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. 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 16 o 18
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