 48 h o d ug ea men a e p esen ed (n= he numbe o independen biological eplica es). Table 2. Cy o oxici y o zampanolide (ZMP) in di e en cell lines. Cell Line Sou ce o ZMP IC50 ±SEM (nM) Du a ion (h) 1A9 na u al 8.2 ±0.1 72 1A9 syn he ic 4.6 ±1.3 72 1A9 [1] na u al 14.3 ±2.4 72 HL-60 [1] na u al 4.3 ±1.1 48 D551 syn he ic 7.3 ±1.2 72 HUVEC syn he ic 0.6 ±0.1 72 HUVEC syn he ic 1.0 ±0.4 120 IC 50 alues o zampanolide in di e en cell lines de e mined using he MTT (3-(4,5-dime hyl hiazol- 2-yl)-2,5-diphenyl e azolium b omide) cell p oli e a ion assay. Du a ion is he ime in which each cell line was ea ed wi h zampanolide be o e MTT was added; nis he numbe o independen biological eplica es. 2.2. Ac ion o Zampanolide on Cells wi h β-Tubulin Mu a ions The e ec o mu an ubulins on he ac i i y o ZMP was in es iga ed using a collec ion o 1A9 cell lines ha we e gene a ed by ea men o ex ended pe iods o ime o s ep-wise inc eases in an MSA, esul ing in single amino acid mu a ions in β 1- ubulin [ 9 – 11 ]. The spon aneous, s able mu a ions we e ei he loca ed a he axoid si e o a he laulimalide/pelo uside si e on ubulin (Table 3). The esis ance a ios (IC 50 mu an /IC 50 pa en ) a e g aphed in Figu e 2, and he IC 50 alues a e p esen ed in Table 3. The ac ual alues o he esis ance a ios a e p esen ed in Supplemen a y Da a Table S1. The e was some c osso e in he speci ici y o he mu a ions gene a ed by high concen a ions o PTX o epo hilone A, wi h he PTX10 and A8 cell lines being esis an o bo h PTX and ixabepilone. B10, he mu an cell line gene a ed by high concen a ions o epo hilone B, also showed signi ican c osso e wi h bo h PTX and ixabepilone showing educed po ency in ha cell line. A simila c osso e was seen o he 1A9-L4 cell line gene a ed in he p esence o high concen a ions o laulimalide which was esis an o bo h laulimalide and pelo uside. None o he mu an axoid si e cell lines showed any majo esis ance o zampanolide, al hough he esis ance a io o PTX22 was 2.4 ± 0.2 (p- alue jus g ea e han he cu -o o signi icance o p< 0.05) and he esis ance a io o B10 was 3.2 ± 0.6 (p< 0.02). In . J. Mol. Sci. 2017,18, 971 5 o 18 Table 3. IC50 alues o MSAs in 1A9 pa en al cells and β- ubulin mu an cell lines. Cell Line Resis ance o Pacli axel Ixabepilone Zampanolide Pelo uside 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 a e age 72 h IC 50 alues o di e en MSAs in he pa en al 1A9 cell line and cloned mu an 1A9 cell lines a e p esen ed as he mean IC 50 alue ± SEM (n= 3 o mo e biological eplica es). The speci ic mu a ions o each cell line a e: PTX10 Phe272Val; PTX22 Ala374Th ; A8 Th 276Ile; B10 A g284Gln; 1A9-R1 Ala298Th ; 1A9-L4 A g308His(70%)/Cys(30%). Resis ance a ios a e p esen ed in Figu e 2and Supplemen a y Da a Table S1. PTX = pacli axel, EPO = epo hilone, PLA = pelo uside A, and LAU = laulimalide. In . J. Mol. Sci. 2017, 18, 971 5 o 18 Figu e 2. Resis ance a ios o MSAs in β- ubulin mu an cell lines. β-Tubulin mu an cell lines and he pa en al 1A9 cell line we e ea ed wi h se ial dilu ions o MSAs o 3 days, and he IC50 alues we e calcula ed. Resis ance a ios (mu an cell IC50/pa en al cell IC50) o (A) Pacli axel; (B) Ixabepilone; (C) Laulimalide; (D) Pelo uside A, and (E) zampanolide a e p esen ed as he mean ± SEM, n ≥ 3 independen expe imen s. The speci ic IC50 alues a e included in Table 3. A one-sample S uden ’s - es was ca ied ou o de e mine i he esis ance a ios we e signi ican ly di e en om 1.0 (* p < 0.05; ** p < 0.01; *** p < 0.001). Table 3. IC50 alues o MSAs in 1A9 pa en al cells and β- ubulin mu an cell lines. Cell Line Resis ance o Pacli axel Ixabepilone Zampanolide Pelo uside 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 a e age 72 h IC50 alues o di e en MSAs in he pa en al 1A9 cell line and cloned mu an 1A9 cell lines a e p esen ed as he mean IC50 alue ± SEM (n = 3 o mo e biological eplica es). The speci ic mu a ions o each cell line a e: PTX10 Phe272Val; PTX22 Ala374Th ; A8 Th 276Ile; B10 A g284Gln; 1A9-R1 Ala298Th ; 1A9-L4 A g308His(70%)/Cys(30%). Resis ance a ios a e p esen ed in Figu e 2 and Supplemen a y Da a Table S1. PTX = pacli axel, EPO = epo hilone, PLA = pelo uside A, and LAU = laulimalide. Figu e 2. Resis ance a ios o MSAs in β - ubulin mu an cell lines. β -Tubulin mu an cell lines and he pa en al 1A9 cell line we e ea ed wi h se ial dilu ions o MSAs o 3 days, and he IC 50 alues we e calcula ed. Resis ance a ios (mu an cell IC 50 /pa en al cell IC 50 ) o ( A ) Pacli axel; ( B ) Ixabepilone; ( C ) Laulimalide; ( D ) Pelo uside A, and ( E ) zampanolide a e p esen ed as he mean ± SEM, n ≥ 3 independen expe imen s. The speci ic IC 50 alues a e included in Table 3. A one-sample S uden ’s - es was ca ied ou o de e mine i he esis ance a ios we e signi ican ly di e en om 1.0 ( *p< 0.05 ; ** p< 0.01; *** p< 0.001). In . J. Mol. Sci. 2017,18, 971 6 o 18 An a emp was made o gene a e a ZMP- esis an cell line by cul u ing 1A9 cells o app oxima ely one yea in g adually inc easing concen a ions o ZMP, simila o he p ocedu e used o gene a e he PTX-, epo hilone-, pelo uside-, and laulimalide- esis an 1A9 cell lines. The p e ea men wi h ZMP, howe e , ailed o gene a e a ZMP- esis an cell line and ac ually led o a cell line ha was sligh ly mo e sensi i e o ZMP ( esis ance a io o 0.59). Despi e no being esis an o ZMP, he cells acqui ed signi ican esis ance o PTX ( esis ance a io o 11.2), sugges ing a mu a ion in β - ubulin a o nea he axoid si e. Howe e , he e was no esis ance o ixabepilone ( esis ance a io 0.49), no o pelo uside A and laulimalide ( esis ance a ios o 0.66 and 0.40, espec i ely). ZMP has been shown by bo h Flu ax compe i ion expe imen s [ 2 , 39 ] and X- ay c ys allog aphy [ 15 ] o bind a he axoid si e, ye axoid si e amino acid mu a ions had li le e ec on i s in e ac ions wi h ubulin. We p e iously showed ha a high concen a ion o PTX could compe e o bound Flu ax-2 bu no a a low concen a ion, whe eas because ZMP binds co alen ly o he axoid si e [ 2 ], bo h high and low concen a ions o ZMP could displace he Flu ax-2 [ 2 , 39 ] (Figu e 3). Pelo uside A, as expec ed, was unable o displace Flu ax-2 because i binds a a dis an , non- axoid si e on β - ubulin [ 16 , 40 ]. In he p esen s udy, we he e o e es ed o he MSAs o see i hey we e e ec i e in displacing Flu ax and ound ha o he axoid si e agen s, including doce axel, ixabepilone, and discode molide, could displace Flu ax simila o PTX, bu laulimalide, simila o pelo uside A, could no (Figu e 3). In . J. Mol. Sci. 2017, 18, 971 6 o 18 An a emp was made o gene a e a ZMP- esis an cell line by cul u ing 1A9 cells o app oxima ely one yea in g adually inc easing concen a ions o ZMP, simila o he p ocedu e used o gene a e he PTX-, epo hilone-, pelo uside-, and laulimalide- esis an 1A9 cell lines. The p e ea men wi h ZMP, howe e , ailed o gene a e a ZMP- esis an cell line and ac ually led o a cell line ha was sligh ly mo e sensi i e o ZMP ( esis ance a io o 0.59). Despi e no being esis an o ZMP, he cells acqui ed signi ican esis ance o PTX ( esis ance a io o 11.2), sugges ing a mu a ion in β- ubulin a o nea he axoid si e. Howe e , he e was no esis ance o ixabepilone ( esis ance a io 0.49), no o pelo uside A and laulimalide ( esis ance a ios o 0.66 and 0.40, espec i ely). ZMP has been shown by bo h Flu ax compe i ion expe imen s [2,39] and X- ay c ys allog aphy [15] o bind a he axoid si e, ye axoid si e amino acid mu a ions had li le e ec on i s in e ac ions wi h ubulin. We p e iously showed ha a high concen a ion o PTX could compe e o bound Flu ax-2 bu no a a low concen a ion, whe eas because ZMP binds co alen ly o he axoid si e [2], bo h high and low concen a ions o ZMP could displace he Flu ax-2 [2,39] (Figu e 3). Pelo uside A, as expec ed, was unable o displace Flu ax-2 because i binds a a dis an , non- axoid si e on β- ubulin [16,40]. In he p esen s udy, we he e o e es ed o he MSAs o see i hey we e e ec i e in displacing Flu ax and ound ha o he axoid si e agen s, including doce axel, ixabepilone, and discode molide, could displace Flu ax simila o PTX, bu laulimalide, simila o pelo uside A, could no (Figu e 3). Figu e 3. Compe i ion o Flu ax-2 binding o cellula mic o ubules by di e en mic o ubule- s abilizing agen s. Figu e 3. Compe i ion o Flu ax-2 binding o cellula mic o ubules by di e en mic o ubule- s abilizing agen s. In . J. Mol. Sci. 2017,18, 971 7 o 18 HL-60 human p omyelocy ic leukemic cells we e co- ea ed wi h a combina ion o a mic o ubule-s abilizing agen (MSA) and Flu ax-2 (FTX) o 16 h, s ained wi h (4 0 ,6-diamidino-2- phenylindole) (DAPI), and he luo escence o he cells was examined in a con ocal mic oscope. Taxoid si e ligands, when in excess a 200 e sus 50 nM FTX, inhibi FTX binding since mos o he axoid si es a e occupied by he non- luo escen axoid si e ligand, hus no g een luo escen mic o ubles (MTs) a e seen. When FTX is in excess o he axoid si e ligands, he MTs luo esce g een since FTX occupies mos o he binding si es on he MTs. Rega dless o he concen a ion o laulimalide (LAU) o pelo uside A (PEL), he MTs always luo esce g een since simul aneous binding o hese wo MSAs a he LAU/PEL si e and Flu ax-2 (FTX) a he axoid si e can occu . When zampanolide (ZMP) is in excess o e FTX, no g een luo escence o he MT is appa en , as was seen wi h he o he axoid si e ligands. In con as , when FTX is in excess, no g een luo escence is p esen , e en a a low concen a ion o ZMP (25 nM). This lack o g een luo escence when FTX is in excess indica es ha ZMP can ou -compe e FTX because i co alen ly occupies he axoid binding si e, p e en ing FTX om displacing i . The PTX, PEL, and ZMP images ha e been p e iously published [ 39 ] and a e shown wi h pe mission om he publishe Else ie . Abb e ia ions a e: PTX = pacli axel; DTX = doce axel; IXA = ixabepilone; DSC = discode molide, and FTX = Flu ax-2. 2.3. Lack o Syne gis ic In e ac ions be ween ZMP and O he MSAs MSAs ha bind o di e en si es on ubulin ha e been shown o syne gize oge he i he app op ia e concen a ions o he wo compounds a e combined, as shown in Figu e 4. CI alues o Figu e 4a e p esen ed in Supplemen a y Da a Table S2. Fo example, in 1A9 cells, pelo uside A in combina ion wi h ixabepilone had a g ea e e ec on cell g ow h han he sum o he wo compounds. PTX, howe e , did no syne gize wi h ixabepilone, because PTX and ixabepilone bind a he same si e, he axoid si e. The excep ion o he ule is discode molide, a axoid si e MSA ha has p e iously been shown o syne gize wi h PTX [ 20 – 22 ], despi e being able o compe e o Flu ax binding (Figu e 3). The 1A9 o a ian ca cinoma cells we e he e o e ea ed wi h ZMP in combina ion wi h h ee o he axoid si e MSAs and wo laulimalide/pelo uside si e MSAs o de e mine i ZMP could syne gize wi h ei he g oup o compounds (Figu e 5). CI alues o Figu e 5a e p esen ed in Supplemen a y Da a Table S3. No syne gy was seen be ween ZMP and ei he a axoid si e MSA o a laulimalide/pelo uside si e MSA. Some an agonism was obse ed be ween PTX and ZMP, and laulimalide and ZMP. 2.4. Zampanolide E ec on Cell Mig a ion in Cul u e To asce ain whe he ZMP has he po en ial o p e en me as asis o cance cells, i s abili y o inhibi cell mig a ion in a wound sc a ch assay was ca ied ou . The abili y o human umbilical ein endo helial cells (HUVEC) o D551 human ib oblas s o epai a sc a ch made in he monolaye wi h a pipe e ip was es ed in he absence and p esence o ZMP and doce axel (Figu e 6). Bo h compounds signi ican ly inhibi ed wound eco e y in bo h cell lines in a concen a ion-dependen manne . Doce axel was sligh ly mo e po en han ZMP a inhibi ing cell mig a ion. In . J. Mol. Sci. 2017,18, 971 8 o 18 In . J. Mol. Sci. 2017, 18, 971 8 o 18 Figu e 4. Syne gis ic in e ac ions be ween MSAs. The Combina ion Index (CI) is g aphed o combina ions o MSAs in 1A9 cells gi en wo a a ime: (A) Pacli axel (PTX) and discode molide (DSC); (B) Pacli axel and doce axel (DXT); (C) Pacli axel and ixabepilone (IXA), and (D) pelo uside A (PELA) and ixabepilone a e p esen ed as he mean CI alue ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; one-sample S uden ’s - es compa ed o 1.0. Values less han 1.0 indica e syne gy be ween he wo compounds; alues g ea e han 1.0 indica e an agonism; alues equal o 1.0 indica e addi i i y. B A D C Figu e 4. Syne gis ic in e ac ions be ween MSAs. The Combina ion Index (CI) is g aphed o combina ions o MSAs in 1A9 cells gi en wo a a ime: ( A ) Pacli axel (PTX) and discode molide (DSC); ( B ) Pacli axel and doce axel (DXT); ( C ) Pacli axel and ixabepilone (IXA), and ( D ) pelo uside A (PELA) and ixabepilone a e p esen ed as he mean CI alue ± SEM. * p< 0.05; ** p< 0.01; *** p< 0.001; one-sample S uden ’s - es compa ed o 1.0. Values less han 1.0 indica e syne gy be ween he wo compounds; alues g ea e han 1.0 indica e an agonism; alues equal o 1.0 indica e addi i i y. In . J. Mol. Sci. 2017,18, 971 9 o 18 1 A B D C E Figu e 5. Syne gis ic in e ac ions o ZMP wi h selec ed MSAs. The Combina ion Index (CI) is g aphed o ZMP gi en in combina ion wi h o he MSAs: ( A ) Pacli axel (PTX); ( B ) Ixabepilone (IXE); ( C ) Pelo uside A (PEL); ( D ) Laulimalide (LAU), and ( E ) discode molide (DSC). Da a a e p esen ed as he mean CI alue ± SEM. * p< 0.05; ** p< 0.01; *** p< 0.001; one-sample S uden ’s - es compa ed o 1.0. 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