Bessa e al.Cell Dea h and Disease (2018) 9:23
DOI 10.1038/s41419-017-0154-9 Cell Dea h & Disease
ARTICLE Open Access
Disco e y o a small-molecule p o ein
kinase Cδ-selec i e ac i a o wi h
p omising applica ion in colon cance
he apy
Cláudia Bessa
1
, Joana Soa es
1
, Liliana Raimundo
1
,JoanaB.Lou ei o
1
, Célia Gomes
2
, Flá io Reis
2
,MiguelL.Soa es
3
,
Daniel San os
4
, Che na Du eja
5
,SaumyaR.Chaudhu i
5
, Cyn hia Lopez-Habe
6
,Ma celoG.Kazanie z
6
, Jo ge Gonçal es
7
,
Ma ia F. Simões
8,9
, Pa ícia Rijo
8,9
and Lucília Sa ai a
1
Abs ac
P o ein kinase C (PKC) isozymes play majo oles in human diseases, including cance . Ye , he poo unde s anding o
isozymes-specific unc ions and he limi ed a ailabili y o selec i e pha macological modula o s o PKC isozymes ha e
limi ed he clinical ansla ion o PKC- a ge ing agen s. He e, we epo he fi s small-molecule PKCδ-selec i e
ac i a o , he 7α-ace oxy-6β-benzoyloxy-12-O-benzoyl oyleanone (Roy-Bz), which binds o he PKCδ-C1-domain. Roy-
Bz po en ly inhibi ed he p oli e a ion o colon cance cells by inducing a PKCδ-dependen mi ochond ial apop o ic
pa hway in ol ing caspase-3 ac i a ion. In HCT116 colon cance cells, Roy-Bz specifically igge ed he ansloca ion o
PKCδbu no o he pho bol es e esponsi e PKCs. Roy-Bz caused a ma ked inhibi ion in mig a ion o HCT116 cells in a
PKCδ-dependen manne . Addi ionally, he impai men o colonosphe e g ow h and o ma ion, associa ed wi h
deple ion o s emness ma ke s, indica e ha Roy-Bz also a ge s d ug- esis an cance s em cells, p e en ing umo
dissemina ion and ecu ence. No ably, in xenog a mouse models, Roy-Bz showed a PKCδ-dependen an i umo
e ec , h ough an i-p oli e a i e, p o-apop o ic, and an i-angiogenic ac i i ies. Besides, Roy-Bz was non-geno oxic, and
in i o i had no appa en oxic side e ec s. Collec i ely, ou findings e eal a no el p omising an icance d ug
candida e. Mos impo an ly, Roy-Bz opens he way o a new e a on PKC biology and pha macology, con ibu ing o
he po en ial edefini ion o he s uc u al equi emen s o isozyme-selec i e agen s, and o he e-es ablishmen o
PKC isozymes as easible he apeu ic a ge s in human diseases.
In oduc ion
P o ein kinase C (PKC) is a amily o se ine- h eonine
kinases g ouped based on hei dis inc egula ion in o
“con en ional”(cPKCs α,βΙ,βΙΙ,γ), “no el”(nPKCs δ,ε,η,
θ), and “a ypical”(aPKCs ι/λ,ζ). PKC isozymes sha e a
conse ed N- e minal egula o y egion comp ising he C1-
domain and C2-domain, and a C- e minal ca aly ic egion
esponsible o ATP binding and phospho ans e ase
ac i i y. The C1-domain is he pho bol es e and dia-
cylglyce ol (DAG) binding si e in cPKCs and nPKCs, whe e
i is duplica ed in andem (C1a and C1b). The C2-domain in
cPKCs binds calcium, whe eas in nPKCs i is p ima ily a
calcium-un esponsi e phospho- y osine binding mo i . On
he o he hand, aPKCs bind nei he pho bol es e /DAG no
calcium
1–3
. PKC isozymes a e in ol ed in he ac i a ion o
© The Au ho (s) 2018
Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion
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Co espondence: Pa ícia Rijo (p1609@uluso ona.p ) o Lucília Sa ai a
([email p o ec ed])
1
UCIBIO/REQUIMTE, Labo a ó io de Mic obiologia, Depa amen o de Ciências
Biológicas, Faculdade de Fa mácia, Uni e sidade do Po o, Po o, Po ugal
2
Labo a o y o Pha macology and Expe imen al The apeu ics, Ins i u e o
Biomedical Imaging and Li e Sciences (IBILI), Facul y o Medicine, & CNC.IBILI
Resea ch Conso ium, Uni e si y o Coimb a, Coimb a, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Edi ed by M. Daugaa d
O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion
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mul iple signaling pa hways, and hence hey a e ecognized
he apeu ic a ge s o se e al human diseases. Thei wide-
anging e ec s in c ucial p ocesses o umo igenesis and
me as a ic dissemina ion jus i y he e o s o de elop PKC-
a ge ed d ugs o cance ea men . Ye , he pha macolo-
gical modula ion o PKCs in an icance he apy has p o ed
gene ally ine ec i e in clinical ials. This can be explained
by he complex biological unc ions egula ed by PKC iso-
zymes, bo h edundan and opposi e, and hei significan
exp ession he e ogenei y in di e en cance ypes. Un o -
una ely, he e is a e y limi ed a ailabili y o isozyme-
selec i e PKC modula o s, which ep esen a majo
limi a ion o achie ing he apeu ic success. As such, he
gene a ion o isozyme-selec i e PKC modula o s has been
o high p io i y. Howe e , his has been a majo challenge
om a pha macological s andpoin , as PKCs a e highly
ela ed among hem, as well as o o he s uc u ally ela ed
kinases
1–3
.
Whe eas PKCs ha e been gene ally iewed as oncogenic
kinases, se e al labo a o ies ha e es ablished ha PKC
ac i i y is o en los in cance due o loss-o - unc ion
mu a ions, hus suppo ing a po en ial unc ion o PKC
isozymes as umo supp esso s a he han umo p o-
mo e s
3
. Mo eo e , specific PKC isozymes induce an i-
p oli e a i e o p o-apop o ic e ec s in cance cells upon
ac i a ion
4–7
. This led esea che s o belie e ha cance
he apies should be ocused on es o ing a he han inhi-
bi ing hei ac i i y, and may jus i y he ine ec i eness o
mo e han h ee decades o clinical ials using non-selec i e
PKC inhibi o s. The e o e, he selec i e PKC ac i a ion may
be a aluable he apeu ic s a egy in cance ea men
3–7
.
Fo long, PKC signaling has been closely associa ed wi h
in es inal ca cinogenesis
5,8,9
, one o he mos p e alen
cance s and a leading cause o cance mo ali y wo ld-
wide
10
. Pa icula ly, clinical da a ha e e ealed educed
p o ein le els o PKCδin colon cance issues compa ed
o no mal issues
11
. In ac , se e al s udies co obo a ed
he no ion ha PKCδbeha es as umo supp esso in
colon cance
11–15
. The e o e, besides hei po en ial
applica ion in o he cance ypes, i is highly concei able
ha PKCδ-selec i e ac i a o s may be he apeu ically
beneficial o pa ien s wi h colon cance . He e, we iden-
ified he compound 7α-ace oxy-6β-benzoyloxy-12-O-
benzoyl oyleanone (Roy-Bz) as a PKCδ-selec i e ac i a o
wi h p omising a ge ed an icance ac i i y in colon can-
ce . Impo an ly, Roy-Bz may also ep esen a aluable
he apeu ic al e na i e in o he human pa hologies
in ol ing an impai men o PKCδ-signaling pa hway.
Resul s
Roy-Bz is a selec i e ac i a o o PKCδ ha binds o he
C1-domain
In o de o iden i y no el PKC ac i a o agen s, se e al
na u al di e penoids isola ed om plan species belonging
o he Lamiaceae amily and semi-syn he ic de i a i es
we e es ed using a p e iously de eloped yeas PKC
sc eening assay
16
. In his assay, PKC ac i a o s induce a
significan g ow h inhibi ion in mammalian PKC-
exp essing yeas , which is p opo ional o he deg ee o
PKC ac i a ion, ha ing no e ec on con ol yeas (emp y
ec o )
16
. Using his app oach, i was obse ed ha
whe eas 0.1–30 μM pho bol 12-my is a e 13-ace a e
(PMA; an es ablished ac i a o o cPKCs and nPKCs)
inhibi ed he g ow h o yeas -exp essing cPKCs (αand βI)
and nPKCs (δand ε), 0.1–30 μM o Roy-Bz (Fig. 1a) only
inhibi ed he g ow h o PKCδ-exp essing yeas wi hou
significan e ec s on yeas exp essing o he PKCs (Fig. 1b).
In e es ingly, in PKCδ-exp essing yeas , while PMA-
induced g ow h inhibi ion was associa ed wi h G2/M-
phase cell cycle a es , Roy-Bz-induced g ow h inhibi ion
was media ed by apop o ic cell dea h, as demons a ed by
he inc ease in DNA agmen a ion wi h p ese a ion o
plasma memb ane in eg i y (Fig. 1d, e). Addi ionally, Roy-
Bz-induced g ow h inhibi ion was comple ely abolished in
yeas exp essing a PKCδin which he C1-domain has been
dele ed (ΔC1-PKCδ; Fig. 1c) (Fig. 1b). In ac , a simila
esul was ob ained wi h PMA, which is known o bind
PKCs a he C1-domain (Fig. 1b). These esul s indica e
ha Roy-Bz is a PKCδ-selec i e ac i a o and, like PMA, i
should ha e he C1-domain as he p edic ed binding si e.
To confi m he PKC isozyme selec i i y obse ed in he
yeas assay, we nex pe o med an in i o kinase assay using
ecombinan human cPKCs (a mix o PKCα,β,andγ),
nPKCδ,nPKCε,andaPKCζ. All ecombinan PKCs we e
e ec i ely ac i a ed a 10
−4
µM PMA/a achidonic acid
(ARA) (posi i e con ols: PMA o cPKCs and nPKCs; ARA
o aPKCζ)(Fig.1 ). Rema kably, 10
−7
–10
−3
μMRoy-Bz
only inc eased he ac i i y o PKCδ(Fig. 1g). Roy-Bz ac i-
a ed PKCδwi h an EC
50
(concen a ion equi ed o induce
50% e ec ) alue o 58.8 ±3.6 nM (n=5), simila o ha o
PMA (61.9 ±4.3 nM, n=5). Thus, he in i o kinase assays,
in acco dance wi h he yeas assays, e ealed a ema kable
selec i i y o Roy-Bz o PKCδ. Besides, hese esul s show
ha Roy-Bz is as e ec i e as PMA o ac i a e PKCδ.
Nex , he po en ial binding mode o Roy-Bz o PKCδ
was explo ed by molecula docking s udies (Fig. 1h, i).
Like 13-ace ylpho bol (PRB; posi i e con ol), which
es ablishes di ec s ong hyd ogen bond in e ac ions wi h
Gly253 and Th 242 (hyd ogen bond dono s), and Leu251
(hyd ogen bond accep o ) in he C1-domain o PKCδ,
Roy-Bz also in e ac ed wi h Gly253 and Th 242, bu
exchanged Leu251 wi h Gln257, all ac ing as hyd ogen
dono s o ca bonyl oxygen unc ional g oups. I is in e -
es ing o no e ha like in he case o PRB, hese in e ac-
ions also s abilized he molecule om bo h sides o he
cle . The es ima ed au odock ee ene gy o binding o
PRB was −7.54 kcal/mol and o Roy-Bz was −7.87 kcal/
mol, and a clus e ing analysis o he molecula docking
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 2 o 16
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Fig. 1 (See legend on nex page.)
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 3 o 16
O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion
esul s showed o he h ee poses simila o he op- anked
( he mos popula ed one; in a o al o 10 poses and 6
di e en clus e s). These p edic ed binding models sup-
po he Roy-Bz binding o he PKCδ-C1-domain.
The ansloca ion o PKC om he cy osol o mem-
b anes, ei he plasma memb ane o in e nal memb anes,
is a well-known hallma k o PKC ac i a ion
1,17
. As such,
we e alua ed he impac o Roy-Bz on he ansloca ion o
(see figu e on p e ious page)
Fig. 1 Roy-Bz selec i ely ac i a es PKCδ h ough binding o he C1-domain. a Chemical s uc u e o Roy-Bz. bDose– esponse cu es o he
inhibi o y e ec o PMA and Roy-Bz on he g ow h o yeas exp essing mammalian PKC isozymes and con ol yeas (emp y ec o ), o 42 h ea men ;
g ow h o yeas incuba ed wi h ehicle was se as 100%. cSchema ic ep esen a ion o dele ion o C1-domain a amino acids 231–283 in he PRKCD gene.
d,eE ec o 10 μMPMAandRoy-Bzon(d) cell cycle p og ession and (e) DNA agmen a ion (TUNEL+)/loss o plasma memb ane in eg i y (PI+) o PKCδ-
exp essing yeas (and con ol yeas in (e)), o 42 h ea men . ,gIn i o kinase assay wi h ecombinan PKCs; inc ease o PKC ac i i y by 10
−4
μMPMA/
ARA and 10
−4
μMRoy-Bz;gdose– esponsecu es o heinc easeo PKCac i i y by Roy-Bz; kinase ac i i y o endogenous PKC ac i a o phospha idylse ine
was se as 1; wo-way analysis o a iance (ANOVA) (p< 0.01). h,iMolecula docking s udies o Roy-Bz in e ac ion wi h he C1-domain o PKCδ.hTop
anked docking pose o Roy-Bz wi h Gly253, Th 242, and Gln257 highligh ed h ough a CPK ep esen a ion. The hyd ogen bonds be ween Roy-Bz and hese
esidues a e also displayed h ough a do ed blue line. The in e ac ion be ween he alpha-hyd ogen o T p252 and he ca bonyl (CH•••O) is also ep esen ed
and he esidue is highligh ed in lico ice. All esidues wi hin 4.5 Å o Roy-Bz a e ep esen ed and he molecula su ace o he pocke is displayed h ough a
mesh ha changes om g een in lipophilic egions o pink (hyd ophilic). iScheme o PRB and Roy-Bz in e ac ions. The pocke p oximi y con ou is
ep esen ed by g ay do ed lines, he ligand exposu e is depic ed in iole and he ecep o exposu e in blue. Di ec in e ac ions a e ep esen ed by a
do ed o g een line along wi h he in e ac ion ene gy (kcal/mol) es ima ed by MOE. In b,d– , da a a e mean ± SEM o fi e independen expe imen s;
alues significan ly di e en om con ol yeas b,eo ehicle d, :*p < 0.05, **p < 0.01, unpai ed S uden ’s - es
Fig. 2 Roy-Bz selec i ely ansloca es PKCδ o he pe inuclea egion in human umo cells. HCT116 cells ans ec ed wi h pEGFP-N1-PKCα,
pEGFP-N1-PKCδ,o pEGFP-N1-PKCεwe e ea ed wi h PMA, Roy-Bz, o ehicle o 1 h; PKC localiza ion was de e mined by con ocal mic oscopy. aImages
a e ep esen a i e o h ee independen expe imen s; scale ba = 10 µm and magnifica ion = ×100. bQuan ifica ion o cells wi h PKC ansloca ion; da a a e
mean ± SEM o h ee independen expe imen s; alues significan ly di e en a e indica ed (***p< 0.001), unpai ed S uden ’s - es
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 4 o 16
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Fig. 3 (See legend on nex page.)
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 5 o 16
O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion

PKCα, PKCδ, and PKCεin human HCT116 colon cance
cells using GFP- used PKCs (Fig. 2a, b). As p e iously
epo ed in LNCaP p os a e cance cells
18
, also in
HCT116 cells he ea men wi h PMA igge ed he
subcellula edis ibu ion o he h ee PKC isozymes.
The e was a clea ansloca ion o he plasma memb ane
o PKCα, PKCδand PKCε, and as epo ed in o he
models
18
, PKCδalso ansloca ed o a pe inuclea com-
pa men . When we assessed he abili y o Roy-Bz o
ansloca e PKCs, he e we e ob ious di e ences wi h
ega d o PMA. Indeed, Roy-Bz was unable o ansloca e
PKCαo PKCε, while i selec i ely ansloca ed PKCδ o
he pe inuclea egion (Fig. 2a, b). A dose-dependen
analysis o PKCδ ansloca ion in HCT116 cells e ealed
an EC
50
alue o 0.19 ±0.07 μM(n=3).
Al oge he , hese esul s s ongly indica e ha Roy-Bz is
a selec i e PKCδac i a o , ac ing a he C1-domain.
Roy-Bz inhibi s he p oli e a ion o colon cance cells
The e ec o Roy-Bz on p oli e a ion o human colo -
ec al cance cells was e alua ed using a sul o hodamine B
(SRB) assay (Fig. 3a, b). T ea men o HCT116, HT-29, and
SW-837 cells (wi h simila PKCδexp ession le els; Sup-
plemen a y Fig. S1) wi h Roy-Bz o 48 h esul ed in a
dose-dependen inhibi ion o cell g ow h (IC
50
alues o
0.58 ±0.05 μM o HCT116, 1.50 ±0.06 μM o HT-29,
and 1.08 ±0.03 μM o SW-837; n=5). The p onounced
inhibi o y e ec o Roy-Bz on cell p oli e a ion/ iabili y o
colo ec al cance cells was u he demons a ed by
assessing he colony- o ming abili y (Fig. 3c). In ac , e en
in he mos esis an SW-837 cells, 0.3 μM Roy-Bz educed
he colony- o ming abili y by ~75% ela i e o ehicle.
In e es ingly, while in HT-29 and SW-837 cells he Roy-
Bz g ow h inhibi o y e ec was associa ed wi h G2/M-
phase cell cycle a es (Fig. 3d; pa icula ly p onounced in
HT-29) and apop osis (Fig. 3e), in HCT116 cells he Roy-
Bz-induced g ow h inhibi ion was only media ed by
apop osis. In ac , Roy-Bz had no e ec on cell cycle
p og ession (Fig. 3d; simila esul s we e ob ained o 8,
16, and 48 h ea men , da a no shown) and ma kedly
inc eased Annexin V-posi i e cells (Fig. 3e). Con e sely,
in HCT116 cells, PMA, wi h an IC
50
alue abou 14- old
highe han ha o Roy-Bz (7.83 ±1.67 μM, n=5; Fig. 3a),
inhibi ed cell g ow h h ough induc ion o S-phase cell
cycle a es and apop osis (Supplemen a y Fig. S2).
In HCT116 cells, he induc ion o apop osis by Roy-Bz
was u he ein o ced by he occu ence o caspase-3 and
PARP clea age, an inc ease in p o-apop o ic p53 and Bax
le els, and a educ ion in he le els o an i-apop o ic
p o eins Bcl-2 and su i in (Fig. 3 , g). The in ol emen o
he mi ochond ial pa hway in Roy-Bz-induced apop osis
was also e idenced by he inc ease o mi ochond ial
eac i e oxygen species (ROS) gene a ion and Δψ
m
dis-
sipa ion (Fig. 3h), as well as he elease o cy och ome c
(cy c) o cy osol (Fig. 3i).
In an a emp o explo e he an i umo ac i i y o Roy-
Bz in a sys em ha mo e closely esembles he in i o
ea u es o he umo mic oen i onmen and is highly
en iched in a small popula ion o cance s em cells
(CSCs), a colonosphe e cul u e model was gene a ed om
HCT116 cells. In ac , he sphe oid- o ma ion (colono-
sphe e) assay is ecognized as a aluable ool o assess-
men and expansion o s em cells in colon cance
19
.In
addi ion, acco ding o se e al s udies, colon CSCs can
mainly be iden ified by he exp ession o cell bioma ke s,
such as CD44 and ALDH
20–23
. Ac ually, a posi i e co -
ela ion be ween he CD44 dec ease and he he apeu ic
esponse o pa ien s wi h colo ec al cance has been
ecen ly iden ified
22,23
. To es he e ec o Roy-Bz in
colonosphe e g ow h, 3-day-old sphe oids we e ea ed
wi h Roy-Bz o up o 96 h (Fig. 3j). A significan educ-
ion in colonosphe e diame e by Roy-Bz ela i e o
ehicle was obse ed bo h a 72 h (a 3 and 3.5 μM) and
96 h (a 2–3.5 μM). Acco dingly, 3.5 μM Roy-Bz ma kedly
educed he exp ession le els o he s emness ma ke s
CD44 and ALDH2 a e 96 h ea men (Fig. 3k). O no e,
a p onounced educ ion o ma ix me allop o einase 9
(MMP-9) and inc ease o E-cadhe in, p ominen playe s
(see figu e on p e ious page)
Fig. 3 Roy-Bz inhibi s he g ow h o colon cance cells. a,bDose– esponse cu es o he g ow h o (a) HCT116, (b) HT-29 and SW-837 cells
ea ed wi h 0.11–10 μM Roy-Bz o PMA o 48 h; da a a e mean ± SEM o fi e independen expe imen s; g ow h ob ained wi h ehicle was se as
100%. cColony o ma ion assay o HCT116, HT-29, and SW-837 cells ea ed wi h Roy-Bz; images co espond o a ep esen a i e expe imen o ou ;
g aphs ep esen mean ± SEM o ou independen expe imen s. dCell cycle a es and eAnnexin V-posi i e cells we e de e mined o 24 h
ea men wi h 1 μM Roy-Bz; da a a e mean ± SEM o ou independen expe imen s. ,gWes e n blo analysis o ( ) caspase-3 and (g) PARP clea age,
Bax, p53, Bcl-2, and su i in exp ession le els, o 24 h ea men wi h Roy-Bz in HCT116 cells. hMi ochond ial ROS gene a ion ( o 24 h) and Δψ
m
dissipa ion ( o 16 h) igge ed by 0.5 μM Roy-Bz ea men in HCT116 cells; da a a e mean ± SEM o ou independen expe imen s. iMi ochond ial
cy c elease in HCT116 cells o 24 h ea men wi h Roy-Bz. jE alua ion o 3-day-old HCT116 sphe oids diame e o 72 and 96 h ea men wi h Roy-
Bz o ehicle. kCD44, ALDH2, MMP-9, and E-cadhe in exp ession le els o 96 h ea men wi h 3.5 μM Roy-Bz. lE alua ion o sphe oids diame e
o med a e 48 h ea men o seeded HCT116 cells wi h Roy-Bz o ehicle. In jand l, b igh field imaging o sphe oids (scale ba = 50 μm,
magnifica ion = ×100); g aphs ep esen mean ± SEM o ou independen expe imen s. In ,g,iand kimmunoblo s ep esen one o h ee
independen expe imen s; loading con ol o cy osolic (GAPDH) and mi ochond ial (COX IV) ac ions. In c,d,e,h,jand l, alues significan ly di e en
om ehicle (*p < 0.05, **p < 0.01, ***p < 0.001), unpai ed S uden ’s - es
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 6 o 16
O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion
in ex acellula ma ix homeos asis and me as asis
24
, we e
also obse ed unde hese ea men condi ions (Fig. 3k).
The e ec i eness o Roy-Bz in colonosphe e o ma ion
was also checked by e alua ing he colonosphe e diame e
a e 48 h ea men wi h Roy-Bz added a he seeding
ime o HCT116 cell suspension (Fig. 3l). The esul s
Fig. 4 (See legend on nex page.)
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 7 o 16
O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion
showed a no able dose-dependen educ ion in colono-
sphe e o ma ion abili y by Roy-Bz, wi h an abolishmen
o colonosphe e o ma ion a 1 μM Roy-Bz.
Al oge he , hese esul s e idence a po en inhibi o y
ac i i y o Roy-Bz in he g ow h o colon cance cells,
including in CSCs, sugges ing a po en ial e ec o he
d ug agains cance chemo esis ance, dissemina ion, and
ecu ence.
Roy-Bz p o-apop o ic and an i-mig a o y ac i i y in
HCT116 cance cells is media ed by PKCδ-selec i e
ac i a ion
To e alua e he in ol emen o PKCδin Roy-Bz-
induced apop osis, a PKCδs able knockdown model
was gene a ed upon ans ec ion o he me as a ic colon
cance HCT116 cells wi h a PKCδRNAi plasmid. As
con ol, we used HCT116 cells ans ec ed wi h non-
a ge con ol RNAi. PKCδsilencing was confi med by
Wes e n blo (Supplemen a y Fig. S3). The impac o
0.1–6μM Roy-Bz on he iabili y o PKCδ-deple ed and
con ol HCT116 cells was he ea e e alua ed by a ypan
blue assay. As shown in Fig. 4a, silencing PKCδdec eased
Roy-Bz-induced cell dea h compa ed o con ol HCT116
cells. Acco dingly, he deple ion o PKCδin HCT116 cells
abolished Roy-Bz-induced Annexin V-posi i e cells
(Fig. 4b), PARP clea age, and inc ease o Bax and p53
exp ession le els (Fig. 4c).
The impac o Roy-Bz on MMP-9 and E-cadhe in
exp ession le els in colonosphe e cul u es led us o
u he in es iga e he e ec o Roy-Bz on he mig a ion
o HCT116 cells and i s dependence on PKCδ.Usinga
wound healing assay, we ound ha Roy-Bz caused a
p onounced inhibi ion o mig a ion. No ably, he inhibi o y
e ec o Roy-Bz was no obse ed in HCT116 cells
subjec o PKCδ-knockdown. I is impo an o no e ha
a he concen a ion o Roy-Bz used in hese assays
(0.25 μM), he e we e no significan e ec s on cell iabili y
(Fig. 4d, e). These esul s we e also ecapi ula ed using a
chemo axis cell mig a ion assay, in which he an i-mig a o y
e ec o Roy-Bz obse ed in con ol HCT116 cells was
significan ly p e en ed by RNAi silencing o PKCδexp es-
sion (Fig. 4 ).
I has been p e iously es ablished in ce ain cellula
models ha PKCδunde goes a p o eoly ic clea age by
caspases ha gene a es a ca aly ically ac i e agmen
(PKCδ-CF)
6,7
. In e es ingly, we ound ha in HCT116
cells, Roy-Bz caused a ime-dependen inc ease in PKCδ-
CF le els, wi hou a isible in e e ence in o al PKCδ
exp ession le els (Fig. 4g).
Since i was p e iously epo ed ha ac i a ion o PKCδ
is a equi ed ac o o his one H3 phospho yla ion on
Se -10 (pSe -10 his one H3), which is a c ucial e en in
apop osis
25
, we nex assessed he e ec o Roy-Bz on
pSe -10 his one H3 le els in HCT116 cells. To his end,
HCT116 cells we e synch onized in G1-phase by hyd o-
xyu ea, in o de o exclude mi o ic pSe -10 his one H3,
ollowed by ea men wi h 1 μM Roy-Bz o up o 8 h. We
obse ed ha 1 μM Roy-Bz caused a p onounced inc ease
o pSe -10 his one H3 le els ha was no iceable a 8 h.
This e ec was abolished by PKCδ-knockdown (Fig. 4h, i),
again ein o cing he concep ha Roy-Bz is a po en
induce o apop o ic cance cell dea h ia ac i a ion o
PKCδ.
Al oge he , hese esul s showed ha Roy-Bz is a po en
p o-apop o ic and an i-mig a o y agen in human colon
cance cells h ough PKCδ-selec i e ac i a ion.
Roy-Bz is non-geno oxic in human cance and no mal cells
and has in i o PKCδ-dependen an i umo ac i i y wi h
no appa en oxic side e ec s
The geno oxici y o Roy-Bz in human cance cells was
e alua ed by checking come -posi i e cells and his one
H2AX phospho yla ion on Se ine-139 (γH2AX) as ma -
ke s o DNA damage (single/double-s and b eaks). Ou
esul s showed ha , con e sely o 25 μM e oposide
(ETOP; posi i e con ol), 48 h ea men wi h Roy-Bz
ailed o induce DNA damage in HCT116 cells, as
demons a ed by he negligible numbe o cells wi h mo e
han 5% o DNA in ail (Fig. 5a, b), by he low ail momen
(Fig. 5a, c), as well as by he absence o inc eased γH2AX
(see figu e on p e ious page)
Fig. 4 P o-apop o ic and an i-mig a o y ac i i y o Roy-Bz is media ed by selec i e ac i a ion o PKCδin HCT116 cance cells. a Cell iabili y
dose– esponse cu es we e de e mined by ypan blue assay o con ol (Mock) and PKCδ-knockdown (PKCδRNAi) HCT116 cells ea ed wi h 0.1–6
μM Roy-Bz o 24 h; cell iabili y o ehicle was se as 100%. bAnnexin V-posi i e cells we e de e mined in con ol and PKCδ-knockdown HCT116 cells
a e 24 h ea men wi h 1 μM Roy-Bz o ehicle. cWes e n blo analysis o PARP clea age, p53 and Bax exp ession le els o 16 h ea men wi h 1
μM Roy-Bz o ehicle in con ol and PKCδ-knockdown HCT116 cells; GAPDH (loading con ol). d,eWound healing assay o con ol and PKCδ-
knockdown HCT116 confluen cells ea ed wi h 0.25 µM Roy-Bz o ehicle, a di e en ime-poin s; in d, scale ba = 50 µm and magnifica ion = ×100;
in e, quan ifica ion o wound closu e in fi e andomly selec ed mic oscopic fields. Chemo axis mig a ion assay o con ol and PKCδ-knockdown
HCT116 cells o 24 h ea men wi h 0.25 µM Roy-Bz; mig a o y cells we e quan ified by fluo escence in ensi y, which was se as 100% o un ea ed
cells. gWes e n blo analysis o ull-leng h PKCδ(PKCδ-FL) and clea ed PKCδ agmen (PKCδ-CF) in HCT116 cells ea ed wi h 1 μM Roy-Bz o up o
24 h. h,iLe els o his one H3 phospho yla ion on Se -10 (pSe -10) in con ol and PKCδ-knockdown HCT116 cells ea ed wi h 1 μM Roy-Bz o up o 8
h; his one H3 (loading con ol); in i, quan ifica ion o pSe -10 le els no malized o his one H3, alues o asynch onized cells we e se as 1. In c,g, and
h, immunoblo s ep esen one o h ee independen expe imen s; in a,b,e, , and ida a a e mean ± SEM o h ee independen expe imen s; alues
significan ly di e en a e indica ed (*p< 0.05, **p < 0.01, ***p< 0.001), unpai ed S uden ’s - es
Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 8 o 16
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Fig. 5 (See legend on nex page.)
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